MXPA00009215A - GFR&agr;3 AND ITS USES - Google Patents

Abstract

The present invention relates to nucleotide sequences, including expressed sequence tags (ESTs), oligonucleotide probes, polypeptides, vectors and host cells expressing, and immunoadhesions and antibodies to mammalian GFR&agr;3, a novel&agr;-subunit receptor of the GDNF (i.e. GFR) receptor family. It further relates to an assay for measuring activation of a&agr;-subunit receptor by detecting tyrosine kinase receptor activation (i.e., autophosphorylation) or other activities related to ligand-induced&agr;-subunit receptor homo-dimerization or homo-oligomerization.

Description

QRFALFA3 AND ITS USES Technical Field of the Invention The present invention relates generally to the identification and isolation of a novel DNA and to the recombinant production of novel polypeptides which are characterized by the presence of sequences of GFRα3, a receptor of subunit a. It also relates in addition to an assay for measuring the ligand-induced activation of a subunit receptor by detecting the autophosphorylation of a kinase domain of a receptor tyrosine kinase (TKP) receptor (rPTK) fusion using an enzyme-linked immunosorbent assay, kinase receptor activation (KIRA ELISA) or by any other means to detect the homodimerization of the subunit a.

Introduction Background Neurotrophic factors such as insulin-like growth factors, Rf.123110 nerve growth factor, brain-derived neurotrophic factor, ciliary neurotrophic factor neurotrophin-3, -4/5 and -6, GDNF , and neurturin has been proposed as a potential means to improve the survival of specific neuronal cells, for example, as a treatment for neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer's disease, seizures, epilepsy, Huntington's disease, Parkinson's disease, and peripheral neuropathy. It may be desirable to provide additional therapy for this purpose. The neurotrophic factors of the protein, or neurotrophins, which have an influence on the growth and development of the vertebrate nervous system, are believed to play an important role in promoting the differentiation, survival, and function of various groups. of neurons in the brain and the periphery. Neurotrophic factors are thought to have important signaling functions in neural tissues, based in part on the precedent established with nerve growth factor (NGF). NGF supports or supports the survival of neurons of the sympathetic, sensory and basal forebrain nervous system, both in vitro and in vivo. The administration of exogenous NGF rescues neurons from cell death during development. In contrast, the removal or sequestration of endogenous NGF by the administration of anti-NGF antibodies promotes such cell death (Heumann, J. Exp. Biol., 132: 133-150 (1978); Hefti, J. Neurosci., 6 : 2155-2162 (1986), Thoenen et al., Annu. Rev. Physiol., 60: 284-335 (1980)). Additional neurotrophic factors related to NGF have been identified accordingly. These include the brain-derived neurotrophic factor (BDNF) (Leibrock, et al., Nature, 341: 149-152 (1989), neurotrophin-3 (NT-3) (Kaisho, et al., FEBS Lett., 266: 187 ( 1990)), Maisonpierre, et al., Science, 247: 1466 (1990), Rosenthal, et al., Neuron, 4: 767 (1990)), and neurotrophin 4/5 (NT-4/5) (Berkmeir, et al. ., Neuron, 7: 857-866 (1991)). Neurotrophins, similar to other growth factors of polypeptides, affect their target or target cells through interactions with cell surface receptors. According to the common understanding, two classes of transmembrane glycoproteins act as receptors for known neurotrophins. Equilibrium binding studies have shown that neuronal neurotrophin response cells have a common low molecular weight (65,000-80,000 daltons), a low affinity receptor typically referred to as a p75LNGFR or p75, and a high molecular weight receptor (130,000-150,000 Daltons). High affinity receptors are members of the trk family of receptor tyrosine kinases. The receptor tyrosine kinases are known to serve as receptors for a variety of protein factors that promote profiling, differentiation, and cell survival. In addition to the trk receptors, examples of other receptor tyrosine kinases include epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). Typically, these receptors expand on the cell membrane, with one portion of the receptor that is intracellular and in contact with the cytoplasm, and another portion of the receptor that is extracellular. The binding of a ligand to the extracellular portion of the receptor induces tyrosine kinase activity in the intracellular portion of the receptor, with the resulting phosphorylation of several intracellular proteins involved in cellular signaling pathways. The neurotrophic factor derived from the glial cell line ("GDNF") and Neurturin ("NTN") are two structurally related, recently identified, potent survival factors for neurons in the central nervous and sympathetic sensory systems (Lin et al. Science 260: 1130-1132 (1993) Henderson et al., Science 266: 1062-1064 (1994) Buj-Bello et al., Neuron 15: 821-828 (1995) Kotzbauer et al., Nature 384: 467-470 (1996) ). Recently, GNDF was shown to have an intermediation in its actions through the multiple component receptor system of a ligand binding glycosyl-phosphatidyl inositol (GPI) protein (designated GDNFRa).; also designated GFR-a-1) and Ret tyrosine kinase of the transmembrane receptor (Treanor et al., Nature 382: 80-83 (1996) Jing et al., Cell 85: 1113-1124 (1996) Trupp et al., Nature 381: 785-789 (1996) Durbec et al., Nature 381: 789-793 (1996)). The NTN signal is transmitted by means of GFRa2, which is also associated with Ret. Proteins and receptors bound to the membrane can play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, for example, proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and / or from the immediate environment. This information is frequently transmitted by secreted polypeptides (for example, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones) which are, in turn, received and interpreted. by various receptors of the cells or proteins bound to the membrane. Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules similar to selectins. and integrins. For example, the transduction of signals that regulate cell growth and differentiation is regulated in part by the phosphorylation of several cellular proteins. Protein tyrosine kinases, the enzymes that catalyze this process, can also act as growth factor receptors. Examples include the fibroblast growth factor receptor and the growth factor receptor. The membrane-bound proteins and the receptor molecules have various industrial applications, including as pharmaceutical and diagnostic agents. The receptor immunoadhesins, for example, can be used as therapeutic agents to block the interaction of the ligand-receptor. The membrane-bound proteins can also be used to select the potential peptide or small molecule inhibitors of the relevant receptor / ligand interaction. The aberrant expression or uncontrolled regulation of any of these receptor tyrosine kinases can lead to different malignancies and pathological disorders. Therefore, there is a need to identify means to regulate, control and manipulate receptor tyrosine kinases ("RTKs"), their ligands, or their receptor molecules of the subunit a, eg, the subunit receptors to GPI-linked. , to which they are associated, to provide new and additional means for the diagnosis and therapy of disorders related to the receptor tyrosine kinase pathway and cellular processes. The present application provides physicians and researchers with such means to provide novel molecules that are specific to interact with certain receptor genes and their gene products. These compounds and their methods of use, as provided herein, allow exquisite therapeutic control and specificity. Accordingly, it is an object of the present invention to provide an improved therapy for the prevention and / or treatment of neurological conditions and other conditions in the. which certain neurotrophic signaling pathways play a role.

Brief Description of the Invention Applicants have identified a family of cDNAs encoding a novel human polypeptide or its homologs, designated in the present application as "GFR3." GFR3 is a receptor for the subunit a, a receptor that forms a complex with a receptor of the beta subunit in response to ligand binding Subunits A provide the binding component of the ligand and the beta subunit provides the transduction activity of the catalytic signal, such as tyrosine kinase activity. of GRAP form a complex with a receptor of the beta subunit referred to as Ret This hetero-complex leads to signal transduction The present invention is based in part on the novel discovery that the a subunit can be dimerized on the binding ligand , and also the dimerization can activate a kinase activity of a kinase catalytic domain fused to the ligand binding domain of the receiver of the subunit a.

In one embodiment, the invention provides an isolated nucleic acid molecule having at least about 65% sequence identity with (a) the sequence of nucleic acids encoding a GFRα3 polypeptide comprising the amino acid sequence 27 to 400 of SEQ ID NO: 15, amino acids 27 to 369 of the SEQ ID NO: 17 or amino acids 27 to 374 of SEQ ID NO: 5 or (b) the complement of the nucleic acid molecules of (to) . In another embodiment, the above nucleic acid sequence comprises a ligand binding domain of a GFRα3 polypeptide of amino acids 84 to 360 of SEQ ID NO: 15, amino acids 84 to 329 of SEQ ID NO: 17, or the amino acid sequence 110 to 386 of SEQ ID NO: 20, or its complementary nucleic acids. The isolated nucleic acid comprises a sequence encoding GFRα3 which hybridizes preferentially under stringent conditions to the nucleic acid sequences encoding a GFRα3 polypeptide of the invention. The identity of the sequence is preferably at least about 75%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%. In one aspect, the encoded polypeptide has at least about 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and still more preferably at least 95% identity of the sequence with a polypeptide having amino acid residues 27 to 400 of SEQ ID NO: 15, amino acids 27 to 369 of SEQ ID NO: 17, amino acids 27 to 374 of SEQ ID NO: 5, a ligand binding domain of a GFRα3 polypeptide of amino acids 84 to 360 of SEQ ID NO: 15, amino acids 84 to 329 of SEQ ID NO: 17, or amino acid sequence 110 to 386 of SEQ ID NO: 20. Preferably, the identity is from amino acid residues 27 to 400 of SEQ ID NO: 15 and the DNA encoding them. In a further embodiment, the isolated nucleic acid molecule comprises the DNA encoding a GFRα3 polypeptide having amino acid residues 27 to 400 of SEQ ID NO: 15, or its complement for such coding of the nucleic acid sequence, and remains stably bound thereto under highly stringent conditions, and optionally, at least moderate. In another aspect, the invention provides a nucleic acid from the full length protein of clone DNA48613, DNA48614 or murine GFR3 (clone 13), deposited in the ATCC under accession numbers ATCC2009752 (Designation: DNA48613-1268), ATCC209751 (Designation: DNA48614-1268), and ATCC, respectively, on April 7, 1998. In yet another embodiment, the invention provides a vector comprising the DNA encoding the GFRα3 polypeptide. A host cell comprising such a vector is also provided. By way of example, the host cells can be CHO cells, of E. Coli, or yeast. A process for producing the GFRα3 polypeptides is further provided and comprises culturing the host cells under conditions suitable for the expression of GFRα3 and recovery thereof from the culture of the cells. In still another embodiment, the invention provides an isolated GFRα3 polypeptide. In particular, the invention provides GFRα3 polypeptides of the isolated native sequence, which in one embodiment, include an amino acid sequence comprising amino acids 27 to 400 of SEQ ID NO: 15. The natural GFRα3 polypeptides with or without the sequence of the natural signal (amino acids 1 to 26) in SEQ ID NO: 15, and with or without the initiating methionine, are specifically included. In yet another embodiment, a polypeptide comprising a sequence of amino acid residues 27 to 400 of SEQ ID NO: 15, amino acids 27 to 369 of SEQ ID NO: 17 or amino acids 27 to 374 of SEQ ID NO is provided. : 5, a ligand binding domain of a GFRα3 polypeptide of amino acids 84 to 360 of SEQ ID NO: 15, amino acids 84 to 329 of SEQ ID NO: 17 or the sequence of amino acids 110 to 386 of SEQ ID NO: 20. Alternatively, the invention provides a GFRα3 polypeptide encoded by the nucleic acid deposited under the above access numbers. The polypeptide is optionally lacking the hydrophobic sequence associated with anchoring or GPI binding. In yet another embodiment, the invention provides chimeric molecules comprising a GFRα3 polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises a GFRα3 polypeptide fused to an epitope tag sequence or an Fc region of an immunoglobulin. Chimeric molecules can comprise the ligand binding domain of a subunit a receptor, the intracellular catalytic domain of a tyrosine kinase receptor, and a flag epitope. In yet another embodiment, the invention provides an antibody which specifically binds to the GFRα3 polypeptide. _Optionally, the antibody is a monoclonal antibody. In view of the surprising discovery here that the a subunit receptor can be dimerized during ligand binding, and in addition to that such dimerization can activate a kinase domain fused to the a subunit receptor, a method for measuring the activation of the receptor of the subunit a induced by the ligand, ie homo-dimerization or homo-oligomerization. In one embodiment, it is provided in a reliable, sensitive assay, which measures the activation of the receptor of the subunit a induced by the agonist or the ligand, ie the homo-dimerization or the ho-oligomerization, preferably by the measurement of the autophosphorylation of receptor protein tyrosine kinase (rPTK) of a fusion of the polypeptide comprising the ligand binding domain of a subunit receptor and the intracellular catalytic domain of a receptor protein tyrosine kinase. The construct may optionally further comprise a flag epitope to facilitate entrapment and detection of the activated (eg, dimerized, phosphorylated) subunit receptor. The assay is desirably useful for qualitatively and quantitatively measuring receptor activation of the subunit as well as for facilitating the identification and characterization of potential agonists and antagonists for a receptor of the selected subunit. It is a further object of the invention to provide an assay which makes it possible for ligand-receptor interactions to be studied for any receptor of the selected subunit a, and preferably a receptor for the GFRα subunit. This test must have the capability of high performance, that is, the ability to reliably evaluate large numbers of samples in a relatively short period of time (eg, in a day) . The assay ideally does not make use of radioactive materials and is also feasible for automation. In at least one embodiment of the invention, a generic assay is provided which makes it possible for a receptor of the a subunit of interest to be studied, regardless of whether a capture agent specific to the receptor having the desired characteristic is available or not. Furthermore, it is an object of the invention to provide an assay which substantially represents the binding activity of the receptor ligand of the subunit to in situ. This is desirable since it reduces the possibility that altered interactions between the receptor and the ligand may occur as a consequence of the receptor not being bound to the membrane. In one embodiment of the assay, a method is provided for measuring ligand binding by the detection of serine-threonine kinase phosphorylation, phosphorylation of intracellular kinases and phosphatase activity of a catalytic domain fused to the subunit receptor. to. Accordingly, the invention provides an assay for measuring the activation or binding of the ligand of a receptor construction chimera of the subunit a by detecting its homo-dimerization or homo-oligomerization by in turn measuring the activity of the kinase or phosphatase (i.e., by autophosphorylation) of the catalytic domain that is fused to the ligand binding domain of a receptor of the a-subunit of interest. The test can be divided into two main stages, each of which is usually carried out on separate test plates. The first stage of the assay involves the activation of the receptor construction of subunit a, preferably at a KIRA stage of the assay. The second stage of the assay involves measuring the activation of the receptor construction. Conveniently, this is accomplished by using an enzyme-linked immunosorbent assay (ELISA) to measure the activation of receptor construction. The KIRA step of the assay involves a fusion construct of the receptor kinase-receptor subunit a, which is located on the cell membrane of a eukaryotic cell in such a way that the extracellular domain of the subunit receptor a with the external medium of the cell, a transmembrane domain is located on the cell membrane and the domain of the catalytic kinase is located intracellularly. This step of the total assay involves steps (a) to (c) that are given below: (a) The first solid phase (eg, a cavity of a first test plate) is coated with a substantially homogeneous population of cells ( usually a mammalian cell line) so that the cells adhere to the solid phase. Frequently, the cells are adherent and thus adhere naturally to the first solid phase. In one embodiment of the invention, the cells have been transformed with the DNA encoding a polypeptide receptor construct comprising a binding domain of the receptor ligand of the subunit to fused to a domain of the catalytic kinase, or a "receptor construct". Further defined further, such DNA is expressed by the cells in such a way that the receptor or the construction of the receptor is suitably placed in the cell membranes thereof. The construction of the receptor in addition, and preferably, comprises a fusion with a flag polypeptide. The flag polypeptide is recognized by the capture agent, often a capture antibody, in the ELISA part of the assay. The use of a receptor construct as described herein is particularly advantageous since it provides a "generic" assay wherein the autophosphorylation of any domain of the kinase receptor can be measured, without taking into account whether the specific capture agent for the receiver that has the required characteristics is available or not. Frequently, the receptor construct is a fusion protein comprising the ECD of a subunit a receptor, the catalytic ICD (and possibly the transmembrane domain) of another well-characterized tyrosine kinase (e.g., the Rse receptor). (b) A substance to be analyzed is then added to the cavity that the adherent cells have, in such a way that the receptor construction is exposed to (or comes into contact with) the substance to be analyzed. This assay makes it possible to identify agonist and antagonist ligands for the receptor of the a-subunit of interest. To detect the presence of an antagonist ligand which blocks the binding and / or activation of the receptor by an antagonist ligand, the adherent cells are first exposed to the suspected antagonist ligand and then to the agonist ligand (or a mixture of the agonist and antagonist) of the antagonist ligand. so that the competitive inhibition of receptor activation and binding can be measured. Also, the assay can identify an antagonist which binds to the agonist ligand and whereby its ability to bind to and to activate the kinase domain is reduced or eliminated. To detect such an antagonist, the suspect antagonist and the agonist for the receptor are incubated together and the adherent cells are then exposed to this mixture of ligands. (c) Following exposure to the substance to be analyzed, the adherent cells are solubilized using a buffer solution for lysis (which has a solubilization detergent therein) and gentle agitation, whereby it releases the cell lysate which can be subjected to the ELISA part of the assay directly, without the need for concentration or clarification of the cell lysate. Accordingly, this assay provides a significant improvement over the assays described by Knutson and Buck, supra, Klein et al., Supra, and Hagino et al., Supra, since surprisingly it is unnecessary to concentrate the cell lysate prior to ELISA. In addition, unlike the other assays, in the present assay the cells can be used in the buffer solution for lysis using gentle agitation without the need for homogenization, centrifugation or clarification of the cells. The cell lysate thus prepared is then ready to be subjected to the ELISA step of the assay. It has surprisingly been discovered that the first test plate can be stored at freezing temperatures (i.e., in the range of about -20 to -70 ° C) for significant periods of time (at least 6 months) before the ELISA stage of the assay. This is a significant discovery since the KIRA and ELISA stages of the assay can be performed on separate days. The ELISA component of the assay comprises steps (d) to (h), described below. (d) As a first step, the second solid phase (usually a cavity of a microtiter plate of ELISA) is coated with a capture agent (frequently a capture antibody) which binds specifically to the construction of the receptor, preferably to a flag polypeptide optionally present. The coating of the second solid phase is carried out so that the capture agent adheres to the second solid phase. The capture agent is generally a monoclonal antibody, but, as described in the examples herein, polyclonal antibodies can be used. (e) The cell lysate obtained in step (c) of the KIRA step mentioned above of the test is exposed to, or is contacted with, the adhesion capture agent so that the construction of the receptor adheres to ( or be captured in) the second solid phase. Unlike the Klein et al. Assay, the present assay does not require that the ligand for the receptor as well as the kinase inhibitors be present to achieve adequate immobilization of the receptor or construction of the receptor with respect to the second solid phase. (f) A washing step is then carried out, to remove the unbound cell lysate, leaving the recipient or the captured receptor construct. (g) The construction of the captured or adherent receptor is then exposed to, or contacted with, an anti-phosphotyrosine antibody which identifies the phosphorylated tyrosine residues in the tyrosine kinase receptor domain. In the preferred embodiment, the anti-phosphotyrosine antibody is conjugated (directly or indirectly) with an enzyme which catalyzes a color change of a non-radioactive color reagent. Accordingly, phosphorylation of the receptor can be measured by a subsequent color change of the reagent. The enzyme can be bound to the anti-phosphotyrosine antibody directly, or a conjugation molecule (eg, biotin) can be conjugated to the anti-phosphotyrosine antibody and the enzyme can be subsequently linked to the anti-phosphotyrosine antibody by of the conjugation molecule. (h) Finally, the binding of the anti-phosphotyrosine antibody to the construction of the captured receptor is measured, for example, by a color change in the color reagent. The invention also pertains to a reagent of Rse.bandera which is particularly useful for use in the KIRA ELISA assay. The reagent of Rse. Andera is a polypeptide comprising a fusion of a flag polypeptide (usually the gD flag described here) to the carboxyl terminus or end of the intracellular domain of the RPTK rse. In general, the transmembrane domain of Rse and the extracellular domain of another rPTK of interest are also present in the reagent of the fusion polypeptide. The nucleic acid encoding this reagent and a cell transformed therewith are also claimed. In a still further aspect, the invention relates to a set or set which can be used in the KIRA ELISA described above which comprises a capture agent of the antifelant polypeptide (for example a capture antibody) which is usually linked to the second solid phase as described herein, and a receiver construction. Accordingly, the kit or assembly generally provides an ELISA microtiter plate having a capture antibody of the antisense polypeptide that adheres to a cavity thereof. Optionally, the kit or set also provides an anti-phosphotyrosine antibody which is often labeled, or reagents for the labeling of the anti-phosphotyrosine antibody are supplied with the set or set. Sometimes, a homogeneous population of cells which have been transformed with a receptor construction as described herein, is also provided with the game or set. The game or set can also properly include the instructions for carrying out the KIRA ELISA.

Brief Description of the Drawings Figures 1A-B show the nucleotide sequence and deduced amino acid sequence of a natural murine GFRα3 sequence. Figure 2 shows the alignment of amino acid sequences for murine GFRα3 (SEQ ID NO: 5), rat GFRα (SEQ ID NO: 8) and rat GFRα2 (SEQ ID NO: 9) . The peptides of the N-terminal signal are indicated. The C-terminal hydrophobic sequences associated with GPI anchoring or fixation have a superior underlining. The asterisks indicate the amino acids for fixing the GPI anchor. Potential glycosylation sites are marked by shaded boxes. The identical conserved residues are enclosed in a square. Figure 3 shows the comparison of the alignment between the amino acid sequences of human and murine GFRα. The conserved waste is enclosed in squares. Figure 4 shows the comparison of the alignment between human GFR3 (of DNA48613) and its splice variant (of DNA48614). The conserved sequences are enclosed in frames. The deletion sequence of 30 amino acids is indicated. Figures 5A-B show the alignment of the nucleic acid sequence of the DNA sequence (SEQ ID NO: 14) encoding the human GFR3 with the DNAs (SEQ ID NO: 21 and SEQ ID NO: 22) encoding the Human GFRal (SEQ ID NO: 6) and human GFRα2 (SEQ ID NO: 7), respectively. Figure 6 shows the alignment of the amino acid sequence of human GFRα3 (SEQ ID NO: 15), human GFRα (SEQ ID NO: 6) and human GFRα2 (SEQ ID NO: 7). Figure 7 shows Northern blotting of multiple tissues using GFRα3 as a probe.

Figure 8 compares the localization of RNA expression determined by in situ hybridization using the DNA probes specific for GFRal, GFRα2 and GFRα3. Figures 9A-C present the results of ligand binding (rat GDNF, human neurturin (NTN) or human persephin (PSN)) with respect to GFRal receptors labeled with IgG (Figure 9A), GFRa2 (Figure 9B ) or GFRa3 (Figure 9C). Figure 10 shows the proliferation of cells expressing recombinant chimeric GFRα2 in response to NTN or GDNF. Figure 11 shows the autophosphorylation of the GFRα2-Rse of the receptor expressed recombinantly in response to NTN. Figure 12 presents the assay for the stimulation of GFRα2 or GFRα3 receptors by GDNF, NTN or PSN. Figure 13 shows the agonist activity of several anti-gD antibodies in a gD-GFRα-2-Rse KIRA assay.

Detailed Description of the Preferred Modalities I. Definitions The terms "GFRα3", "GFRα3 polypeptide" and "GFRα3 homologue" when used herein, encompass GFRα3 and GFRα3 variants of the natural sequence (which are defined herein further). GFRα3 can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A "GFRα3 of the natural sequence" comprises a polypeptide having the same amino acid sequence as a GFRα3 derived from nature. Such GFRα3 of the natural sequence can be isolated from nature or can be produced by recombinant or synthetic means. The term "GFRα3 of the natural sequence" specifically encompasses the secreted or truncated forms that are naturally present in GFRα3 (e.g., an extracellular domain sequence), the variant forms that are naturally present (e.g. alternatively spliced) and the allelic variants that are naturally present in GFR3. In one embodiment of the invention, the GFRα3 of the natural sequence is a full length or mature natural sequence GFRα3 comprising amino acids 1 to 400 of SEQ ID NO: 15, with or without the sequence of the N-terminal signal , and with or without the start mationin at position 1. The "variant of GFRα3" means an active GFRα3 as defined below having at least 75% identity of the amino acid sequence with respect to (a) a molecule of DNA encoding a GFR3 polypeptideU , with or without its natural signal sequence, or (b) the complement of the DNA molecule of (a). In a particular embodiment, the GFRα3 variant has at least about 80% homology of the amino acid sequence with GFRα3 having the deduced amino acid sequence shown in SEQ ID NO: 15 for a full-length wild type GFRα3. Such variants of GFRα3 include, for example, GFRα3 polypeptides wherein one or more amino acids are added, or deleted, at the N or C terminus or end of the sequence of SEQ ID NO: 15. Preferably, the identity of the nucleic acid or amino acid sequence is at least 75%, more preferably at least 80%, and even more preferably at least about 90%, and still more preferably at least about 95%. "Percent (%) identity of the amino acid sequence" with respect to the GFRα3 sequences identified herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the GFRα3 sequence , after alignment of the sequences and introduction of the gaps or voids, if necessary, to achieve the maximum identity of the percentage sequence, and without considering some conservative substitutions as part of the identity of the sequence. Alignment for the purposes of determining the percent identity of the amino acid sequence can be accomplished in various ways that are within the skill in the art, using publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNSTAR). Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms necessary to achieve maximum alignment over the total length of the sequences being compared. "Percent (%) of identity of the nucleic acid sequence" with respect to the GFRα3 sequences identified herein, is defined as the percentage of the nucleotides in a candidate sequence that is identical to the nucleotides in the GFRα3 sequence, then of the alignment of the sequences and the introduction of the gaps or voids, if necessary, to achieve the identity of the maximum percentage sequence. Alignment for purposes of determining the percent identity of the nucleic acid sequence can be achieved in various ways that are within the skill in the art, for example, using a publicly available computer program such as BLAST programs. , BLAST-2, ALIGN or Megalign (DNSTAR). Those skilled in the art can determine the appropriate parameters by measuring the alignment, including any algorithms necessary to achieve maximum alignment over the total length of the sequences being compared. "Isolated", when used to describe the various polypeptides described herein, means the polypeptide that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of their natural environment are those materials that could typically interfere with the therapeutic or diagnostic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to a sufficient degree to obtain at least 15 residues of the internal or N-terminal amino acid sequence by the use of a rotary cup sequencer, or (2) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, tin and silver. The isolated polypeptide includes the polypeptide in situ within the recombinant cells, since at least one component of the natural environment of GFRα3 will not be present. Neverthelessordinarily, the isolated polypeptide will be prepared by at least one purification step. An "isolated" DNA48613 nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid of DNA48613. An isolated DNA48613 nucleic acid molecule is different in the form or environment in which it is found in nature. The nucleic acid molecules of isolated DNA48613 are therefore distinguished from the nucleic acid molecule of DNA48613 because it exists in natural cells. However, an isolated DNA48613 nucleic acid molecule includes DNA48613 nucleic acid molecules contained in cells that ordinarily express DNA48613 where, for example, the nucleic acid molecule is in a chromosomal location different from that of the cells natural The term "control sequences" refers to the DNA sequences necessary for the expression of a coding sequence operably linked in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers. The nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, the DNA for a presequence or secretory director is operably linked to the DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation or translation. In general, "operably linked" means that the DNA sequences that are linked are contiguous, and, in the case of a secretory director, contiguous and in reading phase. However, breeders do not have to be contiguous. The binding is effected by ligation or binding at convenient restriction sites. If such sites do not exist, adapters or linkers of synthetic oligonucleotides are used according to conventional practice. "Strict quality" of the hybridization reactions can be easily determined by a person of ordinary skill in the art, and in general is an empirical calculation dependent on the length of the probe, the wash temperature, and the concentration of the Salt. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured DNA for annealing when the complementary strands are present in an environment below its melting temperature. The higher the degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures may tend to tighten the reaction conditions, while lower temperatures would make them less stringent. For details and further explanation of the stringent conditions of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). "Strict conditions" or "highly stringent conditions", as defined herein, can be identified by those that: (1) employ a low ionic strength and a high temperature for washing, for example 0.015M sodium chloride / citrate sodium 0.0015M / sodium dodecyl sulfate 0.1% at 50EC; (2) employ during denaturation a denaturing agent, such as formamide, for example, 50% (v / v) of formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50mM Sodium phosphate buffer solution at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42EC; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, DNA from salmon sperm subjected to the action of sound (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 0.1% SDS; or (4) employ a buffer solution of 10% dextran sulfate, 2 x SSC and 50% formamide at 55EC, followed by a wash under stringent conditions consisting of 0.1 x SSC containing EDTA at 55EC.

