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WO1999050298A1 - GFRα-X, NOUVEAU RECEPTEUR DE FACTEUR NEUROTROPHIQUE DERIVE DE CELLULES GLIALES, ET SON UTILISATION - Google Patents

GFRα-X, NOUVEAU RECEPTEUR DE FACTEUR NEUROTROPHIQUE DERIVE DE CELLULES GLIALES, ET SON UTILISATION Download PDF

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WO1999050298A1
WO1999050298A1 PCT/US1999/006631 US9906631W WO9950298A1 WO 1999050298 A1 WO1999050298 A1 WO 1999050298A1 US 9906631 W US9906631 W US 9906631W WO 9950298 A1 WO9950298 A1 WO 9950298A1
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protein
gfrα
nucleic acid
seq
acid molecule
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WO1999050298A9 (fr
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Karen J. Moore
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GDNF GDNF
  • NTN neurturin
  • GDNF was first characterized as promoting survival of cultured dopaminergic neurons of the substantia nigra (Lin et al. (( 1993) Science 260(5111 ): 1130- 1132). GDNF was subsequently found to have potent effects on a wide range of additional neural populations. For example, GDNF was found to promote the survival of primary cultures of spinal motorneurons (Henderson, C.E. et al. (1994) Science 266:1062-1064) and mutated motorneurons in vivo (Li, L.X. et al. (1995) PNAS 92:9771-9775; Oppenheim, R. et al. (1995) Nature 373:344-346; Yan, Q.
  • GDNF has been observed to have pronounced effects on cultures of dissociated neurons from various chick peripheral ganglia— sympathetic, sensory, and enteroceptive (Bujbello, A. et al. (1995) Neuron 15:821-828; Ebendal, T. et al. (1995) Cell Growth & Diff. 7:1081-1086;Trupp, M. et al. (1995) J.Cell. Biol. 130:137-148). GDNF has also been shown to promote the survival and morphologic differentiation of primary cultures of Purki ⁇ je cells (Mount, H.T.J. et al.
  • GDNF GDNF-binding protein-binding protein-binding protein
  • Still other functions of GDNF include the ability to prevent degeneration and promote the phenotype of brain noradrenergic neurons in vivo (Arenas, E. et al. (1995) Neuron 15:1465-1473), to sustain axotomized basal forebrain cholinergic neurons in vivo (Williams, L.R. et al. (1996) J. Pharmacol. Exp. Ther. 277:1140-1151), and to inhibit kainic acid mediated seizures in rat (Martin, D. et al. (1995) Brain Res. 683:172-178).
  • GDNF plays a critical role as a morphogen in the developing excretory and enteric nervous systems. This role is evident in the fact that mice defective in GDNF expression display complete renal agenesis and lack of enteric neurons (Moore, M.W. et al. (1996) Nature 382:76-79; Pichel, J.G. et al. (1996) N ⁇ twre 382:73-76; Sanchez, M.P. et al. (1996) Nature 382:70- 73).
  • ⁇ T ⁇ has been characterized as promoting survival of nodose ganglia sensory neurons, dorsal root ganglia sensory neurons, and superior cervical ganglia sympathetic neurons in vitro (Kotzbauer, P.T. et al. (1996) Nature 384:467-470). ⁇ T ⁇ 's effect on other neural cell types has not yet been determined.
  • GD ⁇ F and ⁇ T ⁇ signal cells e.g., neural cells and other cell types, in many instances, via a multicomponent receptor system formed by a glycosyl- phosphatidylinositol (GPI)-linked ligand binding subunit (the " " subunit) and the tyrosine kinase receptor RET as a signaling (" ⁇ ") subunit.
  • GPI glycosyl- phosphatidylinositol
  • RET tyrosine kinase receptor RET as a signaling
  • ⁇ signaling
  • Binding of these neurotrophic factors to the subunit promotes formation of a physical complex between the ⁇ and ⁇ subunits, thereby inducing tyrosine phosphorylation of the ⁇ subunit.
  • Tyrosine phosphorylation of the ⁇ subunit results in transmission of the GD ⁇ F/ ⁇ T ⁇ signal to the interior of the cell.
  • GD ⁇ F receptor- GD ⁇ FR- ⁇
  • GFR ⁇ Nomenclature Committee GFR ⁇ Nomenclature Committee (1997) Nature 19:485
  • RET receptor tyrosine kinase Jing, S.Q. et al. (1996) Cell 85:1113- 1124; Treanor, J.J.S. et al. (1996) Nature 382:80-83).
  • TrnR-2 The second member of the receptor family, alternatively named TrnR-2, NTNR- ⁇ , RETL2, and GDNFR- ⁇ , which has been renamed GFR -2 by the GFR ⁇ Nomenclature Committee (GFR ⁇ Nomenclature Committee (1997) Nature 19:485), has been shown to bind NTN and to mediate activation of RET by both NTN and GDNF (Baloh, R.H. et al. (1997) Neuron 18:793-802; Bujbello, A. et al. (1997) Nature 387:721-724).
  • GRF ⁇ -3 A third member of the receptor family, GRF ⁇ -3, has been described at recent scientific conferences.
  • the invention is based on the discovery of nucleic acid molecules that encode a fourth member of the Glial Derived Neurotrophic Factor- Alpha Family of Receptors, hereinafter the GFRa-X cDN A, as well as the GFR ⁇ -X protein.