"Moderately strict conditions" can be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York; Cold Spring Harbor Press, 1989, and includes the use of a washing solution and hybridization conditions (e.g., temperature, ionic strength, and% SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37 ° C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6) , 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured, sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at approximately 37-50EC. The skilled artisan will recognize how to adjust the temperature, the ionic strength, etc., as necessary to accommodate factors such as the length of the probe and the like. "rPTK" means a tyrosine kinase of the receptor protein. "ECD", "TM domain" and "ICD" refer to the extracellular domain, the transmembrane domain and the intracellular domain of a rPTK, respectively. "Activation of the Kinase Receptor" or "KIRA" when used throughout this application, refers to the first stage of the currently claimed test wherein a receptor construction bound to the cell (typically with a rPTK ICD domain) is exposed to a potential agonist / antagonist ligand which can (or can not) induce phosphorylation of the tyrosine residues in the intracellular domain of the rPTK portion of the receptor construct. The KIRA is generally carried out on the "first test plate" as defined herein. The U.S. patent No. 5,766,863, and its corresponding WO publication, entitled "Kinase receptor activation assay" are hereby incorporated in their entirety for the teaching of a KIRA assay using a fusion of the recombinantly expressed protein of an extracellular domain. of the receptor and a substitute enzymatic domain, for example the tyrosine kinase domain. The "Enzyme Linked Immunosorbent Assay" refers to the second step of the currently claimed assay and involves measurement of the tyrosine phosphorylation of the kinase domain of the receptor construct. The ELISA is normally carried out in the "second test plate" as described in this application. The ELISA is a "sandwich ELISA" because it involves capturing the construction of the receptor with the second solid phase (usually the cavity of an ELISA microtiter plate). ELISA assays generally involve the preparation of the enzyme-antibody conjugates. The conjugated enzyme segments a substrate to generate a colored reaction product that can be detected spectrophotometrically. In this test, the absorbance of the colored solution in the individual microtitre cavities is proportional to the amount of the phosphotyrosines. An ELISA review is found in Current Protocols in Molecular Biology, Vol. 2, chapter 11 (1991). Although the term "ELISA" is used to describe the second step of the present assay, it is only a preferred embodiment of the invention, since, as described herein, techniques other than enzymatic detection are available to measure the binding of the anti-phosphotyrosine antibody to the activated receptor. The terms "tyrosine kinase", "tyrosine kinase receptor", "receptor protein tyrosine kinase" and "rPTK" are used interchangeably herein and refer to a protein having at least one phenolic acceptance group of the phosphate in your ICD. The protein is usually a receptor since it has a ligand binding ECD, the TM domain and the ICD. The ICD usually comprises a domain of the catalytic kinase and has one or more tyrosine residues of phosphate acceptance. Examples of the tyrosine kinase receptors include the insulin receptor, the insulin-related receptor, the epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptors A and B (PDGF-RA and PDGF-RB), the factor 1 receptor insulin-like growth (IGF-lR), the macrophage colony stimulating factor receptor (M-CSF-R), the HER2 / neu / c-erbB-2 receptor, the HER3 / c-erbB-3 receptor , the Xmrk receptor, the IRR receptor, the bek and fig receptors of the fibroblast growth factor (FGF), the c-kit receptor, the Flk / kDR receptor, the Rse receptor, the Eph, Elk, Eck, Eek receptors , Erk, Cek4 / Mek4 / HEK and Cek5, the Axl receptor, the hepatocyte growth factor receptor (HGF-R), the Fltl VEGF receptor, the SAL-SI receptor, the HpTK 5 receptor, the trkA receptor, the trkB receiver, and the trkC receiver. See, for example, Ullrich. and Schlessinger Cell 81: 203-212 (1990); Fantl and collaborators, Annu. Rev. Biochem. 62: 453-481 (1993); Mark et al., Journal of Biological Chemistry 269 (14): 10720-10728 (1994); and WO 93/15201. The terms mentioned above encompass the chimeric "receptor" molecules or the "receptor constructs" the "a subunit receptor constructs", which comprise at least the extracellular domain of the receptor of the selected subunit a, and the intracellular domain of a kinase receptor (preferably a rPTK), and optionally, the transmembrane domain of the same or of another tyrosine kinase, and optionally also a lateral or fin epitope Of course, the a receptor of interest can provide the domain of the transmembrane if it has one.The terms also encompass variants of the amino acid sequence and the covalent derivatives of the various subunit receptors and the kinase domains of the rPTKs to which they are fused, provided that they are still exhibit the phosphorylation activity of the kinase in the KIRA ELISA, therefore, the variants will have generally altered preservative amino acids. The individual domains of the receptor kinase of the subunit a can be delineated based on the receptors recognized in the relevant family and the hydrophobicity plots. For example, the hydrophobic transmembrane domain can be easily determined and the ECD and ICD, when present, are usually amino terminal and carboxy terminal with respect to the transmembrane domain or GPI anchor or fixation, respectively. Conveniently, the transmembrane domain and the ICD of the Rse receptor can be fused to the ECD of a receptor of the a subunit of interest, typically with the GPI binding or anchoring sequence, whereby a chimeric receptor is formed which is encompassed by the terms denoting a receiver construction as mentioned here. In the preferred embodiment, the a subunit is selected from the group consisting of GFRal, GFRα2, GFRα3, and GFRα4. By "autophosphorylation" is meant activation of the catalytic kinase domain of the rPTK portion of the receptor construct, whereby at least one intrinsic tyrosine residue is phosphorylated. Typically, autophosphorylation will result when a molecule binds to the extracellular domain of the a subunit receptor. Without being limited to any particular mechanism of action, it is thought that the binding of the agonist molecule leads to the oligomerization of the receptor construction which causes the activation of the catalytic kinase domain. By "solid phase" is meant a non-aqueous matrix to which the cells (in the KIRA stage of the assay) or the capture agent (in the ELISA stage of the assay) can adhere. Usually, the solid phase comprises the cavity of a test plate but the invention is not understood to be limited to this embodiment. For example, the solid phase may comprise a discontinuous solid phase of discrete particles. The porous particles may be porous and consist of a number of different materials, for example, polysaccharides (for example agarose), polyacrylamides, polystyrene, polyvinyl alcohol, silicones and glasses. For examples of suitable solid particulate phases, see U.S. Pat. No. 4,275,149. By "cavity" is meant a recess or retention space in which an aqueous sample can be placed. The cavity is provided in a "test plate". The invention usually employs a "first test plate" which is formed from a material (e.g. polystyrene) which optimizes the adhesion of the cells (having the receptor or receptor construction) thereto. In general, the individual cavities of the first test plate will have a large ratio of large surface area with respect to the volume and therefore a suitable shape is a flat bottom cavity (where the cells are adherent). The "second test plate" is generally formed of a material (e.g. polystyrene) which optimizes the adhesion of the capture agent thereto. The "second test plate" is generally formed of a material (for example polystyrene) which optimizes the adhesion of the capture agent thereto. The second test plate may have the same general construction and / or features as the first test plate. Nevertheless, separate plates are used for the KIRA stage of the assay and the ELISA stage of the assay. In the preferred embodiment of the invention, both the first test plate and the second test plate are "microtitre" plates. The term "microtiter plate" when used herein refers to a test plate having between about 30 to 200 individual cavities, usually 96 cavities. Frequently, the individual cavities of the microtiter plate will retain a maximum volume of approximately 250 μl. Conveniently, the first test plate is a 96-well polystyrene or plastic cell culture microtiter plate (such as that sold by Becton Dickinson Labware, Lincoln Park, NJ), which allows for automation. Frequently, approximately 50 μl to 300 μl, more preferably 100 μl to 200 μl, of an aqueous sample comprising the cell culture medium will be added with the cells suspended therein to each well of the first test plate in the stage of KIRA of the essay. It is desirable to sow between about 1 x 10 4 to 3 x 10 5 cells per well, more preferably 5 x 10 4 to 1 x 10 5 cells per well are seeded. Usually, the second test bed will comprise a polystyrene microtiter ELISA plate such as that sold by Nunc Maxisorp, inter Med, Denmark. The term "homogeneous population of cells" refers to a substantial homogeneous population of cells in which at least about 80%, and preferably about 90%, of the cells in the population are of the same cell type. Therefore it is convenient to use a cell line. The cell line is a eukaryotic cell line, typically in the cell line of animals and desirably a mammalian cell line. The cells have, or are transformed to produce, the selected receptor construct. Accordingly, the cell is transformed with a nucleic acid encoding the receptor construct and the nucleic acid is expressed so that the ECD of the receptor is confronted with the external medium of the cell or cell, the transmembrane domain is located in the cell. The cell membrane and the kinase domain is located intracellularly. As a general proposition, a minimum number of approximately 1 x 104 receivers / cell is required. The term "adherent" when used herein to describe the cell or cell, refers to a cell or cell which adheres naturally to the first solid phase (often the cavity of the first test plate), thereby a fairly uniform coating of the cells is formed on the inner surface of the cavity. The uniform coating of the cells is generally formed following the incubation of the cells in the cavities of the first test plate for about 8-16 hours. After incubation, the non-adherent cells and the culture medium of the cells are removed by decanting the first test plate. The incubation is usually carried out at a temperature which is optimal for cell growth, ie, approximately 37 ° C. Examples of adherent cell lines include CHO cells (Urlaub and Chasin, Proc. Nal. Acad. Sci. USA 77: 4216 (1980)), MCF-7 cells (ATCC HB 22), 293 cells (Graham et al., J. Gen virol., 36:59 (1977)), the cell line of the Swiss albino 3T3 fibroblasts (ATCC No. CCL 92) and the macrophage cell line U937 (ATCC No. CRL 1593). A "flag polypeptide" comprises a short polypeptide which has enough residues to provide an epitope (preferably a linear epitope) against which a "capture agent" can be made against it, but is still short enough in such a way that it does not interfere with the activity of the kinase domain or the ligand binding domain. The flag polypeptide is also sufficiently unique so that the capture agent against it does not bind to other reagents in the assay. The selection of a sequence of "unique" flag polypeptides can be effected by comparing the sequence of a proposed flag polypeptide against other sequences in the Genbank or EMBL, for example. Suitable flag polypeptides generally have at least 6 amino acid residues and usually between about 8-80 amino acid residues (preferably between about 9-30 amino acid residues). By "receptor construction" is meant a polypeptide which comprises a fusion of a ligand binding domain of the a subunit receptor and a catalytic domain of the kinase receptor, and optionally a flag polypeptide as defined above. The flag polypeptide is provided at a site in the receptor construct such that: a) the flag polypeptide does not interfere with the binding of the ligand to the receptor; b) the flag polypeptide does not interfere with the autophosphorylation of the receptor and c) the flag polypeptide is presented in a suitable configuration so that it can bind to the capture agent in the ELISA step of the assay. Frequently, the polypeptide flag will be present at the N-terminus of the receptor construct. Alternatively, the flag polypeptide may be present at the C-terminus of the receptor construct. A construction of Rse.gD. The Rse construct described herein is particularly useful, since the ICD (and optionally the transmembrane domain) thereof can be fused to the ECD of a receiver of interest, thereby eliminating the need to establish where it should be located the polypeptide with respect to the receptor of interest. "Rse.gD" refers to a receptor construction which has the ICD domain of the tyrosine kinase of the Rse receptor protein with the glycoprotein D (gD) flag polypeptide of the Herpes Simplex virus fused to the end or terminal of COOH of the same. The "Rse.bandera reagent" refers to a polypeptide which comprises the ICD of the Rse receptor fused at its terminus or COOH terminus to a flag polypeptide (typically the gD flag polypeptide). Sometimes, the TM domain of Rse with the ECD of a receptor of the a subunit of interest will also be present in the reagent of Rse.gD. "Chimera of the ECD / Rse.gD receptor" refers to a fusion of the ECD of a ligand binding domain of the subunit receptor of interest to the TM and ICD domains of the Rse which are COOH fused to the polypeptide gD flag. By "capture agent" is meant a compound or agent which is capable of adhering to the second solid phase, as defined herein, and which is selective of a receptor construction. Accordingly, the capture agent captures the construction of the receptor in the cavities of the second test plate. Usually, the capture agent binds selectively to the flag polypeptide which has been fused to the receptor of interest. The binding of the capture agent is not affected by the presence or absence of the ligand bound to the receptor and does not induce receptor activation during capture. In addition, the capture agent does not spherically block access to the tyrosine (s) phosphorylated by the antiphosphotyrosine antibody. The means for selecting the appropriate capture agents are described herein. In general, the capture agent will comprise an antibody (eg, an affinity purified polyclonal antibody or a monoclonal antibody), but other selective agents, such as streptavidin, which binds selectively to the "tag-strep" polypeptide. , they can also be used (see Schmidt et al., Protein Engineering 6 (1): 109-122 (1993)). Streptavidin can be purchased commercially from Zymed Laboratories, S. San Francisco, CA, for example. Alternatively, the capture agent may comprise protein A (which binds specifically to immunoglobulins). In this embodiment of the invention, the active receptor construction present in the cell lysate is incubated with an antibody which binds specifically thereto, whereby a receptor-antibody complex is formed. This complex can be captured by protein A by virtue of its specific binding to the antibody present in the complex. Protein A can be purchased commercially from Pharmacia Biotech, Inc., Piscataway, New Jersey, for example. In the most preferred embodiment, the capture agent is a monoclonal antibody which binds specifically to a flag polypeptide (which is present in the construction of the receptor). Examples of the appropriate flag polypeptides and their respective capture antibodies include the FL flag HA and its antibody 12CA5, (Field et al., Mol.Cell. Biol. 8: 2159-2165 (1998)); the c-myc flag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies for the same (Evan et al., Molecular and Cellular Biology 5 (12): 3610-3616 (1985)); as well as the glycoprotein D (gD) flag of the Herpes Simplex virus and the 5B6 antibody thereto (Paborsky et al., Protein Engineering 3 (6): 547-553 (1990) and Mark et al., Journal of Biological Chemistry 269 (14): 10720-10728 (1994)). Other flag polypeptides have already been described. Examples include the Flag peptide (Hopp et al., BioTechnology 6: 1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science 255: 192-194 (1992)); a peptide from the a-tubulin epitope (Skinner et al., J. Biol. Chem. 266: 15163-15166 (1991)); and the peptide tag of the T7 gene 10 protein (Lutz-Freyermuth et al., Proc. Nati, Acad. Sci. USA 87: 6393-6397 (1990)). Once the flag polypeptide has been selected as described above, a capture antibody thereto can be generated using the techniques described herein. The term "substance to be analyzed" refers to a compound or composition to be studied, usually to investigate its ability to activate (or prevent activation) of the receptor of the a subunit of interest. The substance to be analyzed may comprise a body fluid (such as plasma or amniotic fluid) or a composition known to contain, or is suspected to contain, a ligand for the tyrosine kinase receptor. The substance to be analyzed can also comprise a cell which has a ligand for the receptor of the a subunit of interest. "Ligand" when used herein refers to a molecule which is capable of binding to the receptor of the extracellular subunit of interest or to a known antagonist thereof. The ligand will usually be an agonist or antagonist for the receptor. By "agonist" is meant a molecule which is capable of activating the intracellular kinase domain of the receptor construction during binding to the receptor portion of the extracellular subunit. Frequently, the agonist will comprise a growth factor (i.e., a polypeptide that is capable of stimulating cell division). Exemplary growth factors include artemin, neurturin, GDNF and persephin. Alternatively, the agonist can be an antibody against the receptor or even its flag sequence as shown here in the Examples. However, other agonists that are not proteins such as small organic molecules are also encompassed by the invention. By "antagonist" is meant a molecule which blocks the agonist action. Usually, the antagonist either: (a) will bind to the receptor portion of the subunit a and thereby blocks the binding and / or activation of the receptor by an agonist therefor (the antagonist can bind to the recipient's ECD, but this it is not necessarily the case) or (b) it will bind to the agonist and thus prevent activation of the receptor by the agonist. This assay facilitates the detection of both types of the antagonist. The antagonist, for example, may comprise a fragment of the peptide comprising the receptor binding domain of the endogenous agonist ligand for the receptor. The antagonist may also be an antibody which is directed against the ECD of the receptor, or against a known agonist for the receptor. However, other molecules that are not proteins are also covered by this term. The term "antibody" is used in the broadest sense, and more specifically may cover the unique anti-GFRα3 monoclonal antibodies (including the agonist, antagonist, and neutralizing antibodies) and anti-GFRα3 antibody compositions with polyepitopic specificity. The term "monoclonal antibody" when used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in smaller quantities.

"Active" or "activity" for the purposes herein refers to the form (s) of the GFR3, or a receptor of the subunit a as indicated by the context, which retains the biological and / or immunological activities of natural or naturally occurring GFR3, or the receptor. A preferred activity is the ability to bind to and affect, for example, block or otherwise modulate, an activity of a natural agonist or ligand. The activity preferably involves the regulation of neuronal function. A "GFRα3 ligand" is a molecule which binds to and preferably activates the natural sequence of GFRα3. The ability of a molecule to bind to GFRα3 can be determined, for example, by the ability of the putative ligand or putative ligand to bind to the coated GFRα3 immunoadhesin on a test plate, for example. The specificity of the binding can be determined by comparing the binding to GFRal or 2. The term "anti-phosphotyrosine antibody" refers to a molecule, usually an antibody, which binds selectively to the phosphorylated tyrosine residues in the kinase domain of a rPTK. The antibody can be polyclonal, but it is desirably a monoclonal antibody. Polyclonal anti-phosphotyrosine antibodies can be made using the techniques described in White and Backer, Methods in Enzymology 201: 65-67 (1991) and monoclonal anti-phosphotyrosine antibodies can be obtained commercially from Upstate Biologicals, Inc. (UBI, Lake Placid, NY), for example. The word "tag" when used herein refers to a detectable compound or composition which is directly or indirectly conjugated to a molecule (such as the anti-phosphotyrosine antibody). The label may be detectable by itself (eg radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical alteration of a compound or substrate composition which is detectable. The preferred label is an enzyme which catalyzes a color change of a non-radioactive color reagent. By "washing" is meant the exposure of the solid phase to an aqueous solution (usually a cell culture medium or buffer) such that the unbound material (eg, non-adherent cells, the capture agent). non-adherent, unbound ligand, receptor construction, cell lysate, or anti-phosphotyrosine antibody) is removed from it. To reduce background interference, it is convenient to include a detergent (eg Triton X) in the wash solution. Usually, the aqueous wash solution is decanted from the cavities of the test plate following the wash. Conveniently, the washing can be accomplished using an automated washing device. Sometimes, several washing steps may be required (for example, between about 1 to 10 washing steps). By "blocking buffer" is meant an aqueous buffer solution, which contains at least one blocking compound which is capable of binding to the exposed surfaces of the second solid phase which are not coated with the blocking agent. capture. The blocking compound is usually a protein such as bovine serum albumin (BSA), gelatin, casein, or milk powder and does not cross-react with any of the reagents in the assay (for example, anti-phosphotyrosine antibodies and detection reagents). The blocking buffer solution is generally provided at a pH of between about 7 to 7.5 and suitable buffering agents include phosphate and TRIS. By "buffer solution for lysis" is meant an aqueous buffered solution, comprising a solubilizing detergent, one or more inhibitors. the protease and at least one phosphatase inhibitor (such as sodium orthovanadate). The term "solubilization detergent" refers to a non-ionic detergent, miscible in water, which lyses the cell membranes of the eukaryotic cells but does not denature or activate the receptor construction. Examples of suitable non-ionic detergents include Triton-X 100, Tween 20, CHAPS and Nonidet P-40 (NP40) available from Calbiochem, La Jolla, California, for example. Many other non-ionic detergents are available in the art. Examples of suitable protease inhibitors include phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin, aprotinin, 4- (2-aminoethyl) -benzenesulfonyl-bestatin fluoride hydrochloride, chemostatin, and benzamidine. Condoms (for example, thimerosal) and one or more compounds which maintain the isotonicity of the solution (for example, sodium chloride (NaCl) or sucrose) and a buffer solution (for example, Tris or PBS) are also Presents usually. Generally, the pH of the lysis buffer is in the range of about 7 to 7.5. Usually, following the addition of the lysis buffer to the first test plate, the first test plate is "gently agitated" and this expression refers to the act of physically stirring the first test plate (usually using a circular motion) at a substantially low speed.

The gentle agitation does not involve the mechanical alteration of the cells (for example homogenizing or centrifuging the cells). The exemplary agitation speeds of the order of 200 to 500 rpm, preferably 300 to 400 rpm in the orbital warmer, for example.

II Compositions and Methods of the Invention A. Total length GFR3 The present invention provides newly identified and isolated nucleotide sequences that encode the polypeptides referred to in the present application as GFRα3. In particular, the Applicants have identified and isolated the cDNA encoding a GFRα3 polypeptide, as described in further detail in the following Examples. Using computer programs of alignment of the BLAST, BLAST-2 and FastA sequences, the Applicant has found that a GFRα3 of the full length natural sequence (SEQ ID NO: 15) has 34% identity of the amino acid sequence with GFRal and GFRa2. Accordingly, it is currently believed that GFRα3 described in the present application is a newly identified element of the GFR protein family and may possess a neuronal cell activation function typical of the GFR protein family. However, the limited distribution of GFRα3 compared to GFRal and GFRα2 makes it and its agonists preferred molecules to avoid undesirable side effects when administered. The neurotrophic factor derived from the glial cell line ("GDNF") and Neurturin ("NTN") are two potent structurally related survival factors for neurons of the central nervous and sensory systems (Lin et al., Science 260: 1130 1132 (1993)); Henderson et al. Science 266: 1062-1064 (1994); Buj-Bello et al., Neuron 15: 821-828 (1995); Kotzbauer et al., Nature 384: 467-470 (1996)). GDNF was shown to have a mediating action through a multicomponent receptor system composed of a ligand-binding glycosyl-phosphatidyl inositol (GPI) protein (designated GDNFRa or GFRal) and the Ret of the tyrosine kinase of the transmembrane (Treanor et al., Nature 382: 80-83 (1996); Jing et al., Cell 85: 1113-1124 (1996); Trupp et al., Nature 381: 785-789 (1996); Durbec et al., Nature 381 : 789-793 (1996)). The NTN signal is transmitted by GFRa2, which is also associated with Ret. Described here is the isolation, sequencing, and tissue distribution of a protein bound to GPI and its gene, designated GFRα3, which is known to modulate the response to a novel ligand in the NTN family and GDNF. In the case of cellular responses to NTN, the cells require the presence of GFRα2. The ligand bound to GFRα2 induces Ret phosphorylation of the tyrosine kinase receptor. These findings identify Ret and GFrRa2, respectively, as the signaling and binding components of a receptor ligand for NTN and related ligands. This defines a novel neurotrophic differentiation factor receptor family of receptors that contain a shared transmembrane protein tyrosine kinase (Ret) and a component of the ligand-specific GPI-linked protein (GFRa). The neurotrophic factor derived from the glial cell line ("GDNF") (Lin et al., Science, 260: 1130-1132 (1993); WO 93/06116, which is incorporated herein in its entirety), is a factor of potent survival for dopaminergic elements of the mecencephalon (Lin et al., Science, 260: 1130-1132 (1993); Strdmberg et al., Exp. Neurol., 124: 401-412 (1993); Beck et al., Nature, 373 : 339-341 (1995), Kearns et al., Brain Res., 672: 104-111 (1995), Tomac et al., Nature, 373: 335-339 1995)), the spinal motor elements (Henderson et al., Science , 266: 1062-1064 (1994), Oppenheim et al, Nature, 373: 344-346 (1995), Yan et al, Nature, 373: 341-344 (1995)) and noradrenergic neurons (Arenas et al., Neuron. , 15: 1465-1473 (1995)), which degenerate into Parkinson's disease (Hirsch et al., Nature, 334: 345-348 (1988); Hernykiewicz Mt. Sinai J. Med., 55: 11-20 ( 1988)), sclerosis lat amyotrophic erythrocyte (Hirano, Amiotrophic Lateral Sclerosis and Other Motor Neuron Diseases, P. Rowland, ed. (New York: Raven Press, Inc.) pp. 91-101 (1991)), and the disease of Alzheimer's disease (Marcynuik et al., J. neurol. Sci., 76: 335-345 (1986); Cash et al., Neurology, 37: 42-46 (1987); Chan-Palay et al., Comp. Neurol., 287: 373-392 (1989)) respectively. Based on genetically engineered mice lacking GDNF, additional biological roles have been reported for GDNF: the development and / or survival of enteric, sympathetic, and sensory neurons and the renal system, but not for catecholaminergic neurons in the central nervous system (CNS) (Moore et al., Nature 382: 76-79 (1996); Pichel et al., Nature 382: 73-76 (1996); Sánchez et al., Nature 382: 70-73 (1996) ). Despite the physiological and clinical importance of GDNF, little is known about its mechanism of action.

Cytokine receptors are frequently grouped into complexes of multiple subunits. Sometimes, the a subunit of this complex is involved in the binding of the similar or analogous growth factor and the β subunit may contain a capacity to transduce a signal to the cell. Without wishing to be limited by theory, these receptors have been assigned to three subfamilies depending on the complexes formed. Subfamily 1 includes the receptors for EPO. The stimulating factor of granulocyte colonies (G-CSF), interleukin-4 (IL-4), interleukin-7 (IL-7), growth hormone (GH), and prolactin (PRL). The binding of the ligand to the receptors belonging to this subfamily is thought to lead to homodimerization of the receptor. Subfamily 2 includes the receptors for IL-3, the macrophage-granulocyte-colony stimulating factor (GM-CSF), interleukin-5 (IL-5), interleukin-6 (IL-6), the factor Leukemia inhibitor (LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF). The receptors of subfamily 2 are heterodimers that have a subunit a for ligand binding, and the β subunit (either the shared β subunit of the IL-3 receptors, GM-CSF, and the IL-5 or the subunit gpl30 of the IL-6, LIF, OSM, and CNTF receptors) for signal transduction. Subfamily 3 contains only the interleukin-2 receptor (IL-2). The ß and? Subunits of the IL-2 receptor complex are cytokine receptor polypeptides which are associated with the α subunit of the unrelated Tac antigen. The present invention is based on the discovery of GFRα3, a protein of the GFR family, whose natural ligand is unknown. The experiments described here demonstrate that this molecule is a receptor which appears to play a role in mediating responses to a ligand of the novel GDNF family. In particular, this receptor has been found to be present in a variety of tissue populations and cells, including neurons, thus indicating that GFRα3 ligands, such as agonist antibodies, can be used to stimulate proliferation, growth, survival, differentiation, metabolism, or regeneration of cells containing GFRα3 and Ret.

B. Variants of GFRa3 In addition to the full length natural sequence GFRα3 described herein, it is contemplated that variants of GFRα3 can be prepared. The GFRα3 variants can be prepared by introducing the appropriate nucleotide changes into the GFRα3 DNA, or by the synthesis of the desired GFRα3 polypeptides. Those skilled in the art will appreciate that changes in amino acids can alter the posttranslational processes of GFRα3, such as changing the number or position of glycosylation sites or altering the anchoring or binding characteristics of the membrane. Indeed, a splicing variant of GFRα3 is encoded by DNA48614 and a variant of murine by SEQ ID NO: 4. Other variants include the gD-RSE chimeras and labeled with IgG made as described in the Examples. Variations in GFRα3 of the natural total length sequence or in several domains of GFRα3 described here, can be made, for example, using any of the techniques and guidelines for the conservative and non-conservative mutations described, for example, in U.S. Pat. No. 5,364,934. The variations may be a substitution, deletion or insertion of one or more codons encoding GFRα3 leading to a change in the amino acid sequence of GFRα3 when compared to GFRα3 of the natural sequence. Optionally, the variation is by the substitution of at least one amino acid with any other amino acid in one or more of the domains of GFRα3. Guidance in determining which amino acid residue can be inserted, substituted or deleted, without adversely affecting the desired activity, can be found by comparing the GFRα3 sequence with that of known, homologous protein molecules, and minimizing the number of changes of the amino acid sequence made in regions of high homology. The amino acid substitutions may be the result of replacing an amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, ie, the replacements of conservative amino acids. The insertions or deletions may optionally be in the range of 1 to 5 amino acids. The allowed variation can be determined by systematically inserting, deleting or substituting the amino acids in the sequence and testing the resulting variants for activity in the in vitro assay described in the Examples below. The variants may be those encoded by an isolated nucleic acid molecule having at least about 65% sequence identity with respect to (a) a nucleic acid sequence encoding a GFRα3 polypeptide comprising the sequence of amino acids 27 at 400 of SEQ ID NO: 15, amino acids 27 to 369 of SEQ ID NO: 17 or amino acids 27 to 374 of SEQ ID NO: 5 or (b) the complement of the nucleic acid molecules of (a) ). In addition, the variants can be encoded by the sequences of the nucleic molecules comprising a ligand binding domain of a GFRα3 polypeptide of amino acids 84 to 360 of SEQ ID NO: 15, amino acids 84 to 329 of SEQ ID NO: 15. NO: 17 or the sequence of amino acids 110 to 386 of SEQ ID NO: 20, or their complementary nucleic acids. These nucleic acid molecules preferably comprise a GFRα3 coding sequence which hybridizes preferentially under stringent conditions to the nucleic acid sequences encoding a GFRα3 polypeptide of the invention. The identity of the sequence is preferably at least about 75%, more preferably at least 85%, even more preferably at least 90%, still more preferably at least 95%. Typically, the polypeptide has at least about 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and still more preferably at least about 95% sequence identity with a polypeptide that has the amino acid residues 27 to 400 of SEQ ID NO: 15, amino acids 27 to 369 of SEQ ID NO: 17, amino acids 27 to 374 of SEQ ID NO: 5, a ligand binding domain of a GFRα3 polypeptide of amino acids 84 to 360 of SEQ ID NO: 15, amino acids 84 to 329 of SEQ ID NO: 17, or amino acid sequence 110 to 386 of SEQ ID NO: 20. Preferably, the identity is for amino acid residues 27 to 400 of SEQ ID NO: 15 and the DNA encoding them. The isolated nucleic acid molecule can contain a DNA encoding a GFRα3 polypeptide having amino acid residues 27 to 400 of SEQ ID NO: 15, or is complementary to such encoding of the nucleic acid sequence, and remains Stably linked to it under highly stringent conditions, and optionally, at least moderate. The protein can be encoded by the nucleic acid encoding the full-length protein of clone DNA48613, DNA48614 or murine GFR3 (clone 13), deposited in the ATCC under accession numbers ATCC 209752 (Designation: DNA48613-1268), ATCC 209751 (Designation: DNA48614-1268), and ATCC, respectively, on April 7, 1998, or one that hybridizes thereto under strict conditions. Variants can be made using methods known in the art such as oligonucleotide-mediated mutagenesis (site-directed), alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Cárter et al., Nucí Acids Res., 13_: 4331 (1986); Zoller et al., Nucí. Acids Res., 1_0: 6487 (1987)), cassette mutagenesis (Wells et al., Gene, 3_4: 315 (1985)), restriction selection mutagenesis (Wells et al., P. Soc. London SerA, 317: 415 (1986)) or other known techniques can be performed on the cloned DNA to produce the variant DNA of GFRα3. The analysis of amino acids by scanning can also be used to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are the relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred amino acid of choice among this group because it removes the side chain beyond beta-carbon and is less likely to alter the conformation of the variant's backbone. Alanine is also typically preferred because it is the most common amino acid. In addition, it is frequently bound in both stacked and exposed positions (Creighton, The Proteins, (W.H. Freman &Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)). If the alanine substitution does not produce adequate amounts of the variant, an isoteric amino acid can be used.

C. Modifications of GFRa3 Covalent modifications of GFRα3 are included within the scope of this invention. One type of covalent modification includes reacting the amino acid residues located as targets of GFRα3 with an organic derivatizing agent that is capable of reacting with the selected side chains or the N- or C-terminal residues of GFRα3. Derivatization with bifunctional agents is useful, for example, for the cross-linking of GFRα3 to a support matrix or surface insoluble with water for use in the purification method of GFRα3 antibodies, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis (succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3 - ((p-azidophenyl) dithio ) propioimidate. Other modifications include the deamidation of the glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, the hydroxylation of proline and lysine, the phosphorylation of the hydroxyl groups of the seryl or threonyl residues, the methylation of the a-amino groups of the side chains of lysine, arginine, and histidine (TE Creigton, Proteins: Structure and Molecular Properties, WH Freeman &Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and the amidation of any C-terminal carboxyl group. Another type of covalent modification of the GFRα3 polypeptide included within the scope of this invention comprises altering the natural glycosylation configuration of the polypeptide. The "alteration of the natural glycosylation configuration" is proposed for the purposes herein that mean the deletion of one or more carbohydrate portions found in the GFRα3 of the natural sequence, and / or the addition of one or more glycosylation sites that they are not present in the GFRα3 of the natural sequence, and / or the alteration of the ratio and / or the composition of the sugar residues fixed to the glycosylation site (s). The addition of the glycosylation sites to the GFRα3 polypeptide can be effected by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues with respect to the GFRα3 of the natural sequence (for the glycosylation sites linked to 0). The amino acid sequence of GFRα3 can optionally be altered by changes in the level of the DNA, particularly by mutation of the DNA encoding the GRFα3 polypeptide at the preselected bases such that codons are generated which will result in the amino acids desired. Other means for increasing the number of carbohydrate moieties on the GFRα3 polypeptide is by the chemical or enzymatic binding of the glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87/05330 published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981). The removal of portions of the carbohydrates present on the GFRα3 polypeptide can be effected chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as targets or targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of the carbohydrate moieties on the polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Met. Enzimol., 138: 350 (1987). Another type of covalent modification of GFRα3 comprises the binding of the GFRα3 polypeptide to one of a variety of non-proteinaceous polymers, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The GFRα3 of the present invention can also be modified in a manner to form a chimeric molecule comprising the GFRα3 fused to another heterologous amino acid or polypeptide sequence. In one embodiment, such a chimeric molecule comprises a fusion of GFRα3 with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is usually placed at the amino- or carboxyl- terminus of GFR3. The presence of such tagged forms of the epitope of GFRα3 can be detected using an antibody against the polypeptide of the tag. Also, the provision of the epitope tag makes it possible for GFRα3 to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of GFRα3 with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such fusion could be to the Fc region of an IgG molecule. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) labels; the polypeptide of the HA HA tag and its 12CA5 antibody (Field et al., Mol.Cell. Biol., 8-2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies for the same (Evan et al., Molecular and Cellular Biology, 5: 3610-3616 (1985)); and the glycoprotein D (gD) label of the Herpes Simplex virus and its antibody (Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)). Other tag polypeptides include the Flag peptide (Label) (Hopp et al., BioTechnology, 6: 1204-1210 (1988)); the KT3 epitope peptide (Martin et al., Science, 255: 192-194 (1992)); an α-tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266: 15163-15166 (1991)); and the peptide tag of the T7 gene 10 protein (Lutz-Freyermuth et al., Proc. Nati, Acad. Sci. USA, 87: 6393-6397 (1990)).

D. Preparation of GFRa3 The description below mainly relates to the production of GFRα3 by culturing cells transformed or transfected with a vector containing the GFRα3 nucleic acid. Of course, it is contemplated that alternative methods, which are well known in the art, can be employed to prepare GFRα3. For example, the sequence of GFRα3, or portions thereof, can be produced by the direct synthesis of the peptides using solid phase techniques (see, for example, Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co. , San Francisco, CA (1969), Merrifield, J. Am. Chem. Soc., 5: 2149-2154 (1963)). The synthesis of the protein In vitro can be carried out using manual or automatic techniques. Automated synthesis can be effected, for example, by using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Several portions of GFRα3 can be chemically synthesized separately and combined using chemical or enzymatic methods to produce full length GFRα3. 1. Isolation of GFRα3 that encodes DNA The DNA encoding GFRα3 can be obtained from a cDNA library prepared from the tissue believed to possess the GFRα3 mRNA and to express it at a detectable level. Accordingly, human GFRα3 DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as that described in the Examples. The gene encoding GFRα3 can also be obtained from a genomic library or by the synthesis of oligonucleotides. Libraries can be selected with probes (such as antibodies to GFRα3 or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. The selection of the cDNA or the genomic library with the selected probe can be carried out using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboraory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding GFRα3 is to use the PCR methodology (Sambrook et al., Supra, Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)). The examples given below describe techniques for selecting a cDNA library. The sequences of oligonucleotides selected as probes must be of sufficient length and sufficiently unambiguous so that false positive results are minimized. The oligonucleotide is preferably labesuch that it can be detected during hybridization to the DNA in the library that is selected. Labeling methods are well known in the art, and include the use of radiolabels similar to ATP labewith 32P, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringent conditions and highly stringent conditions, are provided in Sambrook et al., Supra. Sequences identified in such library selection methods can be compared and aligned with other deposited and available sequences in public databases such as GenBank or other private sequence databases. The identity of the sequence (either at the amino acid or nucleotide level) within the defined regions of the molecule or through the full length sequence can be determined through sequence alignment using computer programs such as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT which employ several algorithms to measure homology. The nucleic acid having the protein coding sequence can be obtained by screening the selected DNA or the genomic libraries using the amino acid sequence described herein for the first time, and, if necessary, using primer extension methods. conventional, as described in Sambrook et al., supra, to detect the precursors and process intermediate mRNA compounds that may not have been reverse transcribed in the cDNA. 2. Selection and Transformation of Host Cells The host cells are transfected or transformed with the cloning or expression vectors described herein for the production of GFRα3 and cultured in a modified conventional nutrient medium as appropriate to induce the promoters, select the transformants, or amplify the genes encoding the desired sequences. The culture conditions, such as the medium, temperature, pH and the like, can be selected by the artisan skilled in the art without undue experimentation. In general, the principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., Supra. Transfection methods are known to the artisan with ordinary experience, for example, CaP0 and electroporation. Depending on the host cell used, the transformation is effected using standard techniques appropriate for such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., Supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell wall barriers. The infection with Agrobacterium tumefaciens is used for the transformation of certain plant cells, as described by Shaw et al., Gene, 2_3: 315 (1983) and WO 89/05859 published on June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van derEb, Virology, 52: 456-457 (1978) can be employed. The general aspects of host system transformations of mammalian cells have been described in U.S. Pat. No. 4,399,216. Transformations in the yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130: 946 (1977) and Hsiao et al., Proc.Nati.Acad. Sci. (USA), 76: 3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, the fusion of bacterial protoplasm with intact cells, or polycations, for example, polybrene, polyornithine, can also be used. For various techniques for the transformation of mammalian cells, see Keown et al., Methods in Enzymology, 185: 527-537 (1990) and Mansour et al., Nature, 336: 348-352 (1988). Suitable host cells for cloning or expression of the DNA in the vectors herein include the highest prokaryote, yeast or eukaryotic cells. Suitable prokaryotes include but are not 'limited to eubacteria, such as large-negative or gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Several strains of E. coli are publicly available, such as strain MM294 from E. coli K12 (ATCC 31,446); E. coli X1776 (ATCC 31,537); Strain W3110 from E. coli (ATCC 27,325) and K5 772 (ATCC 53,635). In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors encoding GFRα3. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Suitable host cells for the expression of glycosylated GFRα3 are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of mammalian host cell lines include COS cells and the Chinese hamster ovary (CHO). More specific examples include the kidney CVl line of the monkey transformed by SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Nati, Acad. Sic. USA, 77: 4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23: 243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and the mammary tumor of the mouse (MMT 060562, ATCC CCL 51). The selection of the appropriate host cell is considered to be within the skill of the art. 3. Selection and Use of a Replicable Vector Nucleic acid (eg, cDNA or genomic DNA) encoding GFRα3 can be inserted into a replicable vector for cloning (amplification of DNA) or for expression. Several vectors are publicly available. The vector, for example, may be in the form of a plasmid, cosmid, viral particle, or phage. The sequence of the appropriate nucleic acid can be inserted into the vector by a variety of methods. In general, the DNA is inserted into an appropriate restriction endonuclease site (s) using techniques known in the art. The vector components include in general, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. . The construction of suitable vectors containing one or more of these components employ standard binding or ligation techniques which are well known to one skilled in the art. GFRα3 can be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the protein or mature polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the GFRα3 DNA that is inserted into the vector. The sequence of the signal may be a sequence of the prokaryotic signal selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or stable enterotoxin II directors with heating. For yeast secretion, the sequence of the signal may be, for example, the invertase director of the yeast, the director of the _alfa factor (including the directors of the a factor of Saccharomyces and Kluyveromyces, the latter described in US Pat. No. 5,010,182), or the director of acid phosphatase, the director of glucoamylase of C. albicans (EP 362,179 published April 4, 1990), or the signal described in WO 90/13646 published November 15, 1990 In the expression of mammalian cells, the sequences of the mammalian signal can be used to direct the secretion of the protein, such as the signal sequences from the secreted polypeptides of the same species or related species, as well as of the viral secretory directors. The expression and cloning vectors contain a nucleic acid sequence that makes it possible for the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication of plasmid pBR322 is suitable for most gram-negative bacteria, origin 2: plasmid is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. The expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate, or tetracycline, (b) auxotrophic complement deficiencies, or (c) critical supply nutrients not available from the complex medium, eg, the gene encoding D-alanine racemase for Bacilli An example of selectable markers suitable for mammalian cells are those that make it possible to identify cells competent to receive the GFRα3 nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Nati Acad. Sci. USA, 77: 4216 (1980). A suitable selection gene for use in yeast is the trpl gene present on the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979); Kignsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10: 157 (1980)). The trpl gene provides a selection marker for a mutant strain of the yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PPPEP4-1 (Jones, Genetics, 85:12 (1977)). The expression and cloning vectors usually contain a promoter operably linked to the GFRα3 nucleic acid sequence to direct the synthesis of the mRNA. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the b-lactamase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)), alkaline phosphatase. , a tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al., Proc. Nati. Acad. Scie. USA, 80: 21-25 (1983)). Promoters for use in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operably linked to DNA encoding GFRα3. Examples of promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.

Enzvme Reg., 7: 149 (1968); Holland, Biochemistry, 17: 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other promoters of the yeast, which are inducible promoters that have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocitochrome C, acid phosphatase, the degrading enzymes associated with the nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and the enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in the expression of the yeast are further described in EP 73,657. Transcription of GFRα3 from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, poultry poxvirus (UK 2,211,504 published on 5 July 1989), the adenovirus (such as Adenovirus 2), the bovine papilloma virus, the bird sarcoma virus, the cytomegalovirus, a retrovirus, the hepatitis B virus and the Simian Virus 40 ( SV40), heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, and heat shock promoters, provided that such promoters are compatible with the host cell systems. The transcription of a DNA encoding GFRα3 by the higher eukaryotes can be increased by inserting an enhancer sequence into the vector. The enhancers are cis-acting elements of the DNA, usually approximately 10 to 300 bp, which act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, a virus enhancer of the eukaryotic cell will be used. Examples include the SV40 enhancer on the last side of the replication origin (100-270 bp), the cytomegalovirus initial promoter enhancer, the polyoma enhancer on the last side of the origin of replication, and the adenovirus enhancers. The enhancer can be spliced into the vector at a 5 'or 3' position with respect to the GFRα3 coding sequence, but is preferably located at a 5 'site from the promoter. Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells of other multicellular organisms) will also contain the sequences necessary for the termination of transcription and for the stabilization of mRNA. . Such sequences are commonly available from the 5 'and, occasionally, 3' untranslated regions of the eukaryotic or viral DNAs or cDNAs. These regions contain the transcribed nucleotide segments as the polyadenylated fragments in the untranslated portion of GFRα3 encoding the mRNA. Still other methods, vectors, and host cells suitable for adaptation to the synthesis of GFRα3 in the recombinant vertebrate cell culture are described in Gething et al., Nature, 293: 620-625 (1981); Mantei et al., Nature, 281: 40-46 (1979); EP 117,060; and EP 117,058. 4. Detection of Gene Amplification / Expression The amplification and / or expression of the gene can be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of the mRNA (Thomas, Proc. Nati, Acad. Sci., 77: 5201-5205 (1980)), spot spotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies capable of recognizing specific duplexes can be used, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn can be labeled and the assay can be carried out where the duplex is bound to a surface, so that during the duplex formation on the surface, the presence of the antibody bound to the duplex can be detected. The expression of the gene, alternatively, can be measured by immunological methods, such as immunohistochemical staining of cells or sections of the tissue and assay of the cell culture or body fluids, to directly quantify the expression of the gene product. Antibodies useful for dyeing and / or immunohistochemical testing of the fluids of the sample can be either monoclonal or polyclonal, and can be prepared in any mammal. Conveniently, the antibodies can be prepared against a GFRα3 polypeptide of the wild-type sequence or against a synthetic peptide based on the DNA sequences provided herein or against the exogenous sequence fused to the GFRα3 DNA and encoding an epitope of the specific antibody.5. Purification of the Polypeptide The forms of GFRα3 can be recovered from the culture medium or from the host cell lysates. If they are attached to the membrane, they can be released from the membrane using a suitable detergent solution (for example Triton-X 100) or by enzymatic cleavage. Cells used in the expression of GFRα3 can be altered by various physical or chemical means, such as the freeze-liquefy cycle, sound application, mechanical disruption or disruption, or cell lysate agents. It may be desired to purify GFRα3 from the proteins or polypeptides of the recombinant cell. The following procedures are exemplary of the proper purification procedures: by fractionation on an ion exchange column; the precipitation with ethanol; the reverse phase HPLC; chromatography on silica or on an ion exchange resin such as DEAE; chromatofocusing, SDS-PAGE; precipitation with ammonium sulfate; filtration in a gel using, for example Sephadex G-75; Sepharose columns of protein A to remove contaminants such as IgG; and the metal chelation columns to join the tagged forms of the epitope of GFR3. Various methods of purifying the protein can be employed and such methods are known in the art and are described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The selected purification step (s) will depend, for example, on the nature of the production process used and the particular GFRα3 produced.

E. Uses for GFRa3 The nucleotide sequences (or their complement) that encode GFRα3 have several applications in the art of molecular biology, including uses as hybridization probes, in the assignment of coordinates to chromosomes and genes and in the generation of RNA and DNA antisense The GFRα3 nucleic acid will also be useful for the preparation of the GFRα3 polypeptides by the recombinant techniques described herein. GFRα3 of the full length natural sequence (SEQ ID NO: 14), or portions thereof, can be used in the hybridization probes for a cDNA library to isolate the full length gene or to isolate still others genes (for example, those encoding the variants that are naturally present of GFRα3 or GFRα3 of other species) which have the identity of the desired sequence for the GFRα3 sequence described in SEQ ID NO: 15. Optionally, The length of the probes will be from about 20 to about 50 bases. The hybridization probes can be derived from the nucleotide sequence of SEQ ID NO: 14 or from the genomic sequences that include the promoters, enhancer elements and introns of GFRα3 of the wild-type sequence. By way of example, a screening method will comprise isolating the coding region of the GFRα3 gene using the known DNA sequence to synthesize a selected probe of approximately 40 bases. Hybridization probes can be labeled by a variety of labels, including radionucleotides such as 32P or 13S, or enzymatic labels such as alkaline phosphatase coupled to the probe by means of the avidin / biotin coupling systems. Labeled probes having a sequence complementary to that of the GFRα3 gene of the present invention can be used to select the libraries of the human cDNA, the DNA or the genomic mRNA to determine which members of such libraries for the probe hybridize it. Hybridization techniques are described in further detail in the following Examples. The probes can also be used in PCR techniques to generate a set of sequences for the identification of closely related GFRα3 sequences. The nucleotide sequences encoding a GFRα3 can also be used to construct hybridization probes to assign coordinates to the gene which encodes this GFRα3 and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein can be assigned with coordinates to a chromosome and the specific regions of a chromosome using known techniques, such as in situ hybridization, the analysis of the binding against the known chromosomal markers, and the selection of the hybridization with the libraries. When the coding sequences for GFRα3 encode a protein which binds to another protein (eg, where GFRα3 is a receptor), GFRα3 can be used in assays to identify the other proteins or molecules involved in the interaction of the Union. By such methods, inhibitors of the receptor / ligand binding interaction can be identified. The proteins involved in such binding interactions can also be used to screen for peptides or inhibitors or small molecule agonists of the binding interaction. Also, the GFRα3 of the receptor can be used to isolate the correlating ligand (s). Selection tests can be designed to find master compounds that mimic the biological activity of a natural GFRα3 or a receptor for GFRα3. Such screening assays will include assays capable of high throughput screening of chemical libraries, making them particularly suitable for identifying candidates for small molecule drugs. The contemplated small molecules include synthetic organic or inorganic compounds. Assays can be carried out in a variety of formats, including protein-protein binding assays, biochemical selection assays, immunoassays and cell-based assays, which are well characterized in the art. The nucleic acids which encode GFRα3 or its modified forms can also be used to generate either transgenic animals or excellent animals which, in turn, are useful in the development and selection of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal that has cells that contain a transgene, such a transgene was introduced into the animal or an ancestor of the animal in a prenatal stage, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, the cDNA encoding GFRα3 can be used to clone the genomic DNA encoding GFRα3 according to established techniques and the genomic sequences used to generate the transgenic animals that contain cells expressing the DNA encoding GFRα3. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,780,009. Typically, the particular cells could be targeted for incorporation of the GFRα3 transgene with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding GFRα3 introduced into the germline of the animal at an embryonic stage can be used to examine the effect of increased expression of the DNA encoding GFRα3. Such animals can be used as test animals for reagents that are thought to confer protection against, for example, the pathological conditions associated with their overexpression. According to this approach of the invention, an animal is treated with the reagent and a reduced indignation of the pathological condition, compared to untreated animals carrying the transgene, could indicate a potential therapeutic intervention for the pathological condition. Non-human homologs of GFRα3 can be used to construct an "excellent" animal with GFRα3 which has a defective or altered gene encoding GFRα3 as a result of homologous recombination between the endogenous gene encoding GFRα3 and altered genomic DNA which encodes GFRα3 introduced into an embryonic cell of the animal. For example, the cDNA encoding GFRα3 can be used to clone the genomic DNA encoding GFRα3 according to established techniques. A portion of the genomic DNA encoding GFRα3 can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to verify integration. Typically, several kilobases. of the unaltered flanking DNA (both of the 5 'and 3' ends) are included in the vector (see for example, Thomas and Capecchi, Cell, 51: 503 (1987) for a description of the homologous recombination vectors). The vector is introduced into an embryonic stem cell line (for example, by electroporation) and the cells in which the introduced DNA has been recombined homologously with the endogenous DNA are selected (see for example, Li et al., Cell, 69: 915 (1992)). The selected cells are then injected into a blastocyst of an animal (eg, a mouse or rat) to form aggregation chimeras (see for example, Bradley, in Teratocarcinomas and Erythronic Stem Cells: A Practical Approach, EJ Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted in a suitable pseudopregnant breeding animal and the embryo brought to term to create an "excellent" animal. Progeny that collect recombinant DNA homologously in their germ cells can be identified by standard techniques and used to reproduce animals in which all of the cells of the animal contain the homologously recombined DNA. The excellent animals may be characterized, for example, by their ability to defend against certain pathological conditions and to verify the development of pathological conditions due to the absence of the GFRα3 polypeptide. The agents which bind to the GFRα3 molecule could be useful in the treatment of diseases or conditions involving the peripheral nervous system. For example, such ligands can be used to treat peripheral neuropathies associated with diabetes, HIV, and treatments with chemotherapeutic agents. Ligands that bind to GFRα3 are expected to be useful in the treatment of neuropathic pain, GFRα3 antagonists are expected to be useful for treating chronic pain of a non-neuropathic nature such as, but not limited to, those which are associated with several inflammatory conditions. The above therapies are consistent with the data of Example 5 in which a strong expression of GFRα3 within the adult and developing sensory ganglia was observed. GFR3 or its agonists or antagonists can be used to treat conditions that involve autonomic nervous system dysfunction including, but not limited to, alterations in blood pressure or heart rate, gastrointestinal function, impotence, and urinary continence. Other indications for ligands that bind to GFRα3 include: post-herpetic neuralgia, herpes zooster, asthma, irritable bowel, inflammatory bowel, cystitis, headache (migraine), arthritis, damage to the spinal cord, constipation, hypertension, mucositis, eyes or dry mouth, fibromyalgia, chronic back pain, or the healing of wounds. These uses are consistent with the expression observed in the glanglia of the sympathetic system. The relative, surprising lack of expression of GFRα3 in many organs, notably including the brain, intestines, and kidney, indicates that the ligand (and other agonists or antagonists) which binds to this receptor, lack some side effects which may be associated with the ligands which bind to GFRal or GFRα2 (GDNF and neurturin). Therefore, ligands which act by means of GFRα3 will be particularly useful for treating disorders of the peripheral nervous system while inducing smaller effects in weight loss, motor functioning, or on the function of the kidney that could make that the ligands act by means of GFRal or GFRα2.

F. Anti-GFRα3 antibodies The present invention further provides anti-GFRα3 antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. 1. Polyclonal Antibodies 4 The anti-GFRα3 antibodies can comprise polyclonal antibodies. Methods of preparation of polyclonal antibodies are known to the person skilled in the art. Polyclonal antibodies can be enhanced in a mammal, for example, by one or more injections of an immunization agent and, if desired, an adjuvant. Typically, the immunization agent and / or the adjuvant can be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunization agent can include the GFRα3 polypeptide or a fusion protein thereof. The agent may be useful for conjugating the immunization agent with a protein known to be immunogenic in the mammal that is immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and trypsin and soybean inhibitor. Examples of auxiliaries that can be employed include Freund's complete assistant and MPL-TDM assistant (monophosphoryl lipid A, synthetic trehalose dicorinomycolate). The immunization protocol can be selected by one skilled in the art without undue experimentation. 2. Monoclonal antibodies The anti-GFRα3 antibodies, alternatively, may be monoclonal antibodies. Monoclonal antibodies can be prepared using the hybridoma methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunization agent to produce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunization agent. Alternatively, lymphocytes can be immunized in vitro. The immunization agent will typically include the GFRα3 polypeptide or a fusion protein thereof. In general, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986), pp. 59-103 ). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of immortalized, unfused cells. For example, if the original cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine ("HAT medium")., such substances prevent the growth of HGPRT deficient cells. Preferred immortalized cell lines are those that efficiently fuse, support expression of the stable elevated level of the antibodies by the selected antibody-producing cells, and are sensitive to a medium such as the FAT medium. The most preferred immortalized cell lines are the murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and human-mouse heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) p. 51-63). The culture medium in which the hybridoma cells are cultured can then be evaluated for the presence of the monoclonal antibodies directed against GFRα3_. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as the radioimmunoassay (RIA) or the enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody, for example, can be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution methods and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or the ascites fluid by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. The DNA encoding the monoclonal antibodies of the invention can be easily isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, DNA can be placed in the expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not they otherwise produce the immunoglobulin protein, to obtain the synthesis of the monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substitution of the coding sequence for the heavy and light chain constant domains, human, instead of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison et al., Supra) or by covalent attachment to the immunoglobulin coding sequence of all or part of the coding sequence for a polypeptide other than immunoglobulin. Such a polypeptide other than immunoglobulin can be replaced by the constant domains of an antibody of the invention, or can be substituted by the variable domains of a combination site of the antigen of an antibody of the invention to create a chimeric bivalent antibody. The antibodies can be monovalent antibodies. Methods for preparing the monovalent antibodies are well known in the art. For example, one method involves the recombinant expression of the immunoglobulin light chain and the modified heavy chain. The heavy chain is generally truncated at any point in the Fc region to prevent cross-linking of the heavy chain. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. The digestion of the antibodies to produce fragments thereof, particularly the Fab fragments, can be effected using routine techniques known in the art. 3. Humanized Antibodies The anti-GFRα3 antibodies of the invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other binding subsequences. antigen antibodies) which contain a minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (the receptor antibody) in which the residues of a region of complementary determination (CDR) of the receptor are replaced by the residues of a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit that have the desired specificity, affinity and capacity. In some cases, the residues of the Fv structure of the human immunoglobulin are replaced by the corresponding non-human residues. The humanized antibodies may also comprise residues which are found neither in the receptor antibody nor in the imported structure or CDR sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the All FR regions are those of a consensus sequence of human immunoglobulin. The humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 232-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)). Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced therein from a source which is non-human. These non-human amino acid residues are frequently referred to as "significant" residues, which are typically taken from a "significant" variable domain. Humanization can be effected essentially following the method of Winter et al. (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science., 239: 1534-1536 (1988)), substituting rodent CDR or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of the analogous sites in the rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991)).; Marks et al., J. Mol. Biol., 222: 581 (1991)). The techniques of Colé et al. And Boerner et al. Are available for the preparation of human monoclonal antibodies (Colé et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Im. unol., 147 (1): 86-95 (1991)). 4. Bispecific Antibodies Bispecific antibodies are preferably human or humanized, monoclonal antibodies, which have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for GFRα3, the other is for any other antigen, and preferably for a cell surface protein or receptor or receptor unit. Methods for making bispecific antibodies are already known in the art.

Traditionally, the recombinant production of bispecific antibodies is based on the coexpression of two pairs of light chain / immunoglobulin heavy chain, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305-537-539 81983)) . Because of the randomization of immunoglobulin light and heavy chains, these hybridomas. { quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually carried out by the affinity chromatography steps. Similar procedures are described in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10: 3655-3659 (1991). The variable domains of the antibodies with the desired binding specificities (antigen-antibody combining sites) can be fused to the immunoglobulin constant domain sequences. The fusion preferably is with a constant domain of the immunoglobulin heavy chain, comprising at least part of the joint regions, CH2, and CH3. It is preferred to have the first constant region of the heavy chain (CH1) containing the site necessary for light chain binding present in at least one of the fusions.

The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected in a suitable host organism. For further details of generation of bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

. Heteroconjugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two antibodies covalently linked. Such studies, for example, have been proposed for cells of the target immune system for unwanted cells (US Patent No. 4,676,980), and for the treatment of HIV infection (WO 91/00360; WO 92/200373 EP 03089). It is contemplated that antibodies can be prepared in vitro using methods known in the synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or forming a thioether linkage. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate and those described, for example, in U.S. Pat. No. 4,676,980.

G. Uses for Anti-GFRα3 Antibodies The anti-GFRα3 antibodies of the invention have several utilities. For example, anti-GFRα3 antibodies can be used in diagnostic assays for GFRα3, for example, the detection of specific expression in cells, tissues, or serum. Various diagnostic assay techniques known in the art can be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays in either heterogeneous or homogeneous phases (Zola, Monoclonal antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158). The antibodies used in diagnostic assays can be labeled with a detectable portion. The detectable portion must be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable portion can be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or chemiluminescent compound, such as a fluorescent isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase. , beta-galactosidase or horseradish peroxide. Any method known in the art for conjugation of the antibody to the detectable portion can be employed, including those methods described by Hunter et al., Nature, 144: 945. (1962); David et al., Biochemistry, 13: 1014 (1974); Pain et al., J. Immunol. Meth., 40: 219 (1981); and Nygren, J. Histochem. and Citochem. , 30: 407 (1982). The anti-GFRα3 antibodies are also useful for the affinity purification of GFRα3 from natural sources or the culture of the recombinant cells. In this process, antibodies against GFRα3 are immobilized on a suitable support, such as a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is then contacted with a sample containing the GFRα3 to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all of the material in the sample except GFRα3, which is attached to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the GFRα3 from the antibody.

H. Assays to Verify the Activity of Subunit A Induced by the Ligand The compounds and methods of the invention can be used in assays to detect molecules that activate or inhibit the translation of the GFRα3 signal, and can actually be applied to other receptor molecules of the α subunit (eg, GFRal, GFRα2 , GFRa34) that homodimerize and homo-oligomerize during activation by a ligand or other agonist. The assays are based on the surprising fact that μ subunit receptors can be homodimerized or homooligomerized during ligand binding. And further that this dimerization of a subunit a, when fused to an intracellular domain of the kinase of the receptor protein capable of kinase activity, preferably the activity of the tyrosine kinase, leads to the activity of the kinase, for example easily detectable autophosphorylation. Although the methods and constructions herein are described in terms of one or the other receptor of the GFRα subunit described herein, the methods will readily apply to any receptor a in the α-family of the subunit a receptor in which the the a subunit is the ligand binding origin of a multiple subunit signaling complex containing the beta subunit that typically contains a tyrosine kinase activity that is activated during the binding of the subunit to the ligand-activated subunit. beta subunit. Several assays have been developed which measure the activity of the kinase, and in particular the tyrosine kinase activity. Some of these assays measure the ability of a tyrosine kinase enzyme to phosphorylate a polypeptide of the synthetic substrate. For example, a test has been developed which measures the tyrosine kinase activity stimulated by the growth factor by measuring the ability of the kinase to catalyze the transfer of phosphate? from ATP to a substrate of the appropriate acceptor. See Pike, L., Methods of Enzimology 146: 353-362 (1987) and Hunter, Journal of Biological Chemistry 257 (9): 4843-4848 (1982), for example. In this assay, the use of (? -32P) ATP allows the radioactive labeling of the phosphorylated substrate, which is a peptide containing synthetic tyrosine. Others have described protein kinase assays where the incorporation of 32P into a tyrosine kinase receptor, such as the EGF receptor (see Donato et al., Cell Growth Differ., 3: 259-268 (1992)), the receptor of insulin (see Kasuga et al., Journal of Biological Chemistry 257 (17): 9891-9884 (1982) and Kasuga et al., Methods in Enzymology 109: 609-621 (1985)), and the growth hormone receptor of the Liver (see Wang et al., Journal of Biological Chemistry 267 (24): 17390-17396 (1992)), is measured. The construction of a-receptor constructs, including fusions to Rse or other tyrosine kinase domains, vectors for the expression of such constructs, transfected or transformed host cells expressing these constructs, and means for enhancing their expression in the Cell surface is achieved as would be known in the art, using, for example, the techniques described herein for the expression of GFRα3. Some particularly preferred means are provided later. 1. Activation of the Kinase Receptor - KIRA The first step of an assay of the invention involves the phosphorylation of the kinase domain of a receptor construct, wherein the receptor construct is present in the cell membrane of a eukaryotic cell. The construction of the receptor can be derived from a nucleic acid encoding the construction of the receptor (as described herein) that can be transformed into the cell. In one embodiment of the invention, the nucleic acid encoding a -receptor construct is transformed into the cell. Preferred and exemplary techniques for transforming the cell with either the receptor or the nucleic acid of the receptor construction are given below. to. Transformation of cells The present invention provides a substantial improvement over in vitro soluble kinase receptor assays because it is considered to more accurately reflect the activity of the subunit receptor in situ. It has been found that it is possible to transform the eukaryotic cells with a receptor construct (comprising the subunit receptor a and a kinase domain fusion and optionally, a flag polypeptide either amino- or carboxyl-terminal) so that the The receptor construction resembles itself appropriately in the cell membrane and still retains the activity of the kinase which can be detected in the ELISA stage of the assay. This provides a generic assay for measuring the binding activity of the ligand, via the kinase activity of the fusion, of any receptor of the a-subunit of interest that is homodimerized or homooligomerized during the binding of the ligand.

If a suitable capture agent as described herein is available for a selected receptor construct, the cells can be transformed with the nucleic acid encoding the receptor construct alone, without the flag polypeptide. To provide the nucleic acid encoding a receptor construct, the nucleic acid encoding the subunit a receptor is fused at its 3 'end to the nucleic acid encoding the intracellular catalytic kinase domain of a receptor kinase, preferably an rPTK, which includes a transmembrane domain, and optionally the N terminus or the flag polypeptide. Alternatively, the nucleic acid encoding the kinase domain fusion of the a subunit receptor will be fused at its 5 'end to the nucleic acid encoding the carboxyl terminal or end of the flag polypeptide. Therefore, the flag polypeptide is provided at the carboxyl or amino terminus or terminal of the receptor construction. Examples of suitable flag polypeptides are provided above. The selection of other suitable flag polypeptides is possible using the techniques described herein. To generate fusions between the flag reagent.Rse and a receptor of the a subunit of interest, the nucleic acid encoding the ECD (or the minor variant of GPI anchor) of the receptor of the a subunit of interest is fused in its 3 'end to the nucleic acid encoding the amino terminal or end of the flag reagent. Rse. The incorporation of a signal sequence in the expression vector is required since the receptor construction must be transported to the cell membrane where it is placed in such a way that the ECD is facing the external environment of the cell. Therefore, a sequence of the signal suitable for the placement of the receiver construction in such a manner is used. The signal sequence is generally a component of the vector, or it can be a part of the DNA of the receptor construct that is inserted into the vector. If a sequence of the heterologous signal is used, it is from it that it is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

Selection of cells for use in the trial As mentioned above, the cells to be tested are cells preferably transformed with a receptor construct. The suitability of the cells for use in the assay is investigated. If the cell line is transformed with the receptor construct (without the flag polypeptide), it can be easily discovered if the cell line is suitable for use in the assay. As a first step, the transformation and successful expression of the nucleic acid encoding the receptor construction is determined. The strategy found in U.S. Pat. No. 5,766,863, or its corresponding WO publication, entitled "Kinase receptor activation assay" ("Assay of kinase receptor activation") can be followed. To identify whether the ECD of the receptor construct is present on the surface of the cells, flow cytometric analysis can be performed using an antibody to the ECD of the a subunit receptor. The antibody can be made using the techniques for generating the antibodies described herein. The flow cytometric analysis can be carried out using the techniques described in Current Protocols in Immunology, Ed. Coligen et al., Wiley publishers, Vols. 1 and 2, for example. In summary, the flow cytometric analysis involves the incubation of the intact cells (which have the receptor) with the antibodies for the ECD thereof, followed by the washing. The cells bound to the antibody are then incubated with antibodies specific for the anti-antibodies of the conjugated species with a fluorochrome. Following washing, the labeled cells are analyzed by fluorescence activated flow cytometry to detect if the ECD is present on the surface of the cells. In the next step, the capacity of the receiver attached to the cell that is going to be activated is going to be tested. To determine this, the transformed cells are exposed to a known agonist with respect to the receptor (for example the endogenous ligand or an antagonist antibody to the receptor). In the case of GFR3, the natural ligand is artemin. Following exposure, the cells are lysed in a suitable buffer (eg, sodium dodecylbenzenesulfonate in a phosphate-buffered saline solution, SDS in PBS) and subjected to Western blotting with anti-phosphotyrosine antibodies as described in Wang, Molecular and Cellular Biology 5 (12): 3640-3643 (1985); Glenney et al., Journal of Immunological Methods 109: 277-285 1988); Ka ps, Methods in Enzimology 201: 101-110 (1991); Kozma et al., Methods in Enzymology 201: 28-43 (1991); Holmes et al., Science 256: 1205-10 (1992); or Corfas et al., PNAS, USA 90: 1624-1628 (1993), for example.

Assuming that the Western blotting step indicates that the receptor construct can be activated, a KIRA ELISA test run can be performed to further establish whether the transformed cell line can or can not be used in the assay. In the preferred embodiment of the invention, the KIRA ELISA is a "generic" assay since any receptor of the a subunit of interest can be studied regardless of the availability of specific reagents for the receptor (ie, the capture agent) . This embodiment employs a receptor construct that has a flag polypeptide at the terminal or amino or carboxyl terminus of the receptor. If the flag polypeptide is provided at the terminus or terminal of NH2 (see, for example, the gD.trk A, B and C receptor constructs), the procedure for selecting a transformed cell line for use in the assay is similar. to that described above. In this embodiment, the cells are transformed with the receptor-flag polypeptide construction as described here. The successful transformation of the receptor and the flag polypeptide (ie, the receptor construct in this example) is confirmed. To study this, two-dimensional flow cytometric analysis can be performed using the antibodies for both the flag polypeptide and the receptor ECD. Techniques for two-dimensional flow cytometric analysis are described in Current Protocols in Immunology, supra. Cells that have been successfully transformed with the receptor construct having a C-terminal flag polypeptide are also suitable for use in the assay. Following cell transformation, successful transformation of the receptor is determined by flow cytometric analysis using an antibody directed against the ECD of the receptor of interest, for example. The flow cytometric analysis can be carried out substantially as described above. The successful transformation of the complete receptor construct (including the COOH terminal flag polypeptide) is then analyzed. This can be accomplished by lysing the cells (using the techniques for lysate of the cells described herein) and immunoprecipitation of the membrane extract with an antibody against the receptor of the a-subunit of interest. This extract from the immunoprecipitated membrane is then subjected to a Western blot analysis with the antibodies specific for the flag polypeptide. Alternatively, ELISA analysis specific for the α-subunit of the membrane lysate captured by the anti-flag polypeptide can be carried out. In summary, this involves coating the ELISA cavities with the appropriate capture agent for the flag. The cavities are blocked, washed, and the lysate is then incubated in the cavities. The unbound receptor construction is removed by washing. The cavities are then reacted with the antibody or the antibodies specific for the receptor, conjugated either directly or indirectly to the HRPO. The cavities are washed and the HRPO is then exposed to the chromogenic substrate (e.g., TMB). The detection of receptor activation and the test run of KIRA ELISA are essentially the same as those steps described above. Once the useful cells are identified, they are subjected to the KIRA step of the currently claimed test. c. Coating the first solid phase with the cells The first solid phase (for example a cavity of a first test plate) is coated with the cells which have been transformed with respect to the preceding sections. Preferably, an adherent cell line is chosen, so that the cells adhere naturally to the first solid phase. However, the use of an adherent cell line is not essential. For example, non-adherent cells (e.g., red blood cells) can be added to the round bottom cavities of a test plate such as those sold by Becton Dickinson Labware, Lincoln Park, New Jersey, for example. The test plate is then placed in a plate holder and centrifuged so that a microsphere of cells adhering to the base of the cavities is created. The culture supernatants of the cells are removed using a pipette. Accordingly, the use of an adherent cell is clearly advantageous over non-adherent cells since this reduces the variability in the assay (i.e., the cells in the microsphere of the round bottom cavities can be received with the supernatant when the alternative is used). The cells that are to be added to the cavities of the first test plate can be maintained in the tissue culture bottles and used when the cell densities of a confluence of about 70-90% are achieved. Then, generally approximately between I x l04 and 3 x l05 (and preferably 5 x 104 to 1 x 105) cells are seeded by round bottom cavity, using a pipette, for example. It has been found that, contrary to speculation, the addition of the cell concentrations mentioned above is sufficient to make possible the activation of the receptor construction to be measured in the ELISA stage of the assay, without the need to concentrate or clarify the cells or the cell lysate prior to it. Frequently, the cells are diluted in the culture medium prior to planting them in the cavities of the microtiter plate to achieve the desired cell densities. Usually, the cells are cultured in the microtiter plates for a sufficient period of time to optimize the adhesions to the cavities thereof, but not too prolonged in such a way that the cells begin to deteriorate. Accordingly, incubation for about 8 to 16 hours at a temperature which is physiologically optimal for the cells (usually approximately 37 ° C) is preferred. The suitable medium for the culture of the cells is described in Section 1A above. Cultivation in C02 at 5% is recommended.

Following the overnight incubation, the supernatants of the cavity are decanted and the excess supernatant can be further removed by lightly tampering the microtiter plates with an absorbent substrate, for example, a paper towel, but a sponge works equally well. . Accordingly, a substantially homogeneous layer of adherent cells remains on the inner surfaces of the individual cavities of the microtiter plate. These adherent cells are then exposed to the substance to be analyzed. d. Preparation and addition of the substance to be analyzed As mentioned above, the substance to be analyzed can comprise an agonist ligand (or suspect agonist) or antagonist (or suspect antagonist) for the receptor of the a-subunit of interest. The ligand may be an endogenous polypeptide, or a synthetic molecule, such as an organic or inorganic molecule. Usually, the ligand is a polypeptide. This assay is useful for selecting molecules which activate (or antagonize the activation) of the receptor of the a subunit of interest. Therefore, the assay can be used to develop therapeutically effective molecules. Where the ligand is an agonist, the molecule can comprise the natural growth factor eg, artemin, neurturin, GDNF, and persephin. Many of these growth factors are commercially avaie. Alternatively, the growth factor can be made by the synthesis of peptides or recombinant techniques which are described herein. Synthetic small molecule agonists can be similarly generated by those skilled in the art using conventional chemical synthesis techniques. Preferably, agonist or antagonist antibodies are being tested. Where the ligand is present in a biological fluid, the substance to be analyzed can be prepared using techniques which are well known in the art. Body fluid such as blood or amniotic fluid can be used directly, however a concentration may be required. If the substance to be tested, which is to be tested, comprises a particular tissue, the cells thereof can be grown in the cell culture and the supernatant can be tested to verify the secreted ligand.Frequently, the ligand is diluted in an aqueous diluent (such as a cell culture medium) so that a standard curve can be generated. However, the ligand may be present in a cell or a component of the cell (e.g., the cell membrane). In particular, it has been found that the assay can be used to detect the presence of a ligand in the cell membrane of a selected cell line. This is clearly useful for discovering a novel endogenous ligand for a receptor of the known α subunit. The composition of the ligand is added to each cavity which contains the adherent cells using a pipette, for example. At least one control cavity (for example to which the aqueous diluent for the ligand is added) is included in the assay. The adherent cells are usually stimulated for a sufficient period of time to optimize the signal, but not too long in such a way that the signal is reduced as a consequence of the dephosphorylation of the receptor by the endogenous phosphatases. A suitable stimulation period is between about 10 to 60 minutes, preferably about 30 minutes at an optimum physiological temperature for the cells (usually approximately 37 ° C).

Following activation, the supernatants of the cavity are decanted and the plates are then tamped lightly with an absorbent substrate to remove excess supernatant. The assay can be used to detect antagonist ligands for the receptor of interest. Antagonists generally fall into two categories: (a) those that bind to the receptor and thereby block the binding and / or activation of the receptor by an antagonist thereto (the antagonist can bind to the ECD, but this is not necessarily the case) and (b) those that bind to the agonist and therefore prevent activation of the receptor by the agonist. To detect the antagonist molecules of category (a) above, the cells are exposed to the antagonist ligand substantially suspect as mentioned above. __ Next to the antagonist exposure, the supernatants of the cavity are decanted and the plates are lightly tamped . A known agonist (e.g., endogenous growth factor) is then added to the washed cells essentially as described in the preceding paragraphs, after which, the supernatants of the cavity are decanted and the plates are lightly tamped. Alternatively, a composition comprising both the antagonist and the agonist can be added to the adherent cells substantially as described above. The ability of the suspected agonist to block the binding and / or activation of the receptor can be subsequently measured by ELISA as described below. To detect the antagonist molecules of category (b) above, a known agonist is pre-incubated with the suspected antagonist prior to the KIRA step of the assay. This incubation is carried out for a sufficient period of time to enable a complex of the agonist-antagonist to form; from 30 minutes to 12 hours, for example. This complex is then subjected to the assay with the agonist and antagonist that did not form a complex, used as the control. Following exposure to the agonist (and optionally the antagonist), the cells are used, as will be described later). and. Solubilization of cells In this step of the assay, the cells are used to solubilize the construction of the receptor such that they remain activated (i.e., the tyrosine residues remain phosphorylated) for the ELISA step of the assay. Accordingly, the cells are used using a lysis buffer as described above, which serves to solubilize the receptor construction, not to dephosphorylate or denature the receptor construction. Where the microtiter plates are used as mentioned above, approximately 75 to 200 μl of the buffer for lysis is added to each well. The plates are then gently shaken using a plate shaker (for example, such as that sold by Belico Instruments, Vineland, NJ) for about 1 to 2 hours. The stirring can be carried out at room temperature. 2. Immunosorbent Assay Linked by the ELISA Enzyme The second stage of the assay involves a sandwich ELISA performed on the second test plate. To carry out the ELISA, a capture agent is prepared. to. Preparation of the capture agent As mentioned above, the capture agent frequently comprises a polyclonal antibody (usually an affinity purified polyclonal antibody) or monoclonal antibody. Other capture agents are contemplated and described in the previous section of definitions. The capture agent either binds specifically to the receptor, or to the flag polypeptide (ie the antigen). Polyclonal antibodies to the antigen (either the flag receptor or polypeptide) are generally elevated in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the antigen or an antigenic fragment thereof (often the receiver's ECD). of subunit a) and an auxiliary. It may be useful for conjugating the antigen or a fragment containing the target or target amino acid sequence for a protein that is immunogenic in the species to be immunized (e.g., the keyhole limpet hemocyanin). , using a bifunctional or derivative agent. The route and schedule for the administration of the immunogen to the host animal or the cells that produce the antibody cultured therefrom, are generally in accordance with conventional and established techniques for the stimulation and production of antibodies. Although mice are frequently employed as the test model, it is contemplated that any mammalian subject including the human subjects or the cells that produce antibodies obtained therefrom, may be manipulated according to the processes of this invention to serve as the basis for the production of hybrid, mammalian cell lines, including human ones. The animals are typically immunized against the conjugates or immunogenic derivatives by combining 1 mg or 1 μg of the conjugate (for rabbits or mice, respectively) with 3 volumes of the complete Freund's assistant and injecting the solution intradermally at multiple sites. One month later the animals are stimulated with 1/5 to 1/10 of the original amount of the conjugate in Freund's complete adjuvant (or other suitable auxiliary) by subcutaneous injection at multiple sites. 7 to 14 days later the animals are bled and the serum is evaluated to verify the anti-antigen concentration. The animals are stimulated until the concentration stabilizes. Preferably, the animal is stimulated with the conjugate of the same antigen, but conjugated to a different protein and / or through a different cross-linking agent. The conjugates can also be made in a recombinant cell culture as protein fusions. Also, aggregation agents such as alum are used to improve the immune response. After immunization, the monoclonal antibodies can be prepared by the recovery of immune cells (typically spleen cells or lymphocytes from lymph node tissue) of the immunized animals and the immortalization of the cells in the conventional manner, for example, by fusion with the myeloma cells or by the transformation of the Epstein-Barr virus (EB) and the selection of the clones that produce the desired antibody. The hybridoma technique originally described by Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976), and also described by Hammerling et al., In: Monoclonal antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981) has been widely used to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens. It is possible to fuse the cells of one species with another. Nevertheless, it is preferable that the source of the cells that produce the immunized antibody and the myeloma are of the same species. The hybrid cell lines can be maintained in culture in the cell culture medium. The cell lines of this invention can be selected and / or maintained in a composition comprising the continuous cell line in the hypoxanthine-aminopterin-thymidine (HAT) medium. In effect, once the hybridoma cell line is established, it can be maintained in a variety of nutritionally adequate media. In addition, hybrid cell lines can be stored and preserved in any number of conventional ways, including freezing and storage under liquid nitrogen. Frozen cell lines can be revived and cultured indefinitely with the synthesis and secretion of the monoclonal antibody. The secreted antibody is recovered from the tissue culture supernatant by conventional methods such as precipitation, ion exchange chromatography, affinity chromatography, or the like. The antibodies described herein are also recovered from the cultures of the hybridoma cells by conventional methods by purification of IgG or IgM, depending on the case in question, which until now have been used to purify these immunoglobulins from the pooled plasma, by For example, ethanol or ethylene glycol precipitation procedures. The purified antibodies are then filtered under sterile conditions. Where the antibody is a polyclonal antibody, it is generally affinity purified using an affinity column generated from the antigen of interest to provide a substantially specific capture antibody. Affinity chromatography is usually preceded by other purification techniques, such as liquid chromatography. In a further embodiment, antibodies or antibody fragments can be isolated from the antibody phage libraries generated by means of the techniques described in McCafferty et al., Nature, 348: 552-554 (1990), using the flag polypeptide , a receptor of the subunit a, or a fragment thereof, to select an antibody or suitable antibody fragment. Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222-581-597 (1991) describes the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity human antibodies (nM range) by chain rearrangement (Mark et al, Bio / Technol 10: 779-783 (1992)), as well as combinatorial infection and recombination in vivo as a strategy to build very large phage libraries (Waterhouse et al., Nuc Acids Res., 21: 2265-2266 (1993)). Accordingly, these techniques are feasible alternatives for hybridoma techniques of the traditional monoclonal antibody for the isolation of "monoclonal" antibodies which are encompassed by the present invention. The DNA encoding the monoclonal antibodies of the invention is easily isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are capable of specifically binding to the genes encoding the light and heavy chains of murine antibodies. of the hybridoma of the invention serve as a preferred source of such DNA Once isolated, the DNA can be placed in the expression vectors, which are then transfected into the host cells such as simian COS cells, ovarian cells of the Chinese hamster (CHO), or the myeloma cells that do not otherwise produce the immunoglobulin protein, to obtain the synthesis of the monoclonal antibodies in the recombinant host cells.The DNA can also be modified, for example, by the substitution of the coding sequence for the constant domains of the light and heavy chains, human, instead of the homologous murine sequences, Morrison et al., Proc. Nati Acad. Sci. 81, 6851 (1954), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence of a polypeptide other than immunoglobulin. In this manner, "chimeric" or "hybrid" antibodies are prepared in such a way that they have the bng specificity of a monoclonal antibody of the anti-receptor or anti-flag polypeptide herein. Accordingly, the antibody can be made by recombinant DNA methods (Cabilly et al, U.S. Pat. No. 4,816,567). The bng of the capture agent is not effected by the presence or absence of a ligand bound to the receptor and the capture agent does not spherically block access to the phosphorylated tyrosine (s) by the antiphosphotyrosine antibody. In addition, the capture agent, of course, does not activate the receptor of interest. First, once the capture agent (eg an antibody or streptavidin) has been chosen, binding to and either the receptor or flag polypeptide (wherein a receptor construct is to be used in the assay) is confirmed. This can be determined by flow cytometric analysis, immunoprecipitation or ELISA with antigen coating, for example. The flow cytometric analysis has been described above. Immunoprecipitation usually involves lysis of the cells (which have the receptor construction) in a non-ionic detergent (eg 0.5% Triton X-100) in a suitable buffer (e.g. PBS) and the cell lysates thus obtained are then incubated with the capture agent of the anti-flag polypeptide or potential anti-receptor. The immune complexes are precipitated with either (a) the anti-capture agent antibodies in the presence of polyethylene glycol (PEG) which improves the precipitation of the immune complex or with (b) the protein A or the insoluble protein G (e.g. attached to the agarose). The immunoprecipitated material is then analyzed by electrophoresis of the polyacrylamide gel (PAGE). For the ELISA with antigen coating, the ELISA cavities are coated overnight with either the purified receptor, the purified flag polypeptide or the purified receptor construct. The coated cavities are then exposed to the potential capture agent and selected with the conjugated species of HRPO specific for the anti-capture agent antibody. The ability of the capture agent to bind the receptor or flag polypeptide in the presence of a ligand to the receptor is also confirmed. This can be analyzed by the incubation of the receptor construct with a known ligand for the receptor (for example the endogenous growth factor or an agonist antibody thereto). The flow cytometric analysis, the immunoprecipitation or the ELISA with antigen coating can then be carried out substantially as described above to investigate the binding of the capture agent. Assuming that the capture agent is adequate as determined by the two preceding steps, it is then demonstrated that the capture agent does not induce receptor activation (i.e., autophosphorylation) either before or after cell lysis. Accordingly, the receptor construct bound to the cell is exposed to either the potential capture agent or a negative control (eg, a control antibody which does not activate the receptor). Following cell lysis, the receptor construct can be subjected to Western blot analysis using labeled anti-phosphotyrosine antibodies. See, for example, Glenney et al., Journal of Immunological Methods 109: 277-285 (1988); Kamps, Methods in Enzymology 201: 101-110 (1991); Kozma et al., Methods in Enzimology 201: 28-43 (1991); or Holmes et al., Science 256: 1205-10 (1992). To establish whether the capture agent induces receptor activation following the lysis of the cells, a test run of the KIRA ELISA (with both the capture agent and the negative control as described above) can be performed.

Finally, the ability of an anti-phosphotyrosine antibody (for example the biotinylated anti-phosphotyrosine antibody) to bind to the activated receptor in the presence of the potential capture agent is confirmed by a test run in the KIRA ELISA described herein. Assuming that the capture agent satisfies all of the criteria specified above, it has good potential for use in the KIRA ELISA. Once a suitable capture agent has been prepared, the second solid phase is coated therewith. Between about 0.1 and 10 μg / ml of the capture agent can be added to each cavity of the second test plate using a pipette, for example. The capture agent is often provided in a buffer solution at a high pH (eg, between about 7.5 to 9.6) so that it has an increased total charge and therefore exhibits an improved bond to the second test plate. Usually, the capture agent will be incubated in the cavities between about 8 to 72 hours to enable a sufficient coating of the capture agent to be formed on the internal walls of the cavities. This incubation is generally carried out at low temperatures (eg, between about 3-8 ° C) to avoid or reduce the degradation of the capture agent. Following the incubation, the cavities of the plate are decanted and lightly tamped with an absorbent substrate. The non-specific binding is then blocked. To achieve this, a blocking buffer solution is added to the cavities. For example, a blocking buffer solution containing bovine serum albumin (BSA) such as that sold by Intergen Company, Purchase, NY, is suitable. It has been found that the addition of from about 100 to 200 μl of the blocking buffer to each well followed by gentle agitation at room temperature for about 1-2 hours is sufficient for the non-specific binding of blocking. It is also possible to add the blocking buffer solution directly to the cell lysate obtained from the previous step instead of to the second test plate. Following this, the plates coated with the capture agent are washed several times (usually between about 3-8 times) with a wash buffer. The wash buffer can comprise a salt solution buffered with phosphate (PBS) at pH 7.0 to 7.5, for example. However, other wash buffer solutions are available, which can also be used. Conveniently, an automatic dishwashing machine, such as the Sean Washer 300 (Skatron Instruments, Inc., Sterling, VA) can be used for this, and another, for the washing steps of the test.

B. Measurement of tyrosine phosphorylation The activated, solubilized receptor construction is then added to the cavities that have the capture agent that adheres thereto. As a general proposal, approximately 80% of the cell lysate obtained as mentioned under the previous Section can be added to each cavity (ie, approximately 60 to 160 μl depending on the original volume of the cavities). The lysate is incubated with the capture agent for a suitable period of time to enable the recipient construction to be captured in the cavities, for example, from 1 to 3 hours. The incubation can be carried out at room temperature. The unbound cell lysate is then removed by washing with the buffer solution for washing. Following this wash step, an amount of the anti-phosphotyrosine antibody which is equal to, or less than, the amount of the blocking buffer added previously, is added to the cavity. For example, about 50 to 200 μl of an anti-phosphotyrosine antibody preparation having between about 0.3 to 0.5 μg / ml of the antibody in a suitable buffer solution (for example, PBS with a detergent such as those included in the buffer solution for lysis) is added to the cavity. This is followed by a wash step to remove the unbound anti-phosphotyrosine antibody. The tyrosine phosphorylation is then quantified by the amount of binding of the anti-phosphotyrosine antibody to the second solid phase. Many systems for detecting the presence of an antibody are available to those skilled in the art. Some examples are given below. In general, the anti-phosphotyrosine antibody will be labeled either directly or indirectly with a detectable label. Numerous labels are available, which can be grouped generally into the following categories: (a) Radioisotopes, such as S, 1144 / C-, 125I-, H, and 131- The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, supra, for example, and radioactivity can be measured using scintillation counting. (b) Fluorescent labels such as chelates of rare earths (europium chelates) or fluoroscein and its derivatives, rhodamine and its derivatives, sandil, Lissamine, phycoerythrin and Texas Red are available. Fluorescent labels can be conjugated to the antibody using the techniques described in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter (Dynatech). (c) Various labels of the enzyme substrate are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques. For example, the enzyme can catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can then emit a light that can be measured (using a Dynatech ML3000 chemiluminometer, for example) or donate energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; US Patent No. 4,737,456), luciferin-2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthin oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes with antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-antibody conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed. J. Langone &; H. Van Vunakis), Academic Press, New York, 73: 147-166 (1981) and Current Protocols in Immunology, supra. Examples of the substrate-enzyme combinations include, for example: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein hydrogen peroxidase oxidizes a dye precursor (eg, orthophenylene) diamine (OPD) or 3,3 ', 5,5'-tetramethyl benzidine hydrochloride (TMB)). (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as the chromogenic substrate. (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (for example p-nitrophenyl-β-D-galacosidase) or e-methylumbelliferyl-β-D-galactosidase from the fluorogenic substrate. Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980. Sometimes, the label is indirectly conjugated with the antibody. The skilled artisan will be aware of the various techniques to achieve this. For example, the antibody can be conjugated to biotin and any of three broad categories of the aforementioned labels can be conjugated to avidin, or vice versa. Biotin binds selectively to avidin and therefore, the label can be conjugated to the antibody in this indirect manner. See, Current Protocols in Immunology, supra, for a review of techniques involving biotin-avidin conjugation. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (for example digoxin) and one of the different types of labels mentioned above is conjugated to an anti-hapten antibody (e.g. the anti-digoxin antibody). Accordingly, indirect conjugation of the label with the antibody can be achieved. In another embodiment of the invention, the anti-phosphotyrosine antibody does not need to be labeled, and the presence thereof can be detected using a labeled anti-antiphosphotyrosine antibody (for example the anti-mouse phosphotyrosine antibody conjugated with HRPO). In the preferred embodiment, the anti-phosphotyrosine antibody is labeled with an enzymatic tag which catalyzes a color change of a substrate (such as tetramethyl benzimidine (TMB), or ortaphenylene diamine).

(OPD)). Therefore, the use of radioactive materials is avoided. A color change of the reagent can be determined spectrophotometrically at a suitable wavelength (for example 450 nm for TMB and 490 nm for OPD, with a reference wavelength of 650 nm). 3. Intracellular Kinase Activity The assay described herein is also used to measure phosphorylation and / or activation of an intracellular kinase domain (e.g., the form of cytoplasmic tyrosine kinases and / or cytoplasmic serine-threonine kinases) fused to the subunit a receptor. Phosphorylation of these molecules can occur as a consequence of trans-phosphorylation of the intracellular kinase domain by a kinase receptor or "receptor complex" (which comprises one or more kinase receptors that reside in a cell membrane. of intracellular tyrosine kinases include substrate 1 of the insulin receptor (ISR-1), Shc, Ras and GRB2, for example, antibodies to human Shc, human Rad and GRB2 can be obtained commercially from UBI, NY, which they can be used as capture agents for these tyrosine kinases Examples of the intracellular serine-threonine kinases include MEK and MAPK To measure the phosphorylation of the receptor constructs containing the catalytic domains of these kinases, the procedure is essentially as described previously, the chimera referred to as a "kinase construct." In general, a eukaryotic cell will be transformed with the acid cleic that codes for a kinase construct. During expression of the nucleic acid, the construction of the kinase will settle intracellularly (ie, in the cytoplasm). Cells comprising the kinase construction are subjected to KIRA as described above. Exposure to the agonist can lead to trans-phosphorylation of the intracellular kinase construction which can be quantified in the ELISA as elaborated above. The capture agent in the ELISA binds either to the construction of the intracellular kinase or to the flag polypeptide. 4. Serine-Threonine Kinase Activity This assay is also used to measure the phosphorylation and activation of the ICD domain of the serine-threonine kinase fused to the subunit receptor a. The term "serine-threonine kinase" refers to a kinase which phosphorylates a substrate which has at least one alcohol group of phosphate acceptance. Serine threonine kinase is usually a "receptor" since it has a ligand binding ECD, a TM domain and ICD. The ICD usually comprises a catalytic kinase domain and generally has one or more serine and / or threonine phosphate acceptance residues. Examples of the intracellular serine-threonine kinases include MEK and MAPK. The measurement of the phosphorylation of intracellular serine-threonine kinases can be done as described here. Examples of the serine-threo-nina kinase receptors that can provide ICD domains suitable for fusion to create a receptor construct include the activin type II receptor, daf-1 (ActR-II), the receptor of the type IIB of activin (ActR-IIB), the type II receptor of TGF-β (TßR-II), the kinase similar to the activin receptor (ALK) -l, -2, -3, -4 and the receptor of the T type of TGF-β (TßR-1) / AL -5. see Ten Dij e et al., supra. The serine-threonine kinase assay is essentially the same as described above for tyrosine kinases, except that the phosphorylation is quantified using anti-phosphoserine and / or anti-phosphothreonine antibodies. The monoclonal monoclonal anti-phosphoserine and anti-phosphothreonine antibodies can be purchased from Sigma Immuno Chemicals, St. Louis, MO, for example.

. Phosphatase Activity The activity of the phosphatase can be measured similarly using the assay described here. Phosphatase enzymes are capable of dephosphorylating the phosphorylated tyrosine, serine and / or threonine residues (i.e. releasing the inorganic phosphate from the phosphoric esters of such amino acid residues). In general, the phosphatase enzyme is specific for either the tyrosine residues or the serine-threonine residues but can sometimes dephosphorylate the tyrosine, serine and threonine residues. Sometimes the activity of the "endogenous" phosphatase is measured and this refers to the activity of the phosphatase enzyme (s) which exist in nature in a selected cell. To quantitate the activity of the endogenous phosphatase, cells possessing at least one phosphatase are stimulated in the presence and absence of one or more phosphatase inhibitors. Examples of the protein tyrosine phosphatase inhibitors (PTPase) include sodium orthovanadate and sodium molybdate (Sigma Chemical Co., St. Louis, MO). ICN Biochemicals supplies the okadaic acid, which is an inhibitor of the serine-threonine phosphatase. As a general proposition, between about 1-lOμM of the phosphatase inhibitor can be added to each cavity of the assay plate. In all other aspects, the test is carried out essentially as described above. Accordingly, the ability of the endogenous phosphatases to dephosphorylate a kinase in the selected cell can be quantified.

In a preferred embodiment, a phosphatase enzyme of interest can be studied. Examples of protein tyrosine phosphatases (PTPases) include PTP1B, PTPMEG, PTPlc, Yop51, VH1, cdc25, CD45, HLAR, PTP18, HPTPa and DPTP10D. See Zhang and Dixon, Adv. Enzym. 68: 1-36 (1994). Examples of the serine-threonine phosphatases include PP1, PP2A, PP2B and PP2C. See Meth. Enzyme , ed. Hunter & Sefton, Academic Press, New York, 201: 389-398 (1991). These proteins can be purchased commercially or are made using the recombinant techniques described herein. To measure the activity of the phosphatase, the KIRA ELISA can be effected essentially as described above with the following modifications. Following the capture of the kinase or the construction of the kinase (for example the receptor construction) with respect to the second solid phase and the washing step (to remove the unbound cell lysate), the phosphatase of interest is added to the cavities of the second assay plate and incubated with the kinase or the adherent kinase construct. For example, between about 50-200 μl of the phosphatase in a suitable dilution buffer (see Meth. Enzym., Ed.

Hunter & Sefton, Academic press, New York, 201: 416-440 (1991)) can be added to each cavity. This is generally followed by gentle agitation at room temperature (or 37 ° C) for between about 30 minutes to 2 hours to allow the phosphatase to dephosphorylate the kinase. Following washing to remove the phosphatase, the decreased degree of phosphorylation of the kinase relative to the control (ie no added phosphatase) is quantified by ELISA as described here at the beginning. 6. Teams or sets As a matter of convenience, the reagents can be provided in a set or set, ie, a packaged combination of reagents, for combination with the substance to be analyzed to test the capacity of the substance to be analyzed for activate or prevent the activation of a receptor of the subunit a of interest. The components of the game or set will be provided in predetermined relations. Accordingly, a set or set will comprise the second solid phase specific for the assay as well as the capture agent of the anti-flag polypeptide either separately packaged or captured in the second solid phase (e.g., a microtiter plate). Usually, other reagents, such as anti-phosphotyrosine antibody labeled directly or indirectly with an enzymatic label will also be provided in the kit or set. Where the detectable label is an enzyme, the kit or set will include the substrates and cofactors required by the enzyme (for example a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffer solutions (e.g., a blocking buffer and a lysis buffer) and the like. Conveniently, the game or set can also supply the homogeneous population of the cells which have been transformed with the construction of the receptor. The relative amounts of the various reagents can be varied widely to provide the concentrations in the solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents can be provided as dry powders, usually lyophilized, including the excipients which in the solution will provide a reagent solution having the appropriate concentration. The game or set also adequately includes the instructions for carrying out the KIRA ELISA. 7. Uses for the Test This application provides two assays which are useful for the reliable, sensitive and quantitative detection of kinase activation, which reflects the binding of the ligand by a receptor of subunit a, caused by its homodimerization or homooligomerization. The first assay can be used, wherein a receptor-specific capture antibody having the desired characteristics described herein is available or has been prepared. The second assay is a generic assay which makes possible the activation of any receptor construct that is to be measured by the use of a flag polypeptide and a capture agent which binds by specificity thereto. These assays are useful for identifying novel agonists / antagonists for a selected receptor. Also, the assay provides a means to study ligand-receptor interactions (ie, mechanism studies). Also the presence of an endogenous receptor in a selected cell line can be quantified using the assay. The assays are further useful for identifying the presence of a ligand for a selected receptor in a biological sample and, for example, the establishment of whether a growth factor has been isolated following a purification procedure. It is desirable to have a trial for measure the ability of these growth factors to activate their respective receptors. The assay also has clinical applications for detecting the presence of a ligand for a selected receptor (for example the GFRα3 receptor) in a biological sample taken from a human and therefore patients having high or low levels of the ligand can be identified. This is particularly desirable where high or reduced levels of the ligand cause a pathological condition. In consecuense, candidates for the administration of the selected ligand (for example insulin) can be identified through this diagnostic method. It is possible, using the assay described herein, to evaluate the pK of the agonists or antagonists administered to a patient. This assay also facilitates detection of the shed receptor in a biological sample. The assay is also useful for quantifying the phosphatase activity of the endogenous phosphatases or, in the preferred embodiment, a phosphatase of interest. This can be used to select phosphatase inhibitors, for example.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. All patent and literature references cited in the present specification are incorporated herein by reference in their entirety.

EXAMPLES The commercially available reagents referred to in the examples were used in accordance with the manufacturer's instructions unless otherwise indicated. The source of these cells identified in the following examples, and throughout the specification, by ATCC accession numbers in the American type Culture Collection, Rockville, Maryland.

Example 1 Cloning of Mouse GFR3 Using the sequences of the neurturin receptor, now known as GFRα2 ("alpha receptor of the family of neurotrophic factor derived from the glial cell line"), a new potential member of the GFRα family was identified as the mouse EST on a base of public gene data (Accession Numbers W99197 (SEQ ID NO: 1), AA041935 (SEQ ID NO: 2) and AA050083 (SEQ ID NO: 3)). A DNA fragment corresponding to this potentially new receptor was obtained by the Marathon PCR using the E15 mouse cDNA (Clontech, Inc. USA) as a template and the PCR primers derived from the mouse EST. The PCR product was then used to select an E15 library of the lambda gt10 mouse (Clontech, Inc. USA) to obtain a full-length clone. The nucleotide sequence of the full length mouse cDNA is provided as SEQ ID NO: 4 (Figure 1A-1B). The sequence of the proteins (SEQ ID NO: 5, see Figure 1A-1B) encoded by the isolated DNA was designated GFRα3, since it was determined to be a novel protein with the identity of the sequence for GFRal ( initially the GDNF alpha receptor) and GFRa2. { initially the Alpha receptor of Neurturin; NTNRa). A comparison of the 397 amino acid mouse GFRα3 protein sequence (SEQ ID NO: 5) with respect to the rat GFRα (SEQ ID NO: 8) and the rat GFRα2 (SEQ ID NO: 9) is provided in Figure 2. The sequence of mGFRa3 is thought to identify a novel series of homologs that belong to the GFR receptor family. The potential N-linked glycosylation sites are shaded in Figure 2. The hydrophobic sequence involved in the GPI anchor has a superior underline in Figure 2, with the possible GPI binding site indicated by the asterisks. A variant of the mouse GFRα3 DNA contains a deletion of the base "T" at position 290 in Figure 1A, leading to a shift in the structure and truncation of the variant of the protein. The variant DNA however is useful for hybridization, diagnostics, and other uses of DNA (excluding the production of the full-length protein) described throughout this specification. The DNA (positions 89-289) comprising the coding region of GFRα3 immediately upstream of this base finds use in the invention. DNA comprising the immediately downstream sequence (291-1279) provides another useful embodiment of the invention. In situ hybridization studies using the DNA encoding mouse GFRα3 revealed a configuration of expression in peripheral sensory neurons and neurons of the sympathetic system (data not shown).

Example 2 Isolation of the cDNA Clones Encoding Human 6FRa3 To quickly identify if a human homolog of this new receptor could exist, a DNA database of the expressed sequence tag (EST) (A database of EST proprietary, LIFESEC1, Incyte Pharmaceuticals, Palo Alto, CA) was investigated and an EST (INC3574209) was identified having the sequence: GCGCTGNNTGNCNGNANGNGGGGGCGGGAGGTGCCGGTCGAGGGAGCCCCGCTCTCAGA GCTCCAGGGGAGGAGCGANGGGAGCGCGGAGCCCGGCCGCCTACAGCTCGCCATGGTGC GCCCCCTGAACCCGCGACCGCTGCCGCCCGTAGNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNGCCTCTCGCAGCCGGAGACCCCCTTCCCACAGAAAGCCGACTCATGAA CACGTGTCTCCAGGCCAGGAGGAAGTGCCAGGCTGATCCCACCTGC (SEQ ID NO: 10). This sequence had 61% identity with murine GFRα3. To clone the corresponding full-length cDNA, a panel of cDNA libraries was selected with the primers; newa3.F 5 'GCC TCT CGC AGC CGG AGA CC 3' (SEQ ID NO: 11) newa3.R 5 'CAG GTG GGA TCA GCC TGG CAC 3' (SEQ ID NO: 12) The DNA of the libraries was selected by PCR amplification, as per Ausubel et al., Current Protocols in Molecular Biology (1995), with the pair of the PCR primer. A strong PCR product was identified in all libraries analyzed (fetal lung, fetal kidney, and placenta). To isolate a cDNA clone encoding this protein, a pRK5 vector library of the human fetal lung was selected and enriched to verify the positive cDNA clones by the extension of the single-stranded DNA from the plasmid libraries that were grown in a dug- / bung- host using the newa3.R primer. The RNA for the construction of the cDNA libraries was isolated from the human fetal lung tissue. The cDNA library used to isolate the cDNA clones were constructed by standard methods using commercially available reagents (e.g., Invitrogen, San Diego, CA, Clontech, etc.). The cDNA was primed with the DT oligo containing a NotI site blunt-bound to the Sali hemicinized adapters, segmented with NotI, appropriately sized by gel electrophoresis, and cloned in a defined orientation in a suitable cloning vector (pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the site Sfil; see, Holmes et al., Science, 235: 1278-1280 (1991)) at the unique Xhol and Notl sites. To enrich the positive cDNA clones the primer extension reaction contained 10 μl of the Solution PCR cushion (Perkin Elmer, USA), 1 μl dNTP (20 mM), 1 μl of the library DNA (200 ng), 1 μl of the primer, 86.5 μl of H20 and 1 μl of Amplitaq (Perkin Elmer, USA) added after a hot start. The reaction was denatured for 1 minute at 95 ° C, annealed for 1 minute at 60 ° C, and then extended for 15 minutes at 72 ° C. The DNA was extracted with phenol / chloroform, precipitated with ethanol, and then transformed by electroporation into a host bacterium of DH10B. The complete transformation mixture was placed in sheets over 10 sheets and the colonies were allowed to form. The colonies were elevated on nylon membranes and selected with an oligonucleotide probe (newa3): 5 'TCT CGC AGC CGG AGA CCC CCT TCC CAC AGA AAG CCG ACT CA 3' (SEQ ID NO: 13)) derived from Incyte ITS T. The filters were hybridized with the probe overnight at 42 ° C in 50% formamide, 5XSSC, 10xDenhardt's, 0.05M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, and 50 μg / ml DNA of salmon sperm subjected to the action of sound. The filters were then rinsed in 2xSSC, washed in O.lxSSC, 0.1% SDS, and then exposed overnight to Kodak X-ray films. Five positive clones were identified. Pure positive clones were obtained after purification of the colonies and secondary selection. Two of the isolated clones were sequenced. These cDNA sequences were designated DNA48613 (SEQ ID NO: 14) and DNA48614 (SEQ ID NO: 16). Analysis of the amino acid sequence of DNA48613 revealed an open reading frame sequence of 400 amino acids in length (SEQ ID NO: 15) with a peptide of the N-terminal signal of 26 amino acid length predicted. The predicted mature protein is 374 amino acids in length, with a calculated molecular weight of approximately 41 kDa. Potential N-linked glycosylation sites are similar to those in the mouse sequence. The sequences of the human and mouse GFRα3 protein are compared in Figure 3. The deduced amino acid sequence (SEQ ID NO: 17) of DNA48614 and the comparison with SEQ ID NO: 15, revealed that it will be an alternately spliced form of DNA48613, with a deletion of 30 amino acids (positions 127-157 of the amino acids, counting from the starting methionine), as shown in Figure 4. Interestingly, none of the cysteines are deleted in this clone. The clones DNA48613 and DNA48614 and the variant of mGFRa3 (clone 13) have been deposited in the ATCC and are assigned to the deposit of 'ATCC No. 209752 (Designation: DNA48613-1268), 209751 (Designation: DNA48614-1268), and, respectively. A comparison of the nucleic acid sequences encoding DNA48613 with those encoding GFRal and GFRa2 are provided in Figures 5A-B. The sequence of untranslated or untranslated 5 'GFRα3 immediately upstream of the start ATG in the cloned DNA48613 is GCGAGGGGGAGCGCGGAGCCCGGCGCCTACAGCTCGCC (SEQ ID NO: 21). As described below, a comparison of the sequence of the protein encoded by DNA48613 for GFRal and GFRα2 (Figure 6) indicated that the two human proteins are new elements of the GFRα receptor family, and are homologous to the human GFRα3 of murine . Accordingly, DNA48613 encodes a human GFRα3 designated by the protein, and DNA48614 encodes its splicing variant. The amino acid sequence comparisons between the members of the GFRα family are provided in Table 1, based on the analysis of the alignment of the BLAST-2 and FastA sequence of the full-length sequence.

Table 1 Identity of the Sequence Among the Elements of the Family of the FRP Proteins Compared Percent of Identity rGFRal against hGFRal 92% rGFRa2 against hGFRa2 94% rGFRa3 against hGFRa3 77% rGFRa3 against hGFRal 34% rGFRa3 against hGFRa2 34% rGFRal against hGFRa2 48% From the sequence comparisons it can be seen that human GFRα3 is less related to its rodent homologue which is either GFRal or GFRα2. In addition, GFRa3 seems to be related more distantly to GFRal and GFRa2 than GFRal and GFRa2 are to each other.

Example 3 Use of GFRα3 as a Hybridization Probe The following method describes the use of a nucleotide sequence encoding GFRα3 as a hybridization probe. The DNA comprising the coding sequence of GFRα3 (shown in SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 16), or a fragment thereof, is employed as a probe to select the homologous DNAs ( such as those encoding GFRα3 that is naturally present or variants of GFRα3) in human tissue cDNA libraries, human tissue genomic libraries, RNA isolated from tissues, tissue preparations in situ, or the preparations of the chromosomes (such as for the assignment of coordinates to the chromosomes). Hybridization and washing of the filters containing any of the library's DNAs are carried out under the following highly stringent conditions. Hybridization of the radiolabeled GFRα3-derived probe with respect to the filters is carried out in a 50% solution of formamide, 5xSSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt solution, and 10% dextran sulfate at 42 ° C for 20 hours. The filters are washed in an aqueous solution of O.lxSSC and 0.1% of SDS at 42 ° C.

The DNAs having an identity of the desired sequence with the DNA encoding the GFRα3 of the full length natural sequence can then be identified using standard techniques known in the art.

Example 4 Northern Spotting Analysis The expression of GFRα3 mRNA in human tissues was examined by Northern blot analysis. The multiple human tissue RNA spots were hybridized to a 32 P labeled DNA probe spanning the entire coding region of the human GFRα3 cDNA labeled by random priming. The MTN spot of human fetal RNA _ (Clontech, Inc. USA) and the MTN-I and MTN-II spots of human adult RNA (Clontech) were incubated with the DNA probe. The spots were incubated with the Ixl0e6 cpm / ml probe in the hybridization buffer (5XSSC, 10X Denhardt's solution, 0.05M sodium phosphate pH 6.5, 50 μg / ml of salmon sperm DNA subjected to the action of sound, 50% formamide, 0.1% sodium pyrophosphate) overnight at 42 ° C. The spots were washed in 2X SSC at room temperature for 10 minutes followed by 0.2xSSC in 0.1% SDS at 42 ° C for 30 minutes. The spots were exposed to an X-ray film and developed after exposure through the night by analysis of a phosphor imaging compound (Fuji). As shown in Figure 7, transcripts of GFRα3 mRNA were detected. The expression was observed at high levels in the heart, the intestines (pancreas, small intestine, colon), the thymus, the testes and the prostate.

Example 5 Location of GFRα3 in In Situ Hybridization The following tissues were examined for the expression of GFRα3 mRNA in in situ hybridization: embryos of the mouse of 13 days, brain of the embryonic mouse of 15 days and 17 days, the brain of the mouse of 1 day, postnatal, the brain of the adult mouse (with the optic nerve), the spinal cord of the adult mouse, the roots and trigeminal ganglion of the adult mouse, the retina of the adult mouse, and the multi-stage utricle. For in situ hybridization, E13.5 mouse embryos were fixed by immersion overnight at 4 ° C in 4% paraformaldehyde, cytoprotected overnight in 15% sucrose, intercalated in O.T.C., and frozen on liquid nitrogen. The spinal cord of the adult mouse, the trigeminal ganglia, the retina, and the brains of the mouse Pl were interspersed or sandwiched in O.T.C. and they were frozen fresh on liquid nitrogen. The brains of the adult mouse were frozen fresh with dry ice spray. The sections were cut at 16 um, and processed for in situ hybridization for GFRα3 by a method previously described (Phillips et al., Science 250: 290 (1990)). Using 33P-UTP, tagged RNA probes were generated as described (Melton et al., Nucleic Acids Res. 12: 7035 (1984)) using T7 polymerase with a 326 bp fragment encoding mouse GFR3. In the El3 mouse, the GFRα3 mRNA was expressed very strongly in the dorsal root ganglia, in the ganglia of the sympathetic system, and in the peripheral nerves. The vestibular ganglion also exhibited a strong signal. Moderate expression was observed in the pads of whiskers, in the armpit region, and in urinary bladder environments. Moderate expression was also observed in the intermediolateral region of the gray matter of the thoracic spinal cord, the ventromedial hypothalamus, and the cell groups in the dorsal metencephalon. Most of the other regions of the brain were devoid of the demonstrable signal. Many other organs expressed a signal either weak or undetectable, including the lung, heart, liver, intestine, and kidney. In the final stages of development (E15, E17, Pl, adult), the expression of GFRα3 within the CNS was very limited. Most regions of the brain and spinal cord did not demonstrate the hybridization signal above the background level observed in the control sections hybridized to the sense strand control probe. The exceptions to this were the cell groups found in the metencephalon. In the adult, a subpopulation of trigeminal ganglion neurons was labeled very strongly, although no labeling was observed either in the satellite cells or in the roots of the nerves. The optic nerve also failed to demonstrate the detectable signal. In sections of the heart of the adult mouse, there was spread the signal over the atrial and ventricular myocytes with the focal areas of the increased signal associated with the cardiac conduction system. A comparison of the labeling with GFRal, GFRα2 and GFRα3 is shown in Figure 8. The expression of GFRα3 is very limited and localized compared to the other receptors. The primers containing the sense sequence GCCCGCGACCTACACTGCTG '(designated grfpl; SEQ ID NO: 22) and the antisense sequence CTGTGGGGAGCGGCGGCG (designated gfrp2.r.c; SEQ ID NO: 23) were used to generate a 671 bp hybridization probe of mouse GFR3. The primers containing the sense sequence CCTGAACCTATGGTAACTGG (SEQ ID NO: 24) and the antisense sequence ACCCAGTCCTCCCTACC (SEQ ID NO: 25) were used to generate a 378 bp hybridization probe of mouse GFR3. Human fetal tissues in weeks E12-E16 that were examined include the placenta, umbilical cord, liver, kidney, adrenal glands, thyroid, lungs, heart, large vessels, esophagus, stomach, the small intestine, the spleen, the thymus, the pancreas, the brain, the spinal cord, the walls of the body, the pelvis and the lower limb. The adult tissues examined included the kidney (normal and in the final stage), the adrenal glands, the myocardium, the aorta, the lung, the skin, the eye (including the retina), the bladder, the liver (normal, cirrhotic, and with acute failure), renal carcinoma, and soft tissue sarcoma. The non-human primate tissues examined included the salivary gland of the chimpanzee, stomach, thyroid, parathyroid, skin and thymus. Hybridization to the antisense strand probe of 378 base pairs was detected in the DRGs of the adult and fetal human, the peripheral nerves (as seen in the body wall and the lower limb of the fetus) and the mesenteric nerves in the fetus. No expression was observed in the spinal cord or the fetal brain. No expression was observed in the neuroblastomas examined. Using the 671 base pair antisense probe, hybridization of GFRα3 mRNA was detected at the beginning and end in the adult rat in the peripheral, trigeminal, and E14 ganglion nerves of the skin and the musculoskeletal system: E17 in the skin , the dorsal root ganglia, the peripheral nerves, the cartilage, the skeletal muscle system, and the brain; E19 in the ganglia _ of the dorsal root, the peripheral nerves, the corneum stratum of the skin, the teeth, the musculoskeletal system, the cartilage, the liver and the intestine. No specific signal was detected in the pancreas of the fetal or adult rat. In all the examples in this section, the corresponding sense probes fail to hybridize as one might have expected.

Example 6 Expression of GFRα3 in E. coli The DNA sequence encoding GFRα3, for example human GFRα3, is initially amplified using the selected PCR primers. The primers must contain the restriction enzyme sites corresponding to the restriction enzyme sites on the selected expression vector. A variety of expression vectors can be employed. An example of a suitable vector is pBR322 (derived from E. coli, see Bolivar et al., Gene, 2:95 (1977)) which contains the resistance genes against ampicillin and tetracycline. The vector is digested with the restriction enzyme and dephosphorylated. The sequences amplified by PCR are then ligated into the vector. The vector will preferably include the sequences coding for an antibiotic resistance gene, a trp promoter, a polyhis director (including the first six STII codons, the polyhis sequence, and the enterokinase cleavage site), the mammalian GFRα3 coding region, the lambda transcriptional terminator, and an argU gene.

The binding or ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., Supra. Transformants are identified by their ability to grow on LB plates and then antibiotic resistant colonies are selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing. The selected clones can be grown overnight in a liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture can be used subsequently to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is activated. After culturing the cells for several more hours, the cells can be collected by centrifugation. The cellular microsphere obtained by centrifugation can be solubilized using several agents known in the art and the GFRα3 protein of the solubilized mammal can then be purified using a metal chelation column under conditions that allow tight binding of the protein.

Example 7 Expression of GFRα3 in mammalian cells This example illustrates the preparation of a glycosylated form of mammalian GFRα3 by recombinant expression in mammalian cells. The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the vector of expression. Optionally, the GFRα3 DNA is ligated into pRK5 with the selected restriction enzymes to allow insertion of GFRα3 DNA using binding or ligation methods such as those described in Sambrook et al., Supra. The resulting vector is called pRK5-GFRa3. In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) were grown to confluence in tissue culture plates in a medium such as DMEM supplemented with fetal bovine serum and optionally, components and / or antibiotics of the nutrients. Approximately 10 μg of the pRK-5-GFRα3 DNA are mixed with approximately 1 μg of the DNA encoding the VA RNA gene (Thimmappaya et al., Cell, 31: 543 (1982)) and dissolved in 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 m CaCl2. To this mixture are added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaP0, and a precipitate is allowed to form for 10 minutes at 25 ° C. The precipitate is suspended and added to the 293 cells and allowed to settle for approximately four hours at 37 ° C. The culture medium is removed by aspiration and 2 ml of 20% glycerol in PBS are added for 30 seconds. The 293 cells are then washed with the serum free medium, a fresh medium is added and the cells are incubated for approximately 5 days. Approximately 24 hours after the transfections, the culture medium is removed and replaced with the culture medium (alone) or the culture medium containing 200 μCi / ml 35S-cysteine and 200 μCi / ml 35S-methionine. After 12 hours of incubation, the conditioned medium is collected, concentrated on a rotary filter, and loaded onto a 15% SDS gel. The processed gel can be dried and exposed to the film for a selected period of time to reveal the presence of the GFRα3 polypeptide of the mammal. The cultures containing the transfected cells may undergo further incubation (in the serum free medium) and the medium is tested in the selected bioassays.

In an alternative technique, mammalian GFRα3 can be introduced into 293 cells temporarily using the dextran sulfate method described by Somparyrac et al., Proc. Nati Acad. Sci., 12: 7575 (1981). The 293 cells are grown to the maximum density in a rotating vessel and 700 μg of pRK-5-GFRα3 DNA is added. The cells are first concentrated from the spinner by centrifugation and washed with PBS. The dextran-DNA precipitate is incubated on the cell microsphere for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with the tissue culture medium, and 5 μg / ml bovine insulin and 0.1 μg are reintroduced into the rotating vessel containing the tissue culture medium. / ml of bovine transferrin. After about 4 days, the conditioned medium is centrifuged and filtered to remove the cells and debris. The sample containing the expressed mammalian GFRα3 can then be concentrated and purified by any selected method, such as dialysis and / or column chromatography. In another embodiment, mammalian GFRα3 can be expressed in CHO cells. PSUi-GFRα3 can be transfected into CHO cells using known reagents such as CaP0 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with the culture medium (alone) or the medium containing a radiolabel such as 35S-methionine. After determining the presence of the GFRα3 polypeptide of the mammal, the culture medium can be replaced with the serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is collected. The medium containing the GFRα3 of the expressed mammal can then be concentrated and purified by any selected method. The mammalian GFRα3 tagged with the epitope can also be expressed in the CHO cells of the host. The GFRα3 of the mammal can be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse a frame with a tag of the selected epitope such as a poly-his tag on an expression vector. The mammalian GFRα3 insert labeled with poly-his can then be subcloned into an SV40 driven vector containing a selection marker such as DHFR for the selection of the stable clones. Finally, the CHO cells can be transfected (as described above) with the vector driven with SV40. The labeling can be done, as described above, to verify the expression. The culture medium containing the mammalian GFRα3 labeled with the expressed poly-His can then be concentrated and purified by any selected method, such as by affinity chromatography of the Ni2 + chelate.

Example 8 Expression of GFRα3 in the Insect Cells Infected with the Baculovirus The following method describes the recombinant expression of GFRα3 in insect cells infected with Baculovirus. GFRα3 was fused upstream of an epitope tag contained within a Baculovirus expression vector. Such epitope tags include the poly-his tags and the immunoglobulin tags (similar to the IgG Fc regions). The amino acid sequence of the GFRα3-IgG fusion is provided in SEQ ID NO: 18. A variety of plasmids can be employed, including plasmids derived from commercially available plasmids such as pVL1393. { Novagen). Briefly, the GFRα3 sequence encoding the extracellular domain) was amplified by PCR with the primers complementary to the 5 'and 3' regions. The 5 'primer incorporates flanking restriction enzyme sites (selected). The product was then digested with these restriction enzymes selected and subcloned into the expression vector. The vector for the expression of GFRα3-IgG in the insect cells was pb.PH. { wherein the expression in the Baculovirus was under the control of the polyhedrin promoter). The recombinant baculovirus was generated by cotransfection of the above plasmid and BaculoGold ™ virus DNA (Pharmingen) in Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4-5 days of incubation at 28 ° C, the released viruses were collected and used for additional amplifications. Viral infection and protein expression were performed as described by O'Reilley et al, "Baculovirus expression vectors: A laboratory Manual", Oxford: Oxford University Press (1994). The purification of GFRα3 labeled with IgG (or labeled with Fc) was carried out using known chromatography techniques, including column chromatography of Protein A or protein G. Alternatively, GFRα3 labeled with expressed polyhis can be purified, for example , by affinity chromatography of the Ni + chelate as follows. The extracts are prepared from the sf9 cells infected with the recombinant virus as described by Rupert et al., Nature 362: 175-179 (1993). Briefly, the Sf9 cells are washed, resuspended, in a buffer solution with application of sound (25 ml Hepes, pH 7.9; 12.5 M MgCl 2; 0.1 mM EDTA; 10% Glycerol, 0.1% NP-40; 0.4 M KCl), and is subjected to the action of sound twice for 20 seconds on ice. The materials subjected to the action of sound are cleared by centrifugation, and the supernatant is diluted 50 times in a charge buffer (50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH 7.8) and filtered through a 0.45 Fm filter. An agarose column of NTA-Ni2 + (commercially available from Qiagen) is prepared with a bed volume of 5 ml, washed with 25 ml of water and equilibrated with 25 ml of the loading buffer. The filtered cell extract is loaded onto the column at 0.5 ml per minute. The column is washed with the baseline A28o with the charge buffer, in such a punctual fraction the collection was started. Next, the column is washed with a secondary washing solution. { 50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes the bound protein not specifically. After reaching baseline A28Q again, the column is developed with an imidazole gradient from 0 to 500 mM in a secondary wash buffer. The fractions of one milliliter are collected and analyzed by SDS-PAGE and silver staining or Western staining with Ni2 + -NTA conjugated with alkaline phosphatase. { Qiagen). The fractions containing the His-tagged GFRα3 were pooled and dialyzed against the charge buffer.

Example 9 Union to GFRa3 To determine if any of the elements of the known GDNF family (ligands GDNF, Neurturin (NTN) or Persefin (PSN)) could bind to GFRα3, each ligand was coated on microtitre plates and incubated with either GFRal-IgG , GFRa2-IgG, or GFRa3-IgG. { SEQ ID NO: 18) prepared as in Example 8. The binding of GFRα-IgG was then detected with a secondary antibody for its IgG portion. GDNF, NTN, and PSN were coated on microtiter plates at 1 μg / ml in a 50 mM carbonate buffer solution, pH 9.6, overnight at 4 ° C. The plates were then washed with PBS / 0.05% Tween 20, then blocked with PBS / 0.05% BSA / Tween 20 for 1-2 hours at room temperature. Various concentrations of chimeric receptors labeled with IgG (GFRal-IgG, GFRa2-IgG, GFRa3-IgG, 1 μg / ml up to 1.95 ng / ml) were added to each well and the plates were incubated for 1 hour at room temperature. The plates were then washed as described above and incubated in the presence of the goat anti-human IgG (Fc) -HRP (1: 1000) for 1 hour at room temperature. After washing, the bound HRP was developed with an OPD substrate for 5 to 10 minutes, followed by reading the plates at 490 nm. The results are shown in Figures 9A-C. GFRal binds to GDNF (Figure 9A), GFRα binds to GDNF and NTN (Figure 9B), but GFRα does not bind to any of these molecules (Figure 9C). GFR3 is therefore an orphan receptor.

Example 10 Assays for GFR3 agonists The GDNF family of the ligands uses a unique receptor system: a ligand-binding protein linked to GPI (component a) and a signaling component, to the Ret of the receptor tyrosine kinase. The mechanism of activation of this multi-component receptor complex is still unknown, but tyrosine kinase receptors are known to be activated during dimerization induced by the ligand. Accordingly, a possible mechanism of activation of GFR is by the binding of the ligand to the component to which it induces the dimerization of component a. The two chains in turn will carry two Ret molecules in the complex, which will lead to the activation of the kinase domains and the phosphorylation of target or tyrosine residues on the receptor and / or other signaling molecules. To demonstrate that the ligands induce the dimerization of component a, the chimeric receptors made from the extracellular domain of rat GFRα2 and the transmembrane and intracellular domain of the TPO receptor (c-mpl) or the receptor tyrosine kinase of Rse were built. These two receptors belong to a different family of receptors but it is known that both they will be activated by the dimerization induced by the ligand or by the dimerization induced by the agonist-antibody. GFRa2-c-mpl. A chimeric receptor made from the epitope tag of gD followed by the extracellular domain of rGFRa2 (minus the GPI signal) followed by the transmembrane and intracellular domain of the TPO receptor was assembled by the recombinant PCR into the pRKtkneo vector under the I controlled the. CMV promoter. The Ba / F3 cells were electroporated with the pRKtkneo-GFRa2-mpl linearized with Notl, and the clones were obtained by limiting the dilutions. Individual clones were analyzed to verify receptor expression by FACS analysis using an anti-gD antibody. The positive clones were selected and further characterized for their ability to proliferate in response to the stimulation of NTN, a ligand for GFRα2. As shown in Figure 10, Ba / F3 cells expressing GFRα2-mpl are able to proliferate in response to NTN stimulation, as assessed by the incorporation of 3 H-thymidine. GFRa2-Rse. A chimeric receptor was constructed with the gD epitope tag followed by the extracellular domain of rat GFRα2 (minus the GPI signal) followed by the transmembrane and intracellular domain of the Rse tyrosine kinase receptor and another tag of the epitope of gD and was assembled by the recombinant PCR into a pSVi vector under the control of the SV40 promoter. The sequence of gD-GFRa2-Rse-gD is presented in SEQ ID NO: 19. The CHO cells were transfected by the lipofectamine method (GIBCO-BRL). The unique transfected CHO clones were collected and analyzed for receptor expression by FACS analysis using an anti-gD antibody. Positive clones of the receptor were then analyzed using a KIRA assay (e.g., U.S. Patent No. 5,709,858) for receptor-induced phosphorylation during NTN stimulation. As shown in Figure 11, NTN stimulation caused the autophosphorylation of the receptor. GFRa3-Rse. Together, the above data indicate that the activation of the GRFs is mediated by dimerization induced by the ligands and that, in addition to their ligands, the various receptors will be susceptible to antibody-mediated activation. Accordingly, in one embodiment, an assay to identify agonist antibodies and a natural ligand. { or other agonists) for a mammalian GFRα3, follows the method described above for GFRα2-Rse. The GFRα3 receptor was constructed with the epitope tag of gD followed by the extracellular domain of murine GFRα3 (minus the GPI signal; preferably human GFR3 is used) followed by the transmembrane and intracellular domain of the Rse tyrosine kinase receptor and a second gD tag using the recombinant PCR in the pSVI vector under the control of the SV40 promoter. The CHO cells were transfected by the lipofectamine method (GIBCO-BRL). The unique transfected clones were collected and analyzed for the expression of the chimeric receptor of GFRα3 by the analysis of FACS using an anti-gD antibody. Positive clones were then analyzed to verify phosphorylation induced by the receptor during treatment with DGNF, NTN or PSN. The results are shown in Figure 12. The results confirmed that GFRα3 is a receptor for a novel ligand of the GDNF family. The sequence of gD-GFRα3-Rse-gD is presented in SEQ ID NO: 20. As is evident from this construction sequence and its homology to other elements of the GFR family, a sufficient ligand binding region is from the amino acid 110 to amino acid 386 of SEQ ID NO: 20, which corresponds to amino acid residues 84 to 360 in SEQ ID NO: 15. The natural ligand for GFRα3 has been identified as artemin (Baloh et al., Neuron 21: 1291-1302 (1998), which has been found to bind to GFRα3 of the present invention and its fusion of gD-GFRα3-Rse-gD. Antibodies raised against GFRα3 (or other candidate agonists) can be selected for agonist activity by using the GFRα3 construct expressed in CHO cells. Alternatively, the antagonists are selected to verify their ability to inhibit agonist function in this assay.

Example 11 Preparation of the Antibodies that join GFR3 This example illustrates the preparation of monoclonal antibodies which bind specifically to GFRα3. Techniques for producing monoclonal antibodies are already known in the art and are described, for example, in Goding, supra. Immunogens that can be employed include purified GFRα3, fusion proteins containing GFRα3, and cells expressing recombinant GFRα3 on the cell surface. The selection of the immunogen can be made by the skilled artisan without undue experimentation. Mice, such as Balb / c, are immunized with the GFRα3 immunogen emulsified in the complete Freund's helper injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in the MPL-TDM helper (Ribi Immunochemical Research, Hamilton, MT) and injected into the hind paws or legs of the animal. The immunized mice are then stimulated or injected 10 to 12 days later with the additional immunogen emulsified in the selected adjuvant. After this, for several weeks, the mice can also be injected or stimulated with additional immunization injections. Serum samples can be obtained periodically from the mice by retro-orbital blood extraction for testing in ELISA assays, to detect GFRα3 antibodies. After a suitable antibody concentration has been detected, the animals "positive" for the antibodies can be injected with a final intravenous injection of GFRα3. Three to four days later, the mice are sacrificed and the spleen cells are collected. Spleen cells are then fused (using 35% polyethylene glycol) to a selected murine myeloma cell line such as P3X63AAAgU. I, available from ATCC, No. CRL 1597. Fusions generate hybridoma cells which can be plated on 96-well tissue culture plates containing the HAT medium (hypoxanthine, aminopterin, and thymidine) to inhibit the proliferation of unfused cells, the hybrids of myeloma, and the hybrids of spleen cells. Hybridoma cells will be selected in an ELISA to verify the reactivity against GFRα3. The determination of the "positive" hybridoma cells that secrete the desired monoclonal antibodies against GFRα3 is within the skill in the art. The positive hybridoma cells can be injected intraperitoneally into the syngeneic Balb / c mice to produce ascites containing the anti-GFRα3 monoclonal antibodies. Alternatively, the hybridoma cells can be grown in containers for tissue culture or in spin bottles. The purification of the monoclonal antibodies produced in the ascites can be effected using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of the antibody to protein A or protein G may be employed.

Example 12 Dimerization Selection Tests Candidate agonists, for example antibodies raised against the a-subunit receptor of the GFR family, eg GFRα3, GFRα2, or GFRα, can be selected to verify their agonist activity using the Rse-receptor-like construct expressed in CHO cells or other suitable cells in a KIRA assay. An exemplary KIRA protocol that uses monoclonal antibodies for the gD region of the gD-GFRα2-Rse construct is presented. The CHO cell culture expressing the gD-GFRα2-Rse fusion protein was prepared and cultured as follows. On day 1, the CHO cells transfected from the culture vessel are preferably 70-90% confluent with a very small amount of floating (disunited) cells. The culture plates were plates for tissue culture, sterile, 96 cavities, flat bottom Falcon (1270) with cover. The solution Disbanding cushion was PBS with 5mMEDTA diluted 1:50 (250 mM of the storage solution). The means of Cell culture was Ham 's F-12 without GHT, DMEM with a low level of Glucose without Glycine: with NaHCO3 (50:50) + 10% FBS Diafiltration, 25 mM HEPES, 2 mM L-Glutamine. On Day 2 the Stimulation Medium was the insect cell medium Excell-401 (JRH Biosciences cat # 14401-78p) plus 0.5% BSA. The buffer solution for lysis was 150mM NaCl with 50 mM HEPES and 0.5% Triton-X 100. The protease inhibitors added to the buffer for lysis before use were the storage solution 100X AEBSF (100 mM) using a 1: 100 dilution, a storage solution of 1000X Aprotinin (liquid) using a 1: 1000 dilution, and the 1,000X storage solution of Leupeptin (50 mM) using a 1: 1000 dilution. The phosphatase inhibitor added to the buffer for lysis before use was the storage solution of Sodium Ortovanidate 50X (100 mM) using a 1:50 dilution. The ELISA format used the following materials. The solid support was the immunoplate 4-39454 of Nunc Maxisorp. The buffer solution for the coating was PBS pH 7.4. The Shock Absorber Solution was PBS with 0.05% Tween 20 pH 7.4. The Locking Buffer Solution was PBS with 0.5% BSA. The Test Buffer solution was PBS with 0.5% BSA, 0.05% Tween 20 and 5 mM EDTA, pH 7.4. The substrate was a set or set of the TMB substrate. { 2 bottles: A: TMB substrate; B: TMB peroxide solution) from Kirkegard and Perry. The stop or stop solution was 1.0 M H3P04. The antigen was the transfected, solubilized "Receptor.dG" of the cavities for the culture of the cells (cell lysate). The antibodies were (Io) 3C8 (anti-gD peptide) concentration 1.0 μg / ml, 1: 1300 dil. 1.3 mg / ml of the storage solution, batch 24564-7 # 1766, (2 °) biotinylated 4G10 (UBI) concentration 1: 1000 from a stock solution at 4 ° C (50 μg / ml) # 100796. The conjugate was Streptavidin / Zimed Concentration HRP 1: 50000, lot # 26246-91 (1: 100 of the solution in frozen storage).

The cells collected by sucking the cell culture supernatant from the tissue culture vessel were rinsed once with sterile PBS, and 10 ml of the buffer was added for cell disunity. The cells were incubated at 37 ° C for ~ 10 minutes until the cells were separated. The disunited cells were transferred to a centrifuge tube and an equal volume of the cell culture medium was added. The counting of the cells was done with a hemocytometer. The cells were centrifuged, the supernatant was removed by aspiration, and the cells were suspended up to 1x106 cells / ml. 100 μl of the cell suspension (final of approximately 1x105 cells / well) was added to each well. The plates were incubated at 37 ° C overnight. Activation of the receiver was carried out as follows. Typically, a material in storage of the ligand, in this example a 2 mg / ml preparation of hNTNFP was used to make a final concentration of NTN in each cavity such as 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.00312, 0.001, and 0.0 ug / ml. The solutions in the titration plates were added to each cavity and incubated for 25 minutes at 37 ° C. About 100 μl of the sample, the control or NBS were added to each well and incubated for 25 minutes at 37 ° C. The plates were mixed gently. To each cavity 130 μl of the Lysing Buffer Solution were added with the protease and phosphatase inhibitors. Cell lysis was allowed to proceed for 30 minutes on the tissue culture plates. For storage cell lysates were placed at -70 ° C. An ELISA was run as follows: To coat the ELISA plates, 100 ul of 1 ° a mAb solution (primary; 3C8 1 μg / ml) were added to each cavity, and it was allowed to coat the cavities at 4 ° C overnight. To perform the assay on the Capture Plates (ELISA), the coating solution was discarded and 150 ul of the blocking buffer was added to each well. The blockade was allowed to continue for 1 hour. The cell lysates were dissolved with gentle agitation. The ELISA plates were rinsed with the wash buffer three times (using a Skatron Sean Washer 300). To each well of the capture ELISA plate, 100 μl of the cell lysate was transferred using fresh or fresh pipette tips for each transfer. Plates were incubated at room temperature for 2 hours with gentle shaking. Diluted biotinylated 4G10 (2 ° Ab; secondary antibody; 4 ° C) 1: 1000 in the assay buffer was prepared. Each cavity was rinsed 10 times with the wash buffer. To each cavity 100 ul of 2 ° Ab was added, followed by incubation at room temperature for 2 hours with gentle agitation. The plates were washed with the buffer solution for washing six times. Streptavidin / HRP diluted 1: 50000 was prepared in the assay buffer. To each well was added 100 μl of the diluted StrAv / HRP, followed by incubation at room temperature for 1 hour with gentle shaking. The plates were washed with the wash buffer six times. To each well was added 100 ul of the substrate solution: 1 volume of the K & P TMB substrate plus 1 volume of the K &P TMB peroxide solution. The color of the reaction was allowed to develop for 15 minutes, followed by the addition of 50 ul of 1.0 M H3P04 to stop the color development. The O.D. (450 nm) mof of each well was read. Figure 13 shows the results of the activation using three different agonist antibodies - in this case the antibodies were raised against the epitope of the gD flag, but were able to induce the oligomerization of the subunit and the activation of the subsequent tyrosine kinase domain (Rse region).

Deposit of the Material The following materials have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA (ATCC): ATCC Material Dep. No. Depository Date mGFRa3 (clone 13) DNA48613 209752 April 7, 1998 DNA48614 209751 7 April 1998 This deposit was made under the provisions of the Budapest Treaty in the International Recognition of the Deposit of Microorganisms for the Purpose of the Patent Procedure and the Regulations under it (Budapest Treaty). This ensures the maintenance of a feasible crop of the deposit for 30 days from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty, and is subject to an agreement between Genentech, Inc. and the ATCC, which ensures the permanent and unrestricted availability of the progeny of the deposit culture to the public during the issuance of the US patent relevant or during the open presentation to the public of any U.S. patent. or foreign, whichever comes first, and ensure the availability of the progeny to one determined by the US Patent and Trademark Commissioner. to be titled in accordance with 35 USC § 122 and the rules of the Commissioner with respect thereto (including 37 CFR § 1.14 with particular reference to 886 OG 638). The assignee of the present application has agreed that if a culture of the materials on deposit could die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced by notification during notification with others thereof. The availability of the deposited material is not constructed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws. The above written specification is considered to be sufficient to make it possible for a person skilled in the art to practice the invention. The present invention will not be limited in scope by the deposited construction, since the deposited mode is proposed as a single illustration of certain aspects of the invention and any constructions that are functionally equivalent are considered within the scope of this invention. The deposit of the material here does not constitute an admission that the written description contained herein is inadequate to make possible the practice of any aspect of the invention, including the best mode thereof, nor shall it be construed as limiting the scope of the claims with respect to the specific illustrations that it represents. Actually, various modifications of the invention in addition to those shown and described will become apparent to those skilled in the art from the foregoing description and are considered to be within the scope of the appended claims.

Sequence Listing < 110 > Genentech, Inc. < 120 > GFRalfa3 and its Uses < 130 > P1268R1PCT < 141 > 1999-03-19 < 150 > US 60 / 079,124 < 151 > 198-03-23 < 150 > US 60/081, 569 < 151 > 1998-04-13 < 160 > 25 < 210 > 1 < 211 > 387 < 212 > DNA < 213 > Mus Musculus < 400 > 1 ctgatttgca gcctgtggtg ggagagaact cgccagcctg tggaagaaga 50 tacacagcaa cgcagcgcgc accaggcatt cccggaacca ccgcagcaca 100 tcccgtctgc tccagaagag gtcttagaag tgagggctgt gacccttccg 150 atcccgagcg gccagttttc aaacctccct tgcccctgct tccttctggc 200 tcaggctgct cctccttagg actttgtggg tccagttttg ccttctgttc 250 tgatggtgat tagcggctca ectccagcgc ttcttcctgt tteccaggac 300 cacccagagg ctaaggaatc agtcattccc tgttgccttc tccaggaagg 350 caggctaagg gttccgaggt gactgagaaa aatgttt 387 < 210 > 2 < 211 > 353 < 212 > DNA < 213 > Mus Musculus < 400 > 2 cgcggcgccc agcgaggcag agcgctgtcg catcccgggc gtccaccegc 50 catggggctc tcctggagcc cgcgacctcc actgctgatg atcctgctac 100 tggtgctgtc gttgtggctg ccacttggag caggaaactc ccttgccaca 150 gagaacaggt t'tgtgaacag ctgtacccag gccagaaaga aatgcgaggc 200 taatcccgct tgcaaggctg cctaccagca cctgggctcc tgcacctcca 2S0 gttaagcagg ccgctgccct tagaggagtc tgccatgtcc gcagactgcc 300 tagaggcagc agaacaactc aggaacagct ctctgataga ctgcagg gc 350 353 cat < 210 > 3 < 211 > 498 < 212 > DNA < 213 > Mus Musculus < 400 > 3 aattcggaac gagggtgaag gagcttcgca agtcccaagg ccctctggaa 50 gtcgctgaag ctgccgtcag ccaatccagt ggactcgcag ccaaatttgt 100 catccactgt cacatccccc agtgggactc cgacaaatgt gaagaacagc 150 tggaagagac catcaaaaac tgcctgtctg cagcagagga caagaagctt 200 aaatccgtcg ccttcccacc gttccccagt ggcagaaact gcttccccaa 250 acagacggcc gcccaggtga ccctcaaggc catctcggct cacttcgacg 300 actcgagetc gtcctcgctg aagaatgtgt acttcctgct cttcgacagc 350 gagacategg catctacgcg caggag tgg ccaaactgga caccaagtag 400 ctctctccag tggcggcgaa ggaggaggat cggcgtgacg tcacaagagc 450 gggggtttta ttttttacaa ggattgcaga agggtgacgg ggcatggg 498 < 210 > 4 < 211 > 1935 < 212 > DNA < 213 > Mus Musculus < 400 > 4 gaatttggcc ctcgaggcca agaattcggc acgaggcgcg gcgcccagcg 50 caggc'agagc gctgtcgcat cccgggcgtc cacccgccat ggggctctcc 100 tggagcccgc gacctccact gctgatgatc ctgctactgg tgctgtcgtt 150 gtggctgcca cttggagcag gaaactccct tgccacagag aacaggtttg 200 tgaacagctg tacccaggcc agaaagaaat gcgaggctaa tcccgcttgc 250 aaggctgcct accagcacct gggetectgc acctccagtt taagcaggcc 300 gctgccctta gaggagtctg ccatgtctgc agactgccta gaggcagcag 350 aacaactcag gaacagctct ctgatagact gcaggtgcca tcggcgcatg 400 aagcaccaag ctacetgtct ggacatttat tggaccgttc accctgcccg 450 aagccttggt gactacgagt tggatgtctc accctatgaa gacacagtga 500 ccagcaaacc ctggaaaatg aatcttagca agttgaacat gctcaaacca 550 gactcggacc tctgcctcaa atttgctatg ctgtgtactc ttcacgacaa 600 gtgtgaccgc ctgcgcaagg ggcatgctca cctacgggga gggatccgct 650 cctctgccta gccagcgcca gcccagctgc gctccttctt tgagaaggca 700 gcagagtccc acgctcaggg tctgctgctg tgtccctgtg caccagaaga 750 tgcgggctgt ggggagcggc ggcgtaacac catcgccccc agttgcgccc 800 tgccttctgt aacccccaat tgcetggatc tgcggagctt ctgccgtg cg 850 gaccctttgt gcagatcacg cctgatggac ttccagaccc actgtcatcc 900 tatggacatc cttgggactt gtgcaactga gcagtccaga tgtctgcggg 950 catacctggg gctgattggg actgccatga ccccaaactt catcagcaag 1000 gtcaacacta ctgttgcctt aagctgcacc tgccgaggca gcggcaacct 1050 acaggacgag tgtgaacagc tggaaaggtc cttctcccag aacccctgcc 1100 tcgtggaggc cattgcagct aagatgcgtt tccacagaca gctcttctcc 1150 caggactggg cagactctac tttttcagtg gtgcagcagc agaacagcaa 1200 ccctgctctg agactgcagc ccaggctacc cattctttct ttctccatcc 1250 ttcccttgat tctgctgcag accctctggt agctgggctt cctcagggtc 1300 ctttgtcctc tccaccacac ccagactgat ttgcagcctg tggtgggaga 1350 gcctgtggaa gaactcgcca gaagacgcag cgtgctacac agcaacccgg 1400 aaccaaccag gcattccgca gcacatcccg tctgctccag aagaggtctt 1450 agaagtgagg gctgtgaccc ttccgatcct gagcggctag ttttcaaacc 1500 tcccttgccc ctgcttcctt ctggctcagg ctgctcctec ttaggacttt 1550 gtgggtccag ttttgccttc tgttctgatg gtgattagcg gctcacctcc 1600 agcgcttctt cctgtttccc aggaccaccc agaggctaag gaatcagtca 1650 ttccctgttg ccttctccag gaaggcaggc ta agggttct gaggtgactg 1700 agaaaaatgt ttcctttgtg tggaaggctg gtgctccagc ctccacgtcc 17S0 ctctgaatgg aagataaaaa cctgctggtg tcttgactgc tctgccaggc 1800 aatcctgaac atttgggcat gaagagctaa agtctttggg tcttgtttaa 1850 ctcctattac tgtccccaaa ttcccctagt cccttgggtc ATGATT AAAE 1900 aaaaaaaaaa attttgactt aaaaa aaaaaaaaaa 1935 < 210 > 5 < 211 > 622 < 212 > PRT < 213 > Mus Musculus < 400 > 5 Glu Phe Gly Pro Arg Gly Gln Glu Phe Gly Thr Arg Arg Gly Wing 1 5 10 15 G n Arg Arg G n Ser Ala Ala Ala Ser Arg Ala Ser Thr Arg His 20 25 30 Gly Wing Leu Leu Glu Pro Wing Thr Ser Thr Wing Asp Aßp Pro Wing 35 40 45 Thr Gly Ala Val Val Val Ala Ala Thr Trp Ser Arg Lys Leu Pro 50 55 60 Cys His Arg Glu Gla Val Cys Glu Gln Leu Tyr Pro Gly Gln Lyiss 65 70 75 Glu Met Arg Gly Ser Arg Leu Gln 31 and Cyß Leu Pro Ala Pro Gly 80 85 90 Leu Leu His Leu Gln Phe Lya G n Ala Ala Ala Leu Arg Gly Val 95 100 105 Cys His Val Cys Arg Leu Pro Arg Gly Ser Arg Thr Thr Gln Glu 110 115 120 Gln Leu Ser Aßp Arg Leu Gln Val Pro Ser Wing His Glu Ala Pro 125 130 135 Ser Tyr Leu Ser Gly His Leu Leu Asp Arg Ser Pro Cys Pro Lys 140 145 150 Pro Trp Leu Arg Val Gly Cys Leu Thr Leu Arg His Ser Asp Gln 155 160 165 Gln Thr Leu Glu Lyß Ser Gln Val Glu Hiß Wing Glp Thr Arg Leu 170 175 180 Gly Pro Leu Pro "Gln lie Cyß Tyr Wing Val Tyr Ser Ser Arg Gln 185 190 195 Val Pro Pro Wing Gln Gly Leu Arg Gly Gly Met Leu Arg Asp Pro 200 205 210 Leu Pro Pro Pro Pro Leu Pro Pro Pro Ala Pro Leu Leu Leu Glu 215 220 225 Gly Ser Arg Val Pro Arg Ser Gly Ser Ala Ala Val Ser Leu Cys 230 235 240 Thr Arg Arg Cyß Gly Leu Trp Gly Ala Ala Ala His His Arg Pro 245 250 255 Gln Leu Arg Pro Wing Phe Cys Asn Pro Gln Leu Pro Gly Ser Wing 260 265 270 Glu Leu Leu Pro Cys Gly Pro Phe Val Gln Zle Thr Pro Asp Gly 275 280 285 Leu Pro Asp Pro Leu Ser Ser Tyr Gly His Pro Trp Asp Leu Cys 290 295 300 Asn Ala Val G n Met Be Wing Gly lie Pro Gly Wing Asp Trp Asp 305 310 315 Cys His Asp Pro Lys Leu His Gln Gln Gly Gln His Tyr Cys Cys 320 325 330 Leu Lys Leu His Leu Pro Arg G n Arg Gln Pro Thr ßly Arg Val 335 340 345 Thr Ala Gly Lys Val Leu Leu Pro Glu Pro Leu Pro Arg Gly Gly 350 355 360 His Cyß Ser Asp Ala Phe Pro Gln Thr Ala Leu Leu Pro Gly Leu 365 370 375 Gly Arg Leu Tyr Phe Phe Ser Gly Wing Wing Ala Glu Gln Gln Pro 380 385 390 Cys Ser Glu Thr Ala Ala G n Ala Thr His Ser Phe Phe Leu Kis 395 400 405 Pro Ser Leu Asp Be Ala Ala Asp Pro Leu Val Ala Gly Leu Pro 410 415 420 Gln Gly Pro Leu Ser Pro Pro Pro His Pro Asp Phe Ala Ala Cyß 425 430 435 Gly Gly Arg Glu Leu Wing Being Leu Trp Lys Lys Thr Gln Arg Wing 440 445 450 Thr Gln Gln Pro Gly Thr Asn Gln Wing Phe Arg Ser Thr Ser Arg 455 460 465 Leu Leu Gln Lys Arg Ser Lys Gly Leu Pro Phe Arg Ser Wing Ala 470 475 480 Be Phe Gln Thr Be Leu Ala Pro Wing Be Phß Trp Leu Arg Leu 485 490 495 Leu Leu Leu Arg Thr Leu Trp Val Gln Phe Cys Leu Leu Phe Trp 500 505 510 Leu Ala Ala His Leu Gln Arg Phe Phe Leu Phe Pro Arg Thr Thr 515 520 525 Gln Arg Leu Arg Asn Gln Ser Phe Pro Val Wing Phe Ser Arg Ly3 530 535 .540 Wing Gly Gly Phe Gly Asp Glu Lys Cys Phe Leu Cys Val Glu Gly 545 550 555 Trp Cys Ser Ser Leu His Val Pro Leu Asn Gly Arg Lys Pro Wing 560 565 570 Gly Val Leu Thr Ala Leu Pro Gly Asn Pro Glu His Leu Gly Met 575 580 585 Lys Ser Ser Leu Trp Val Leu Phe Asn Ser Tyr Tyr Cys Pro Gln 590 595 600 lie Pro Leu Val Pro Trp Val Met lie Lyß His Phe Asp Leu Lys 605 610 615 Lys Lys Lys Lys Lys Lys Lyß 620 622 < 210 > 6 < 211 > 460 < 212 > PRT < 213 > Homo sapiens < 400 > 6 Met Phe Leu Wing Thr Leu Tyr Phe Wing Leu Pro Leu Leu Asp Leu 5 10 is Leu Leu S * > r Glu Wing Val Ser Gly sly Asp Arg Leu Asp Cys Val 20 25 30 Lyß Wing Ser Asp Gln Cys Leu Lyß Glu Gln Ser Cys Ser Thr Lys 35 40 5 Tyr Arg Thr Leu Arg Gln Cys Val Wing Gly Lys Glu Thr Aan Phe 50 55 60 Ser Leu Ala Ser Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala 65 70 75 Met Glu Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys 80 85 90 Arg Gly Met Lys Lys Glu Lys Asn Cys Leu Arg He Tyr Trp Ser 95 100 105 Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro 110 115 120 Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp Zle Phe Arg Val Val 125 130 135 Pro Phe Zle Ser Val Glu His Zle Pro Lys ßly Asn Asn Cys Leu 140 145 150 Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp Zle Cys Lys Lys Tyr 155 160 165 Arg Ser Wing Tyr lie Thr Pro Cys Thr Thr Ser Val Ser Asn Asp 170 175 180 Val Cys Asn Arg Arg Lys Cys His Lyß Ala Leu Arg Gln Phe Phe 185 190 195 Asp Lys Val Pro Wing Lys Kis Ser Tyr Gly Met Leu Pbe Cys Ser 200 205 210 Cys Arg Asp Zle Wing Cys Thr Glu Arg Arg Arg ßln Thr Ilß Val 215 _ 220 225 Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys Pro Asn Cys Leu? Sn 230 235 240 Leu Gln Asp Ser Cys Lys Thr Asn Tyr Zle Cys Arg Ser Arg Leu 245 250 255 Wing Asp Phe Phe Thr Asn Cys Gln Pro Glu Ser Arg Ser Val Ser 260 265 270 Ser Cys Leu Lys Glu Asn Tyr Wing Asp Cys Leu Leu Wing Tyr Ser 275 280 285 Gly Leu lie Gly Thr Val Met Thr Pro Asn Tyr lie Aßp Ser Ser 290 295 300 Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Sar Asn Ser Gly Asn 305 310 315 Asp Leu Glu Glu Cys Leu Lyß Phe Leu Asn Phß Phe Lys Asp Asn 320 325 330 Thr Cys Leu Lys Asn Wing Zle Gln Wing Phe Gly Asn Gly Ser Asp 335 340 345 Val Thr Val Trp Gln Pro Ala Phe Pro Val Gln Thr Thr Thr Ala 350 355 360 Thr Thr Thr Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro 365 370 375 Wing Gly Ser Glu Asn Glu Zle Pro Thr His Val Leu Pro Pro Cys 380 385 390 Wing Asn Leu Gln Wing Gln Lys Leu Lys Ser Asn Val Ser Gly Asn 395 400 405 Thr Hie Leu Cys Zle Ser Asn Gly Asn Tyr Glu Lys Glu Gly Leu 410 415 420 Gly Ala Ser Ser His Zle Thr Thr Lys Ser Met Wing Ala Pro Pro 425 430 435 Ser Cys Gly Leu Ser Pro Leu Leu Val Leu Val Val Thr Ala Leu 440 445 450 Being Thr Leu Leu Being Leu Thr Glu Thr Being < 210 > 7 < 211 > 464 < 212 > PRT < 213 > Homo sapiens < 400 > 7 Met Zle Leu Wing Asn Val Phe Phe Leu Phe Phe Phe Leu Asp Glu 1 5 10 15 Thr Leu Arg Being Leu Wing Being Pro Being Ser Leu Gln Asp Pro Glu 20 25 30 Leu His Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala Aßn Glu 35 40 45 Leu Cys Wing Wing Glu Being Asn Cys Ser Being Arg Tyr Arg Thr Leu 50 55 60 Arg Gln Cys Leu Wing Gly Arg Asp Arg Asn Thr Met Leu Wing Asn 65 70 75 Lys Glu Cys Gln Ala Ala Leu Glu Val Leu Gln Glu Ser Pro Leu 80 85 90 Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu Leu Gln Cys 95 100 105 Leu Gln Zle Tyr Trp Ser Zle His Leu Gly Leu Thr Glu Gly Glu 110 115 120 ßlu Phe Tyr Glu Wing Pro Pro Tyr Glu Pro Val Thr Ser Arg Leu 125 130 135 Be Asp Zle Phe Arg Leu Wing Ser lie Phe Ser Gly Thr Gly Wing 140 145 150 Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 155 160 165 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lyß Leu Arg Ser Ser 170 175 180 Tyr Zle Ser Zle Cys Asn Arg Glu Zle Ser Pro Thr Glu Arg Cys 185 190 _ 195 Asn Arg Arg Lys Cys Kis Lys Wing Leu Arg Gln Phe Phe Asp Arg 200 205 210 Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln 215 220 225 Asp G n Ala Cys Ala Glu Arg Arg Arg Gln Thr Zle Leu Pro Ser 230 235 240 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg 245 250 255 Gly Val Cyß Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp. "260 265 270 Phe Hiß Ala Asn Cyß Arg Ala Ser Tyr Gln Thr Val Thr Ser Cys 275 280 285 Pro Wing Asp Asn Tyr Gln Wing Cys Leu Gly Ser Tyr Wing Gly Met 290 295 300 lie Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr 305 3io 315 Gly Zle Val Val Ser Pro Tr Cys Ser Cys Arg Gly Ser Gly Asn 320 325 330 Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp Phe Thr Glu Asn 335 340 345 Pro Cys Leu Arg Asn Wing Zle Gln Wing Phß Gly Asn Gly Thr Asp 350 355 360 Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gln Ala Thr Gln Ala 365 370 375 Pro Arg Val Glu Lyß Thr Pro Ser Leu Pro Asp? - > p Leu Ser Asp 380 385 390 Be Thr Ser Leu Gly Thr Ser Val lie Thr Thr Cys Thr Ser Val 395 400 405 Gln Glu Gln Gly Leu Lys Wing Asn Asn Ser Lys Glu Leu Ser Met 410 415 420 Cys Phe Thr Glu Leu Thr Thr Asn Zle? L? Pro Gly Ser Asn Lys 425 430 435 Val Zle Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Wing 440 445 450 Ala Leu Thr Val Leu Ser Val Leu Met Leu Lys Leu Ala Leu 455 460 464 < 210 > 8 < 211 > 468 < 212 > PRT < 213 > Rattus norvegicus < 400 > 8 Met Phe Leu Wing Thr Leu Tyr Phe Wing Leu Pro Leu Leu Asp Leu 1 5 10 15 Leu Met Ser Wing Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val 20 25 30 Lys Ala Ser Asp Gln Cys Leu Lys Glu Gln Ser Cys Ser Thr Lys 35 40 45 Tyr Arg Thr Leu Arg Gln Cyß Val Wing Gly Lys Glu Thr Asn Phe 50 55 60 Ser Leu Thr Ser Gly Leu Glu Wing Lys Asp Glu Cys Arg Ser Wing 65 70 75 Met Glu Ala Leu Lys Gln Lys Ser Leu Tyr Asn Cys Arg Cys Lys 80 85 90 Arg Gly Met Lys Lys Glu Lys Asn Cys Leu Arg lie Tyr Trp Ser 95 100 OS Met Tyr Gln Ser Leu Gln Gly Asn Asp Leu Leu Glu Asp Ser Pro 110 115 120 Tyr Glu Pro Val Asn Ser Arg Leu Ser Asp Zle Phe Arg Ala Val 125 130 135 Pro Phe Zle Ser Asp Val Phe Gln Gln Val Glu His? e Ser Lya 140 145 150 Gly Asn Asn 'Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asp Asp 155 160 165 Thr Cys Lys Lys Tyr Arg Ser Wing Tyr Zle Ttir Pro Cys Thr Thr 170 175 180 Ser Mee Ser Asn Glu Val Cyß Asn Arg Arg Lys Cys Hiß Lyß Ala 185 190 195 Leu Arg Gln Phe Phe Asp Lys Val Pro Wing Lys His Ser Tyr Gly 200 205 210 Met Leu Phe Cys Ser Cys Arg Asp lie Wing Cys Thr Glu Arg Arg 215 _ 220 225 Arg Gln Thr Zle Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Arg 230 235 240 Pro Asn Cys Leu Ser Leu Gln Asp Ser Cys Lys Thr Asn Tyr Zlß 245 250 255 Cys Arg Ser Arg Leu Wing Asp Phe Phe Thr Asn Cys Gln Pro Glu 260 265 270 Ser Arg Ser Val Ser Asn Cys Leu Lys Glu Asn Tyr Wing Asp Cys 275 280 285 Leu Leu Ala Tyr Ser Gly Leu lie Gly Thr Val Met Thr Pro Asn 290 295 300 Tyr Val Asp Ser Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys 305 310 315 Ser Asn Ser Gly Asn Asp Leu Glu Asp Cys Leu Lys Phe Leu Asn 320 325 330 Phe Phe Lys Asp Asn Thr Cys Leu Lys Asn Wing Zle Gln Wing Phe 335 340 345 Gly Asn Gly Ser Asp Val Thr Met Trp Gln Pro Pro Pro Wing Val 350 355 360 Gln Thr Thr Thr Wing Thr Thr Thr Thr Wing Phß Arg Val Lys Asn 365 370 375 Lys Pro Leu Gly Pro Wing Gly Ser Glu Asn Glu He Pro Thr His 380 385 390 Val Leu Pro Pro Cys Wing Asn Leu Gln Wing Gln Lys Leu Lys Ser 395 400 405 Asn Val Ser Gly Ser Thr His Leu Cys Leu Ser Asp Ser Asp Phe 410 415 420 Gly Lys Asp Gly Leu Wing Gly Wing Ser Ser His Zle Thr Thr Lys 425 430 435 Ser Met Ala Ala Pro Pro Ser Cys Ser Leu Ser Ser Leu Pro Val 440 445 450 Leu Met Leu Thr Ala Leu Ala Ala Leu «_? Ser Val Ser Leu Ala 455 460 465 < 210 > 9 < 211 > 464 < 212 > PRT < 213 > Rattus Norvegicus < 400 > 9 Mßt lie Leu Ala Aßn Ala Phß Cys Leu Phß Phß Phß Leu Asp Glu 1 5 10 15 Thr Leu Arg Ser Leu Ala Be Pro Pro Ser Leu Gln Gly Ser Glu 20 25 30 Leu His Gly Trp Arg Pro Gln Val Asp Cys Val Arg Wing Asn Glu 35 40 45 Leu Cys Wing Wing Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu 50 55 60 Arg Gln Cys Leu Wing Gly Arg Asp Arg Asn Thr Met Leu Wing Asn 65 70 75 Lys Glu Cys Gln Ala Ala Leu Glu Val Leu Gln Glu Ser Pro Leu 80 85 90 Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu Leu Gln Cys 95 100 105 Leu Gln Zle Tyr Trp Ser Zle His Leu Gly Leu Thr Glu Gly Glu 110 115 120 Glu Phe Tyr Glu Ala Sa Pro Tyr Glu Pro Val Thr Ser Arg Leu 125 130 135 Be Asp lie Phe Arg Leu Wing Ser lie Phe Ser Gly Thr Gly Thr 140 145 150 Asp Pro Wing Val Ser Thr Lys Ser Asn Kis Cys Leu Asp Ala Wing 155 160 165 Lyß Ala Cyß Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser 170 175 180 Tyr Zle Ser Zle Cys Asn Arg Glu lie Ser Pro Thr Glu Arg Cys 185 190 95 Asn Arg Arg Lys Cyß His Lys Wing Leu Arg Gln Phe Phß Asp Arg 200 205 210 Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gln 215 220 225 Asp Gln Ala Cys Ala Glu Arg Arg Arg Gln Thr? Le Leu Pro Ser 230 235 240 Cys Ser Tyr Glu Asp Lyß Glu Lys Pro Asn Cys Leu Asp Leu Arg 245 250 255 Ser Leu Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Wing Asp 260 265 270 Phe His Wing Asn Cys Arg Wing Ser Tyr Arg Thr lie Thr Ser Cys 275 280 285 Pro Wing Asp Asn Tyr Gln Wing Cys Leu Gly Ser Tyr Wing Gly Met 290 295 300 ? le Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser Asn Pro Thr - 305 310 315 Gly Val Ser Val Pro lie Trp Cys Asn Cys Arg Gly Ser Gly Asn 320 325 330 Glu Glu Cys Glu MSST Glu Lys Phe Leu Arg Asp Phe Thr Glu Asn 335 340 345 Pro Cys Leu Arg Asn Wing Zle Gln Wing Phe Gly Asn Gly Thr Asp 350 355 360 Val Asn Met Ser Pro Lys Gly Pro Ser Leu Pro Wing Thr Gln Wing 365 370 375 Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp 380 385 390 Be Thr Ser Leu Gly Thr Ser Val lie Thr Thr Cys Thr Ser lie 395 400 405 G n Glu Gln Gly Leu Lys Wing Asn Asn Ser Lys Glu Leu Ser Met 410 415 420 Cys Phe Thr Glu Leu Thr Thr Asn lie Ser Pro Gly Ser Lys Lys 425 430 435 Val Zle Lys Leu Asn Ser Gly Ser Ser Arg Ala Arg Leu Ser Wing 440 445 450 Ala Leu Thr Ala Leu Pro Leu Leu Met Leu Thr Leu Ala Leu 455 460 464 < 210 > 10 < 211 > 282 < 212 > DNA < 213 > Artificial < 220 > < 221 > unknown < 222 > 7-8, 11, 13, 15, 17, 19, 78, 152-188 < 223 > unknown base < 400 > 10 gcgctgnntg ncngnangng ggggcgggag gtgccggtcg agggagcccc 50 gctctcagag ctccagggga ggagcgangg gagcgcggag cccggccgcc 100 tacagctcgc catggtgcgc cccctgaacc cgcgaccgct gccgcccgta 150 gnnnnnnnnn nruu nnnnn luixuuinnnnn nnnnnnnngc ctctcgcagc 200 cggagacccc cttcccacag aaagecgact catgaacagc cgtctccagg 250 ccaggaggaa gtgccaggct gatcccacct ge 282 < 210 > 11 < 211 > 20 < 212 > DNA < 213 > Artificial < 400 > 11 gcctctcgca gccggagacc 20 < 210 > 12 < 211 > 21 < 212 > DNA < 213 > Artificial < 400 > 12 caggtgggat cagcctggca c 21 < 210 > 13 < 211 > 41 < 212 > DNA < 213 > Artificial < 400 > 13 tctcgcagcc ggagaccccc ttcccacaga aagccgactc a 41 < 210 > 14 < 211 > 1792 < 212 > DNA < 213 > Homo sapiens < 400 > 14 acggtgcgcc ccctgaaccc gcgaccgctg ccgcccgtag tcctgatgtt 50 gctgctgctg ctgccgccgt cgccgccgcc tctcgcagcc ggagaccccc 100 ttcccacaga aagccgaccc atgaacagct gtctccaggc caggaggaag 150 cgccaggctg atcccacctg cagtgctgcc taccaccacc tggattcctg 200 cacctctagc ataagcaccc cactgccctc agaggagcct tcggtccctg 250 ggaggcagca ctgactgcct cagcaactca ggaacagctc tctgataggc 300 tgcatgtgcc accggcgcat gaagaaccag gttgcctgct tggacatcta 350 tcggaccgct caccgcgccc gcagccttgg caactatgag etggatgtcc 400 ccccctatga agacacagtg accagcaaac cctggaaaat gaatctcagc 450 aaactgaaca tgctcaaacc agactcagac ctctgcctca agtttgccat 500 ctcaatgaca gctgtgtact agtgtgaccg gctgcgcaag gcctacgggg 550 aggcgtgctc cgggccccac tgccagcgcc acgtctgcct caggcagctg 600 ctcactttcc tcgagaaggc cgccgagccc cacgcgcagg gcctgctact 650 gtgcccatgc gcccccaacg accggggctg cggggagcgc cggcgcaaca 700 ccatcgcccc caactgcgcg ctgccgcctg tggcccccaa ctgcctggag 750 ctgcggcgcc tctgcttctc cgacccgctt tgcagatcac gcctggtgga 800 tttccagacc cactgccatc ccatggacat cctaggaact tgtgcaacag 8 50 agcagtccag atgtctacga gcatacctgg ggctgattgg gactgccatg 900 acccccaact ttgtcagcaa tgtcaacacc agtgttgcct taagctgcac 950 ctgccgaggc agtggcaacc tgcaggagga gtgtgaaatg ctggaagggt 1000 tcttctccca caacccctgc ctcacggagg ccattgcagc taagatgcgt 1050 tttcacagcc aacccttctc ccaggactgg ccacacccta cctttgctgt 1100 gatggcacac cagaacgaaa accctgccgt gaggccacag ccccgggtgc 1150 cctctctttt ctcctgcacg cttccctttga ttctgctcct gagcctatgg 1200 tagctggact tccccagggc cctcttcccc tccaccacac ccaggtggac 1250 ttgcagccca ggaaaggaca caaggggtga gcagcaggaa ggaggtgcag 1300 tgcgeagatg agggcacagg agaagctaag ggttatgacc cccagatcct 1350 tactggtcca gtcctcattc cctccacccc atcttccactt ctgattcatg 1400 ttggtggcca ctgcccctcc caatttagcc atgtcatctg gtggtgacca 1450 gctccaceaa gcccetttct gagcccttcc tcttgactac caggatcacc 1500 agaatctaat aagttagcct ttctctattg cattccagat tagggtttagg 15S0 gtagggagga ctgggtgttc tgaggcagcc tagaaagtca ttctcctttg 1600 tgaagaaggc tcctgcccec tcgtctcctc ctctgagtgg aggatggaaa 1650 actactgcct gcactgccct gtccccggat cc tgccgaac atctgggcat 1700 caggagctgg agcctgtggg cctcgcttta ttcccattat tgtcctaaag 1750 tctctetggg ctcttggatu atgattaaac ctttgactta ag 1792 < 210 > 15 < 211 > 400 < 212 > PRT < 213 > Homo sapiens < 400 > 15 Met Val Arg Pro Leu Asn Pro Arg Pro Leu Pro Pro Val Val Leu 1 5 10 15 Met Leu Leu Leu Leu Le Pro Pro Pro Pro Leu Pro Leu Ala Ala 20 25 30 Gly Asp Pro Leu Pro Thr Glu Ser Arg Leu Met Asn Ser Cys Leu 35 40 45 Gln Ala Arg Arg Lys Cys Gln Wing Asp Pro Thr Cys Ser Wing Wing 50 55 60 Tyr His His Leu Asp Ser Cys Thr Ser Ser Zle Ser Thr Pro Leu 65 70 75 Pro Ser Glu Glu Pro Ser Val Pro Wing Asp Cys Leu Glu Wing Wing 80 85 90 Gln Gln Leu Arg Asn Being Ser Leu? Le Gly Cys Met Cys His Arg 95 100 105 Arg Met Lys Asn Gln Val Wing Cys Leu Asp lie Tyr Trp Thr Val 110 115 120 Hiß Arg Ala Arg Ser Leu Gly Asn Tyr Glu Leu? Sp Val Ser Pro 125 130 135 Tyr Glu Asp.Thr Val Thr Ser Lys Pro Trp Lys Mss Asn Leu Ser 140 145 150 Lys Leu Asn Met Leu Lys Pro Asp Ser Asp Leu Cys Leu Lyß Phe 155 160 165 Wing Met Leu Cys Thr Leu Asn Asp Lys Cys Asp Arg Leu Arg Lys 170 175 180 Wing Tyr Gly Glu Wing Cys Ser ßly Pro His Cys Gln Arg His Val 185 190 195 Cys Leu Arg Gln Leu Leu Thr Phe Phe ßlu Lys Ala Wing Glu Pro 200 205 210 His Wing Gln Gly Leu Leu Leu Cys Pro Cyß Wing Pro Asn Asp Arg 215 220 225 Gly Cys Gly Glu Arg Arg Arg Asn Thr? e Ala Pro Asn Cyß Ala 230 235 240 Leu Pro Pro Val Wing Pro Asn Cyß Leu Glu Leu Arg Arg Leu Cyß 245 250 255 Phe Ser Asp Pro Leu Cys Arg Ser Arg Leu Val Asp Phß Gln Thr 260 265 270 Hiß Cys His Pro Met Asp lie Leu Gly Thr Cys Ala Thr Glu Gln 275 280 285 Ser Arg Cys Leu Arg Ala Tyr Leu ßly Leu? Le Gly Thr Ala Met 290 295 300 Thr Pro Asn Phe Val Ser Asn Val Asn Thr Ser Val Ala Leu Ser 305 310 315 Cys Thr Cys Arg Gly Ser Gly Asn Leu Gln Glu Glu Cys Glu Met 320 325 330 Leu Glu Gly Phe Phe Ser His Asn Pro Cys Leu Thr Glu Ala? Le 335 340 345 Wing Wing Lys Met Arg Phe Kis Ser G n Leu Phe Ser Gln Asp Trp 350 355 360 Pro His Pro Thr Phe Wing Val Met Wing His Gln? Sn Glu? Sn Pro 365 370 375 Ala Val Arg Pro Gln Pro Trp Val Pro Ser Leu Phß Ser Cys Thr 380 385 390 Leu Pro 'Leu lie Leu Leu Leu Ser Leu Trp 395 400 < 210 > 16 < 211 > 1837 < 212 > DNA < 213 > Homo sapiens < 400 > 16 cccaggaccc tggtgggaga gtgtgtgcgt cgcgctggag ggcgggaggc 50 gggggcggga ggtgccggtc gagggagccc cgctctcaga gctccagggg 100 aggagcgagg ggagcgcgga gcccggcgcc tacagctcgc cacggtgcgc 150 cccctgaacc cgcgaccgct gccgcccgta gccccgacgt tgccgccgct 200 gctgccgccg tcgccgctgc ctctcgcagc cggagacccc cttcccacag 250 aaagccgact catgaacagc tgtctccagg ccaggaggaa gtgccagget 300 gatcccacct gcagcgctgc ctaccaccac ctggattccc gcacctctag 350 cataagcacc ccactgccct cagaggagcc ttcggcccctt gctgaccgcc 400 tggaggcagc acagcaactc aggaacagct ccecgatagg ctgcatgCgc 450 tgaagaacca caecggcgca ggttgcctgc ttggacatct attggaccgt 500 tcaccgtgcc cgcagccttg actcagacct ctgcctcaag tttgccatgc 550 tgtgtactct caatgacaag tgtgaccggc tgcgcaaggc ctacggggag 600 gcgtgctccg ggccccactg ccagcgccac gtctgcctca ggcagctgct 650 cactttcttc gagaaggccg ccgagcccca cgcgcagggc ctgctactgt 700 gcccatgtgc ccccaacgac cggggctgcg gggagcgccg gcgcaacacc 750 atcgccccca actgcgcgct gccgcctgtg gcccccaact gcctggagct 800 gcggcgcccc tgcttctccg acccgctttg cagatcacgc ctggtggatt 850 tccagaccca ctgccatccc atggacatcc taggaacttg tgcaacagag 900 cagtccagat gtctacgagc atacctgggg ctgattggga ctgccatgac 950 ccccaacttt gtcagcaatg tcaacaccag tgttgecttta agctgcacct 1000 gccgaggcag tggcaacctg caggaggagt gtgaaatgct ggaagggttc 1050 ttctcccaca acccctgcct cacggaggcc attgcagcta agatgcgttt 1100 tcacagccaa ctcttctccc aggactggcc acaccctacc tttgctgtga 1150 gaatgaaaac tggcacacca cctgctgtga ggccacagcc ctgggtgccc 1200 tctcttttct cctgcacgct tcccttgatt gcctatggta ctgctcctga 1250 gccggacttc cccagggccc ccttcccctc caccacaccc aggtggaccc 1300 gcagcccaca aggggtgagg aaaggacagc agcaggaagg aggtgcagtg 1350 cgcagacgag ggcacaggag aagccaaggg ttatgacctc cagatcctta 1400 ctggtccagt cctcattccc tccaccccat ctccacttct gattcatgct 1450 gcccctcctt ggtggccaca atttagccat gtcatctggt ggtgaccagc 1500 tccaccaagc ccccttctga gcccttcctc ttgactacca ggatcaccag 1550 gttagccctt aatctaataa ttccagatta ctctattgca gggttagggt 1600 agggaggact gggtgttctg aggcagccca gaaagtcatt ctcctttgtg 1650 aagaaggctc ctgccccctc gtctcctcct ctgagtggag gatggaaaac 1700 tactgcctgc actgccctgt ccccggatcc tgccgaacat ctgggcatca 1750 ggagctggag cctgtgggcc ttgctttatt cctattattg tcctaaagtc 1800 tccccgggct ctcggatcat gattaaacct ttgactt 1837 < 210 > 17 < 211 > 369 < 212 > PRT < 213 > Homo sapiens < 400 > 17 Met Val? Rg Pro Leu? Sn Pro Arg Pro Leu Pro Pro Val Val Leu 1 5 10 15 Met Leu Leu Leu Leu Leu Pro Pro Pro Pro Leu Pro Leu? La? La 20 25 30 Gly? S Pro Leu Pro Thr Glu Ser? Rg Leu Met? Sn Ser Cys Leu 35 40 45 Gln Ala Arg? Rg Lys Cys Gln Ala Asp Pro Thr Cys Ser Ala Ala 50 55 60 Tyr His His Leu? Sp Ser Cys Thr Ser Ser Zle Ser Thr Pro Leu 65 70 75 Pro Ser Glu Glu Pro Ser Val Pro? La? Sp Cys Leu Glu Ala? La 80 85 90 Gln Gln Leu? Rg Asn Ser Ser Leu Sle Gly Cys Met Cys His Arg 95 100 105 Arg Met Lys Asn Gln Val Wing Cys Leu Asp He Tyr Trp Thr Val 110 115 120 His Arg Ala Arg Ser Leu Asp Ser Asp Leu Cys Leu Lys Phe Wing 125 130 135 Met Leu Cys Thr Leu Asn Asp Lys Cya Asp? Rg Leu? Rg Lys? La 140 145 150 Tyr Gly Glu? The Cys Ser Gly Pro Hi? Cy? Gln? Rg His Val Cy? 155 160 165 Leu? Rg Gln -Leu Leu Thr Phe Phe Glu Lys? The? Glu Pro Hi? 170 175 180? The Gln Gly Leu Leu Leu Cys Pro Cys Wing Pro Asn Asp Arg Gly 185 190 195 Cys Gly Glu Arg Arg Arg Asn Thr He Wing Pro? Sn Cys? The Leu 200 205 210 Pro Pro Val Wing Pro Asn Cys Leu Glu Leu Arg Arg Leu Cyi Ph? 215 220 225 Ser Asp Pro Leu Cys? Rg Ser? Rg Leu Val? Sp Phe Gln Thr His 230 235 240 Cys Hiß Pro Met? S He Leu Gly Thr Cys? The Thr Glu Gln Ser 245 250 255 Arg Cys Leu Arg Ala Tyr Leu Gly Leu He Gly Thr Ala Met Thr 260 265 270 Pro Asn Phe Val Ser Asn Val Asn Thr Ser Val Ala Leu Ser Cys 275 280 285 Thr Cys Arg Gly Ser Gly Asn Leu Gln Glu Glu Cys Glu Met Leu 290 295 300 Glu Gly Phe Phe Ser His Asn Pro Cys Leu Thr Glu Wing He Ala 305 310 315 Wing Lys Met Arg Phe Kis Ser Gln Leu Phe Ser Gln? Sp Trp Pro 320 325 330 His Pro Thr Phe? The Val Met Wing Kis Gln Asn Glu? Sn Pro Wing 335 340 345 Val Arg Pro Gln Pro Trp Val Pro Ser Leu Phß Ser Cys Thr Leu 350 355 360 Pro Leu He Leu Leu Leu Ser Leu Trp 365 369 < 210 > 18 < 211 > 628 < 212 > PRT < 213 > Artificial < 400 > 18 Met Val Arg Pro Leu Asn Pro Arg Pro Leu Pro Pro Val Val Leu 5? O 15 Met Leu Leu Leu Leu Leu Pro Pro Pro Pro Leu Pro Leu Ala Wing 20 25 30 Gly Asp Pro Leu Pro Thr Glu Be Arg Leu Met Asn Ser Cys Leu 35 40 45 Gln? La? Rg.Arg Lys Cys Gln Wing Asp Pro Thr Cys Ser? La? La 50 55 60 Tyr His His Leu? Sp Ser Cys Thr Ser Ser Be Ser Thr Pro Leu 65 70 75 Pro Ser Glu Glu Pro Ser Val Pro Wing Asp Cys Leu Glu Wing Wing 80 85 90 Gln Gln Leu Arg Asn Ser Ser Leu He Gly Cys Met Cys His Arg 95 100 105 Arg Met Lyß Asn Gln Val Wing Cys Leu Asp He Tyr Trp Thr Val 110 115 120 His Arg Ala? Rg Ser Leu Gly Asn Tyr Glu Leu Asp Val Ser Pro 125 130 135 Tyr Glu Asp Thr Val Thr Ser Lys Pro Trp Lys Met Asn Leu Ser 140 145 150 Lys Leu? Sn Met Leu Lys Pro? Sp Ser Asp Leu Cys Leu Lys Ph? 155 160 165? The Met Leu Cys Thr Leu? Sn? Sp Lys Cys? Sp? Rg Leu Arg Lys 170 175 180 Wing Tyr Gly Glu Wing Cys Ser Gly Pro His Cys Gln Arg His Val 185 190 195 Cys Leu? Rg Gln Leu Leu Thr Phe Phe Glu Lys? The? Glu Pro 200 205 210 His Wing Gln Gly Leu Leu Cs Pro Cys Wing Pro Asn Asp Arg 215 '220 22K Gly Cys Gly Glu Arg Arg Arg? Sn Thr He? Pro? Sn Cy?? 230 235 240 Leu Pro Pro Val Ala Pro Asn Cyß Leu Glu Leu Arg Arg Leu Cys 245 250 255 Phe Ser Asp Pro Leu Cys Arg Ser? Rg Leu Val? Sp Phe Gln Thr 260 265 270 His Cys His Pro Met? Sp He Leu Gly Thr Cys? The Thr Glu Gln 275 280 285 Ser? Rg Cys Leu? Rg? The Tyr Leu Gly Leu He Gly Thr? The Met 290 295 300 Thr Pro? Sn Phe Val Ser? Sn Val? Sn Thr Ser Val? La Leu Ser 305 310 315 Cy? Thr Cy?? G Gly Ser Gly? Sn Leu Gln Glu Glu Cys Glu Met 320 325 330 Leu Glu Gly- Phe Phe Ser His? Sn Pro Cys Leu Thr Glu? La He 335 340 345 Wing Wing Lys Met Arg Phe His Ser Gln Leu Phe Ser G n Asp Trp 350 355 360 Pro His Pro Thr Phe Wing Val Met Wing His Gln Asn Glu Asn Pro 365 370 375 Ala Val Arg Pro Gln Pro Trp Val Pro Ser Leu Phß Ser Cys Thr 380 385 390 Leu Pro Leu He Leu Leu Leu Ser Leu Trp Pro Asp Lys Thr His 395 400 405 Thr Cye Pro Pro Cys Pro Pro Wing Glu Leu Leu Gly Gly Pro Ser 410 415 420 Val Phe Leu Phe Pro Pro Lyß Pro Lys Asp Thr Leu Met He Ser 425 430 435 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 440 445 450 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val? Sp Gly Val Glu Val 455 460 465 Hiß? Sn? The Lys Thr Lys Pro? Rg Glu Glu Gln Tyr? Sn Ser Thr 470 475 480 Tyr? Rg Val Val Ser Val Leu Thr Val Leu Hi? Gln? Sp Trp Leu 485 490 495 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 500 505 510 Wing Pro He Glu Lys Thr He Ser Lys Wing Lys Gly Gln Pro Arg 515 520 525 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 530 535 540 Lyß Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phß Tyr Pro 545 550 555 Be Asp He Wing Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 560 565 570 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 575 580 585 Phe Leu Tyr Ser Lys Leu Thr Val? Sp Lys Ser? Rg Trp Gln Gln 590 595 600 Gly Asn Val Phe Ser Cyß Ser Val Met His Glu Ala Leu His Asn 605 610 615 His Tyr Thr- Gln Lyß Ser Leu Ser Leu Ser Pro Gly Lyß 620 625 628 < 210 > 19 < 211 > 951 < 212 > PRT < 213 > Homo sapiens < 400 > 19 Met Gly Gly Thr Wing Wing Arg Leu Gly Wing Val Zle Leu Phe Val 1 5 10 15 Val He Val Gly Leu His Gly Val Arg Gly Lys Tyr Ala Leu Ala 20 25 30 Asp Ala Ser Leu Lys Met? La? Sp Pro? Sn? Rg Phe Arg Gly Lys 35 40 45? ßp Leu Pro Val Leu? Sp Gln Leu Leu Glu Pro Ser Ser Leu Gln 50 55 60 Gly Ser Glu Leu His Gly Trp? Rg Pro Gln Val? Sp Cys Val? Rg 65 70 75 Ala? Sn Glu Leu Cys? The? La Glu? Ser? Sn Cys Ser Ser? Rg Tyr 80 85 90? Rg Thr Leu? Rg Gln Cys Leu? The Gly? Rg? Sp? Rg? Sn Thr Met 95 100 105 Leu? La? Sn Lys Glu Cys Gln? The? The Leu Glu Val Leu Gln Glu 110 115 120 Ser Pro Leu Tyr? Sp Cys? Rg Cys Lys? Rg Gly Met Lys Ly? Glu 125 130 135 Leu Gln Cys Leu Gln He Tyr Trp Ser He His Leu Gly Leu Thr 140 145 150 Glu Glu Glu Glu Phe Tyr Glu? The Ser Pro Tyr Glu Pro Val Thr 155 160 165 Ser? Rg Leu Ser Asp Sle Phe? Rg Leu? The Ser He Phe Ser Gly 170 175 1B0 Thr Gly Thr? Sp Pro Wing Val Ser Thr Lys Ser Asn His Cys Leu 185 190 195 Asp? La? La Lys? La Cys? Sn Leu? Sn? Sp? Sn Cys Lys Lys Leu 200 205 210 Arg Ser Ser Tyr He Ser He Cys? Sn? Rg Glu He Ser Pro Thr 215 220 225 Glu? Rg Cys? Sn? Rg? Rg Lys Cys His Lys? The Leu? Rg Gln Ph? 230 235 240 Phß? Sp Arg * Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cy? 245 250 255 Ser Cys Gln Asp Gln Wing Cys Wing Glu Arg Arg? Rg Gln Thr He 260 265 270 Leu Pro Ser Cys Ser Tyr Glu? Sp Lys Glu Lys Pro? Sn Cys Leu 275 280 285? Sp Leu? Rg Ser Leu Cys? Rg Thr? Sp Kis Leu Cys? Rg Ser? Rg 290 295 300 Leu? La? Sp Phe His? The Asn Cys Arg? The Ser Tyr Arg Thr He 305 310 315 Thr Ser Cys Pro Wing Asp? Sn Tyr Gln? The Cys Leu Gly Ser Tyr 320 325 330 Wing Gly Met He Gly Phe Asp Met Thr Pro Asn Tyr Val Asp Ser 335 340 345 Asn Pro Thr Gly He Val Val Ser Pro Trp Cyß? Sn Cys Arg Gly 350 355 360 Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg? Sp Phß 365 370 375 Thr Glu? Sn Pro Cys Leu? Rg? Sn? La? Le Gln? The Phe Gly? ßn 380 385 390 Gly Thr? Sp Val? Sn Met Ser Pro Ly? Gly Pro Ser Leu Pro? La 395 400 405 Thr Gln? The Pro? Rg Val Glu Lys Thr Pro Ser Leu Pro? Sp? Sp 410 415 420 Leu Ser? Sp Ser Thr Ser Leu Gly Thr Ser Val He Thr Thr Cys 425 430 435 Thr Ser He Gln Glu Gln Gly Leu Lyß? The? Sn? Sn Ser Lys Glu 440 445 450 Leu Ser Mett Cys Phß Thr Glu Leu Thr Thr Asn He Zle Pro Gly 455 460 465 Trp Arg Ala Trp Val Pro Val Val Leu Gly Val Leu Thr Ala Leu 470 475 480 Val Thr Ala? La? La Leu Ala Leu He Leu Leu Arg Lys? Rg? Rg 485 490 495 Lys Glu Thr? Rg Phe Gly Gln? The Phe? Sp Ser Val Met? La? Rg 500 505 510 Gly Glu Pro? The Val Kis Phe? Rg? The? The Arg Ser Phe Asn Arg 515 520 525 Glu? Rg Pro-Glu Arg Zle Glu Wing Thr Leu Asp Ser Leu Gly Zle 530 535 540 Being Asp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu He Pro Glu 545 550 555 Gln Gln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu Phe Gly 560 565 570 Ser Val Arg Glu? Gln Leu Lys Gln Glu Asp Gly Ser Phe Val 575 580 585 Lys Val Wing Val Lys Met Leu Lys Wing Asp Zle He Wing Ser Ser 590 595 600 Asp He Glu Glu Phe Leu Arg Glu Ala Wing Cys Met Lys Glu Phe 605 610 615 Asp His Pro His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser 620 625 630 Arg Ala Lys Gly Arg Leu Priesel Pro Met Val? Le Leu Pro Phe 635 640 645 Met Lys His Gly Asp Leu His Wing Phe Leu Leu Wing Ser Arg He 650 655 660 Gly Glu? Sn Pro Ph? ßn Leu Pro Leu Gln Thr Leu He? Rg Ph? 665 670 675 Mßt Val Aap He Wing Cyß Gly Met Glu Tyr Leu Ser Ser Arg Asn 680 685 690 Phß He His Arg Asp Leu Ala Wing Arg Asn Cyß Met Leu Wing Glu 695 700 705? ßp Met Thr Val Cys Val? La? Sp Phe Gly Leu Ser? Rg Lys He 710 715 720 Tyr Ser Gly? Sp Tyr Tyr? Rg Gln Gly Cys? The Ser Lys Leu Pro 725 730 735 Val Lys Trp Leu Wing Leu Glu Ser Leu Wing Asp Asn Leu Tyr Thr 740 745 750 Val Gln Ser? Sp Val Trp Wing Phe Gly Val Thr Met Trp Glu Zle 755 760 765 Met Thr Arg Gly Gln Thr Pro Tyr Wing Gly He Glu Asn Wing Glu 770 775 780 He Tyr Asn Tyr Leu He Gly Gly Asn Arg Leu Lyß Gln Pro Pro 785 790 795 Glu Cys Met Glu Asp Val Tyr Asp Leu Met Tyr Gln Cyß Trp Ser 800 805 810 Ala? Sp Pro Lys Gln? Rg Pro Ser Phe Thr Cys Leu? Rg Met Glu 815 820 825 Leu Glu? Sn He Leu Gly Gln Leu Ser Val Leu Ser? The Ser Gis 830 835 840? S Pro Leu Tyr He Asn He Glu Arg? The Glu Glu Pro Thr Wing 845 850 855 Gly Gly Ser Leu Glu Leu Pro Gly Arg Asp Gln Pro Tyr Ser Gly 860 865 870? The Gly? Gly Ser Gly M? T Gly Wing Val Gly Gly Thr Pro Ser 875 880 885 Asp Cys? Rg Tyr He Leu Thr Pro Gly Gly Leu? Glu Gln Pro 890 895 900 Gly Gln? Glu His Gln Pro Glu Pro Pro Leu? Sn Glu Thr Gln 905 910 915? Rg Leu Leu Leu Leu Gln Gln Gly Leu Leu Pro His Ser Ser Cy? 920 925 930 Wing Asp Wing Ser Leu Lyß Met Wing Asp Pro Asn Arg Phe Arg Gly 935 940 945 Lys Asp Leu Pro Val Leu 950 951 < 210 > 20 < 211 > 888 < 212 > PRT < 213 > Artificial < 400 > 20 Met Gly Gly Thr? La? La? Rg Leu Gly? The Val Zle Leu Phe Val 1 5 10 15 Val He Val Gly Leu Kis Gly Val? Rg Gly Lys Tyr? The Leu? The 20 25 30? Sp? The Ser Leu Lys Met Wing Asp Pro Asn Arg Phe? Rg Gly Lys 35 40 45? Sp Leu Pro Val Leu Asp Gln Leu Leu Glu Ala Gly Asn Ser Leu 50 55 60 Wing Thr Glu Asn Arg Phe Val Asn Ser Cys Thr Gln Wing Arg Lys 65 70 75 Lys Cys Glu Wing Asn Pro Wing Cys Lys Wing Wing Tyr Gln His Leu 80 is 90 Gly Ser Cys Thr Ser Ser Leu Ser Arg Pro Leu Pro Leu Glu Glu 95 100 ios It will be the Met-Ser? The? Sp Cys Leu Glu? The? The Glu Gln Leu Arg 110 115 120 Asn Ser Ser Leu lie Asp Cys Arg Cys His Arg Arg Met Lys His 125 130 135 Gln Ala Thr Cyß Leu Asp lie Tyr Trp Thr Val Hiß Pro Ala Arg 140 145 150 Ser Leu Gly Asp Tyr Glu Leu Asp Val Ser Pro Tyr Glu Asp Thr 155 160 165 Val Thr Ser Lys Pro Trp Lys Met Asn Leu Ser Lyß Leu Asn Met 170 175 180 Leu Lyß Pro Asp Ser Asp Leu Cys Leu Lys Phe Wing Met Leu Cys 185 190 igs Thr Leu His Asp Lys Cys Asp Arg Leu Arg Lys Wing Tyr Gly Glu 200 205 210 Ala Cys Ser Gly He Arg Cys Gln Arg His Leu Cys Leu Ala Gln 215 220 225 Leu Arg Ser Phe Phe Glu Lys Wing Wing Glu Ser His Wing Gln Gly 230 235 240 Leu Leu Leu Cys Pro Cys Pro Pro Glu Asp Wing Gly Cys Gly Glu 245 250 2S5? Rg? Rg? Rg? Sn Thr He Wing Pro Ser Cys Wing Leu Pro Ser Val 260 265 270 Thr Pro Asn Cys Leu Asp Leu Arg Ser Phe Cys Arg Ala Asp Pro 275 280 285 Leu Cys? Rg Ser? Rg Leu Met? S Phe Gln Thr His Cys His Pro 290 295 300 Met Asp He Leu Gly Thr Cys Wing Thr Glu Gln Ser Arg Cys Leu 305 310 315 Arg Ala Tyr Leu Gly Leu He Gly Thr Ala Met Thr Pro Asn Phe 320 325 330 I have Lyß Val Asn Thr Thr Val? The Leu Ser Cyß Thr Cys? Rg 335 340 345 Gly Ser Gly? Sn Leu Gln? Sp Glu Cys Glu G n Leu Glu Arg Ser 350 355 360 Phe Ser Gln Asn Pro Cys Leu Val Glu Ala He Ala Wing Lys Met 365 370 375 Arg Phe His Arg Gln Leu Phß Ser Gln Asp Trp Wing Asp Ser Tbr 380 385 390 Phe Ser Val-Val Gln Gln Gln Asn Ser? Sn Pro? Trp? Rg Ala 395 400 405 Val Val Leu Gly Val Leu Thr? Leu Val Thr? 410 415 420?? Leu Ala Leu He Leu Leu Arg Lys? Rg? Rg Lys Glu Thr 425 430 435? Rg Phe Gly Gln? The Phe ? sp Ser Val Met? the? rg Gly Glu Pro 440 445 450 Ala Val His Phe Arg Ala Ala Arg Ser Phe? Sn Arg Glu Arg Pro 455 460 465 Glu? Rg He Glu? The Thr Leu? Ap Ser Leu Gly lie Ser? Sp ßlu 470"475 480 Leu Lys Glu Lys Leu Glu? Sp Val Leu He Pro Glu Gln Gln Phe 485 490 495 Thr Leu Gly? Rg Met Leu Gly Lys Gly Glu Phe Gly Ser Val? Rg 500 505 S10 Glu? Gln Leu Lys Gln Glu? Sp Gly Ser Phe Val Lys Val? La 515 520 525 Val Lys Met Leu Lys? La? Sp He He? The Ser Ser? Sp He Glu 530 535 540 Glu Phe Leu Arg Glu Wing Wing Cys Met Lys Glu Phe Asp His Pro 545 550 555 His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser? Rg? The Lys 560 565 570 Gly? Rg Leu Pro He Pro Met Val He Leu Pro Phe Met Lys His 575 580 585 Gly? Sp Leu Hiß? The Phe Leu Leu? The Ser? Rg He Gly Glu? ßn 590 595 600 Pro Phe? Sn Leu Pro Leu Gln Thr Leu He? Rg Phe Met Val? Sp 605 610 615 Zle? The Cys Gly Met Glu Tyr Leu Ser Ser? Rg? Sn Phe Zle His 620 625 630? Rg? Sp Leu? La? La? Rg? Sn Cys Met Leu? The Glu? Sp Mett Thr 635 640 645 Val Cys Val Wing Asp Phe Gly Leu Ser? Rg Lys He Tyr Ser Gly 650 655 660 ? Tyr Tyr? rg Glp Gly Cys? Ser Lys Leu Pro Val Lys Trp 665 670 675 Leu? Leu-Glu Ser Leu? La? Sp? Sn Leu Tyr Thr Val Gln Ser 680 685 690? Sp Val Trp Wing Phe Gly Val Thr Met Trp Glu He Met Thr Arg 695 700 705 Gly Gln Thr Pro Tyr Wing Gly He Glu Asn Wing Glu He Tyr Aßn 710 715 720 Tyr Leu? Le Gly Gly Asn Arg Leu Lys Gln Pro Pro Glu Cys Met 725 730 735 Glu Asp Val Tyr Asp Leu Met Tyr Gln Cys Trp Ser Ala? Sp Pro 740 745 750 Lys Gln? Rg Pro Ser Phe Thr Cys Leu? Rg Met Glu Leu Glu? Sn 755 760 765 He Leu Gly Gln Leu Ser Val Leu Ser? The Ser Gln? Sp Pro Leu 770 775 780 Tyr He? Sn He Glu? Rg? The Glu Glu Pro Thr? The Gly Gly Ser 785 790 795 Leu Glu Leu Pro Gly? Rg? Sp Gln Pro Tyr Ser Gly? The Gly? Sp 800 805 810 Gly Ser Gly Mee Gly? The Val Gly Gly Thr Pro Ser? Sp Cys? Rg 815 820 825 Tyr He Leu Thr Pro Gly Gly Leu? Glu Gln Pro Gly Gln Wing 830 835 840 Glu Hi3 Gln Pro Glu Pro Pro Leu Asn Glu Thr Gln Arg Leu Leu 845 850 855 Leu Leu Gln Gln Gly Leu Leu Pro His Ser Ser Cys? La? Sp? La 860 865 870 Being Leu Lys Met? La? Sp Pro? Sn? Rg Phe? Rg Gly Lys? Sp Leu 875 880 885 Pro Val Leu 888 < 210 > 21 < 211 > 37 < 212 > DNA < 213 > Homo sapiens < 400 > 21 gcgaggggag cgcggagccc ggcgcctaca gctcgcc 37 < 210 > 22 < 211 > 20 < 212 > DNA < 213 > Artificial < 400 > 22 gcccgcgacc tccactgctg 20 < 210 > 23 < 211 > 18 < 212 > DNA < 213 > Artificial < 400 > 23 ctgtggggag cggcggcg 18 < 210 > 24 < 211 > 20 < 212 > DNA < 213 > Artificial < 400 > 24 cctgaaccta tggtaactgg 20 < 210 > 25 < 211 > 17 < 212 > DNA < 213 > Artificial < 400 > 25 acccagtcct ccctacc 17 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following

Claims (62)

1. An isolated nucleic acid, characterized in that it comprises a nucleic acid having at least 65% sequence identity with respect to (a) a nucleic acid molecule encoding a GFRα3 polypeptide (a-3 receptor of the factor family neurotrophic derivative of the glial cell line) comprising the amino acid sequence 27 to 369 of SEQ ID NO: 17, or (b) the complement of the nucleic acid molecule of (a).

2. The nucleic acid according to claim 1, characterized in that it comprises a nucleic acid having at least one sequence identity of 65% with respect to (a) the nucleic acid molecule encoding a GFRα3 polypeptide comprising the sequence of amino acids 1 to 379 of SEQ ID NO: 17, or (b) the complement of the nucleic acid molecule of (a).

3. The nucleic acid according to claim 1, characterized in that the nucleic acid has at least 75% identity of the sequence with respect to (a) the nucleic acid molecule encoding the GFRα3 polypeptide comprising the amino acid sequence 27 a 369 of SEQ ID NO: 17, or (b) the complement of the nucleic acid molecule of (a).

4. The isolated nucleic acid according to claim 1, characterized in that it comprises a nucleic acid encoding a GFRα3 polypeptide having the amino acid residues 27 to 369 of SEQ ID NO: 17.

5. The isolated nucleic acid according to claim 1, characterized in that it comprises the DNA encoding a GFRα3 polypeptide having the amino acid residues 1 to 369 of SEQ ID NO: 17.

6. An isolated nucleic acid, characterized in that it comprises the nucleic acid having at least 65% sequence identity with respect to (a) a nucleic acid molecule encoding the same mature polypeptide encoded by the cDNA in ATCC Repository No. 209751 (designation DNA 48614-1269), or (b) the complement of the DNA molecule of (a) or (b).

7. The isolated nucleic acid according to claim 6, characterized in that it comprises the coding sequence of GFRα3 of the cDNA in the ATCC deposit No. 209751 (designation DNA 48614-1268) or a sequence which hybridizes thereto under stringent conditions.

8. An isolated nucleic acid, characterized in that it comprises a nucleic acid having at least 65% identity of the sequence with respect to (a) a nucleic acid molecule encoding a GFRα3 polypeptide comprising amino acid sequence 84 to 329 of SEQ ID NO: 17, or (b) the complement of the nucleic acid molecule of (a).

9. The isolated nucleic acid according to claim 8, characterized in that it comprises a sequence encoding GFRα3 which hybridizes under stringent conditions to (a) a nucleic acid molecule encoding a GFRα3 polygypeptide comprising the amino acid sequence 84 a 329 of SEQ ID NO: 17, or (b) the complement of the nucleic acid molecule of (a).

10. A vector, characterized in that it comprises the nucleic acid according to claim 1.

11. The vector according to claim 10, operably linked to the control sequences recognized by a host cell transformed with the vector.

12. A host cell, characterized in that it comprises the vector according to the claim 10.

13. The host cell according to claim 12, characterized in that the cell is a CHO cell, an E. coli, or a yeast cell.

14. A process for producing the GFRα3 polypeptides, characterized in that it comprises culturing the host cell of claim 12 under conditions suitable for the expression of GFRα3 and recovering the GFRα3 from the cell culture.

15. A polypeptide, characterized in that it comprises a sequence having at least 65% identity of the sequence with amino acid residues 84 to 329 of SEQ ID NO: 17.

16. The polypeptide according to claim 15, characterized in that it is a GFRα3 polypeptide of the isolated natural sequence.

17. The polypeptide according to claim 16, characterized in that it comprises amino acid residues 27 to 369 of SEQ ID NO: 17.

18. A chimeric molecule, characterized in that it comprises a GFRα3 polypeptide according to claim 15, fused to a heterologous amino acid sequence.

19. The chimeric molecule according to claim 18, characterized in that the heterologous amino acid sequence is a tag sequence of the epitope.

20. The chimeric molecule according to claim 18, characterized in that the heterologous amino acid sequence is an Fc region of an immunoglobulin.

21. An antibody, characterized in that it binds specifically to the GFRα3 polypeptide according to claim 15.

22. The antibody according to claim 21, characterized in that it is an antagonist antibody.

23. The use of the antibody according to claim 21 to treat a neuronal disorder of the periphery.

24. A method for measuring the binding of the agonist to a polypeptide comprising a binding domain of the agonist of a receptor of the subunit a, characterized in that it comprises the steps of exposing the polypeptide placed on a cell membrane to a candidate agonist and measuring the homodimerization and the homooligomerization of the polypeptide.

25. The method according to claim 24, characterized in that the receptor of subunit a is a receptor of GRAP.

26. The method according to claim 24, characterized in that the polypeptide further comprises an enzymatic domain activated by dimerization or oligomerization and homodimerization and the homooligomerization is detected by the measurement of the enzymatic activity of the polypeptide.

27. The method according to claim 26, characterized in that the enzymatic domain is the intracellular autocatalytic domain of a receptor tyrosine kinase and the homodimerization or homooligomerization is detected by measuring autophosphorylation of the polypeptide.

28. A method of measuring the autophosphorylation of a polypeptide receptor construct comprising a ligand binding domain of a subunit a receptor, the intracellular catalytic domain of a tyrosine kinase receptor, and a flag epitope, characterized in that it comprises the steps of: (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein, placed on their membranes, the cells have the polypeptide receptor construction; (b) exposing the adherent cells to a substance to be analyzed; (c) solubilizing the adherent cells, whereby the cell lysate thereof is released; (d) coating a second solid phase with a capture agent which binds specifically to the flag epitope so that the capture agent adheres to the second solid phase; (e) exposing the adherent capture agent to the cell lysate obtained in step (c) so that the receptor construction adheres to the second solid phase, (f) washing the second solid phase to remove the unbound cell lysate; (g) exposing the adherent receptor construct to an anti-phosphotyrosine antibody which identifies the tyrosine phosphorylated residues at the tyrosine kinase receptor; and (h) measuring the binding of the anti-phosphotyrosine antibody to the adherent receptor construct.

29. The method according to claim 28, characterized in that the cells are transformed with the nucleic acid encoding the receptor construction prior to step (a).

30. The method according to claim 28, characterized in that the cells comprise a mammalian cell line.

31. The method according to claim 28, characterized in that the cells are adherent.

32. The method according to claim 28, characterized in that the capture agent comprises a capture antibody.

33. The method according to claim 28, characterized in that the first solid phase comprises a cavity of a first test plate.

34. The method according to claim 28, characterized in that the anti-phosphotyrosine antibody is labeled.

35. The method according to claim 34, characterized in that the label comprises an enzyme which is exposed to a coloring reagent and the color change of the coloring reagent is determined in step (h).

36. The method according to claim 28, characterized in that the flag polypeptide is fused to the amino terminal or terminal of the ligand binding domain of the a subunit receptor.

37. The method according to claim 28, characterized in that the flag polypeptide is fused to the carboxyl terminus or terminal of the intracellular catalytic domain of the tyrosine kinase receptor.

38. The method according to claim 28, characterized in that the tyrosine kinase receptor is a Rse receptor, a trk A receptor, a trk B receptor or a trk C receptor.

39. The method according to claim 28, characterized in that the subunit receptor a is a GFRα receptor.

40. The method according to claim 38, characterized in that the receptor construction further comprises the transmembrane domain of the Rse receptor and the flag epitope comprises the gD polypeptide.

41. The method according to claim 28, characterized in that the substance to be analyzed comprises an agonof the subunit a receptor.

42. The method according to claim 28, characterized in that the substance to be analyzed comprises an antagonfor the subunit a receptor.

43. The method according to claim 42, characterized in that the antagoncompetitively inhibits the binding or activation of the subunit receptor a by an agonthereto and step (b) is followed by a step wherein the adherent cells are exposed to the agon .

44. The method according to claim 28, characterized in that the substance to be analyzed is a composition which comprises an antagonand an agonfor the subunit receptor and the assay measures the ability of the antagonto bind to the agonand therefore which reduces the activation of the construction of the polypeptide by the agon

45. A method of measuring the autophosphorylation of a polypeptide receptor construct comprising a ligand binding domain, a subunit a receptor, the intracellular catalytic domain of a tyrosine kinase receptor, and a flag epitope, characterized in that comprises the steps of: (a) coating a cavity of a first test plate with a homogeneous population of adherent cells so that the cells adhere to the cavity, wherein the cells have the polypeptide receptor construction placed on the cell membranes of the same; (b) exposing the adherent cells to a substance to be analyzed; (c) solubilizing the adherent cells, whereby the cell lysate thereof is released; (d) coating a cavity of a second assay plate with a capture agent which specifically binds to the polypeptide receptor construction so that the capture agent adheres to the cavity; (e) exposing the cell lysate obtained in step (c) to the adherent capture agent so that the receptor construction of the polypeptide will adhere to the cavity, (f) washing the cavity to remove the unbound cell lysate; (g) exposing the receptor construct of the adherent polypeptide to an anti-phosphotyrosine antibody which selectively binds to the phosphorylated tyrosine residues in the polypeptide receptor construction; (h) measuring the binding of the anti-phosphotyrosine antibody to the receptor construct of the adherent polypeptide.

46. The method according to claim 45, characterized in that the receptor of the subunit a is a receptor of GFRα.

47. A polypeptide, characterized in that it comprises a ligand binding domain of the subunit a receptor, a flag polypeptide, and an intracellular catalytic domain of a tyrosine kinase receptor.

48. The polypeptide according to claim 47, characterized in that the flag polypeptide comprises the flag epitope of gD.

49. The polypeptide according to claim 47, characterized in that the tyrosine kinase receptor is a Rse receptor.

50. The polypeptide according to claim 49, characterized in that it also comprises the transmembrane domain of the Rse receptor.

51. The polypeptide according to claim 47, characterized in that the subunit receptor a is a GFRα receptor.

52. A set or set, characterized in that it comprises a solid phase coated with a capture agent which binds specifically to a flag polypeptide, and a polypeptide comprising a ligand binding domain of the subunit receptor, a flag polypeptide , and an intracellular catalytic domain of a tyrosine kinase receptor.

53. The kit or assembly according to claim 52, characterized in that the solid phase comprises a cavity of a microtitre plate.

54. A kit or assembly according to claim 52, characterized in that it also comprises a labeled anti-phosphotyrosine antibody.

55. The kit or assembly according to claim 54, characterized in that the label comprises an enzyme.

56. The kit or assembly according to claim 52, characterized in that it further comprises a cell transformed with a nucleic acid encoding a polypeptide comprising a ligand binding domain of the α-subunit receptor, a flag polypeptide, and a catalytic domain intracellular receptor tyrosine kinase.

57. An assay for measuring phosphorylation of a polypeptide receptor construct comprising a ligand binding domain of a subunit a receptor, the intracellular catalytic domain of a kinase receptor, and a flag epitope, characterized in that it comprises the steps of: (a) coating a first solid phase with a homogeneous population of eukaryotic cells so that the cells adhere to the first solid phase, wherein the cells comprise the polypeptide receptor construct; (b) exposing the adherent cells to a substance to be analyzed; (c) solubilizing the adherent cells, whereby the cell lysate thereof is released; (d) coating a second solid phase with a capture agent which binds specifically to the flag polypeptide so that the capture agent adheres to the second solid phase; (e) exposing the adherent capture agent to the cell lysate obtained in step (c) so that the receptor construction adheres to the second solid phase, (f) washing the second solid phase to remove the unbound cell lysate; (g) exposing the adherent kinase construct to an antibody which identifies the phosphorylated residues in the receptor construct; and (h) measuring the binding of the antibody to the adherent receptor construction.

58. The assay according to claim 57, characterized in that the a receptor is a GFRα receptor.

59. The assay according to claim 57, characterized in that the kinase receptor is a serine-threonine kinase receptor.

60. The assay according to claim 57, characterized in that it measures the activity of the phosphatase.

61. The assay according to claim 60, characterized in that the cells further comprise a phosphatase and the assay further comprises the step of exposing the eukaryotic cells to a phosphatase inhibitor prior to step (c).

62. The assay according to claim 60, characterized in that it further comprises the steps between steps (f) and (g) of exposing the adherent kinase construct to a phosphatase and then washing the second solid phase to remove the unbound phosphatase.