  • the first member of the GFRa-X subfamily was identified, as described herein, in a positional cloning process in which the mouse mahogany locus was being sequenced to identify genes involved in obesity.
  • Nucleic acid molecules encoding the GFR ⁇ -X proteins are referred to herein as GFRa-X nucleic acid molecules.
  • the GFR ⁇ -X proteins of the present invention bind to neurotrophic factors, such as GDNF and/or NTN, and mediate signals within cells expressing the GFR ⁇ -X protein.
  • the GFR ⁇ -X protein transmits a signal to the interior of the cell by activation of the RET protein tyrosine kinase signalling pathway.
  • Neurotrophic factors promote survival and function of neural cells of both the central and peripheral nervous systems.
  • modulation of the activity of a molecule involved in transmitting a neurotrophic factor signal to a cell results in modulation of the neurotrophic factor initiated cell function.
  • GFR ⁇ -X function can be used to modulate neurotrophic factor action/activity and thereby treat disorders associated with such functions (or lack thereof).
  • GFR ⁇ proteins are expressed in a variety of cell lineages in the brain (for example Lateral septal neurons, Septohypothalamic neurons, paraventricular thalamic neurons (anterior), superchiasmatic neurons, anterior cortical amygdaloid neurons, piriform cortex, paracentral thalamic neurons, lateral habenular neurons, paraventricular hypothalamic neurons (PVN), amygdaloid nucleus area, arcuate neurons, and ventromedial hypothalamic neurons (VMH)) and during embryogenesis, including, for example, cells of the midbrain, motorneurons, cells of the enteric nervous system, embryonic smooth and striated muscles around the enteric nervous system in the esophagus, gut and stomach, developing nephrons and cells of the pancreatic primordium.
  • modulators of GFR ⁇ -X can be used to modulate neuro
  • one aspect of the invention provides isolated nucleic acid molecules (e.g., cDNAs) comprising a nucleotide sequence encoding a GFR ⁇ -X protein or a fragment thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of GFR ⁇ -X-encoding nucleic acid (e.g., mRNA).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule which encodes the amino acid sequence of SEQ ID NO:2, such as the nucleotide sequence of SEQ ID NO: 1.
  • the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1.
  • the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to a protein comprising an amino acid sequence of SEQ ID NO:2, such that the protein or portion thereof maintains a GFR ⁇ -X activity.
  • the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to bind to a neurotrophic factor and modulate a cellular response.
  • the protein encoded by the nucleic acid molecule is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2.
  • the protein is a full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2, such the naturally occurring full length protein, and all allelic variants and splice variants of human and murine GFR ⁇ -X.
  • the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1.
  • the isolated nucleic acid molecule corresponds to a naturally occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes naturally-occurring alleles and splice variants of human GFR ⁇ -X.
  • antisense nucleic acid molecules i.e., molecules which are complementary to the coding strand of the GFR ⁇ -X cDNA sequence
  • vectors e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention and host cells into which such vectors have been introduced.
  • a host cell is used to produce GFR ⁇ -X protein by culturing the host cell in a suitable medium. If desired, the GFR ⁇ -X protein can then be isolated from the host cell.
  • Yet another aspect of the invention pertains to transgenic non-human animals in which a GFRa-X gene has been introduced or altered.
  • the genome of the non-human animal has been altered by introduction of a nucleic acid molecule of the invention encoding GFR ⁇ -X as a transgene.
  • an endogenous GFRa-X gene within the genome of the non-human animal has been altered, e.g., functionally disrupted, by homologous recombination.
  • Still another aspect of the invention pertains to an isolated GFR ⁇ -X protein or a portion, e.g., a biologically active portion, thereof.
  • the isolated GFR ⁇ -X protein or portion thereof can bind a neurotrophic factor and stimulate a response in a neurotrophic factor responsive cell.
  • the invention also provides an isolated preparation of a GFR ⁇ -X protein.
  • the GFR ⁇ -X protein comprises the amino acid sequence of SEQ ID NO:2.
  • the invention pertains to an isolated full length protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2 (containing additional 5' sequence).
  • the protein is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the entire amino acid sequence of SEQ ID NO:2.
  • the isolated GFR ⁇ -X protein comprises an amino acid sequence which is at least about 60-70% or more homologous to the amino acid sequence of SEQ ID NO: 2 and has one or more of the following activities: 1) it can interact with (e.g., bind to) a neurotrophic factor, e.g., GDNF and/or NTN; 2) it can interact with (e.g., bind to) a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET; 3) it can modulate the activity of a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET; and 4) it can bind a neurotrophic factor and modulate a response in a neurotrophic factor responsive cell, e.g., a neural cell, a cell of the developing digestive tract, or a cell of its associated nervous system innervation, to, for example, beneficially affect the following activities: 1)
  • the isolated GFR ⁇ -X protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence of SEQ ID NO: 1. It is also preferred that the preferred forms of GFR ⁇ -X also have one or more of the GFR ⁇ -X activities described herein.
  • the GFR ⁇ -X protein (or polypeptide) or a biologically active portion thereof can be operatively linked to a non-GFR ⁇ -X polypeptide to form a fusion protein.
  • the GFR ⁇ -X protein of the invention, or portions or fragments thereof, can be used to prepare anti-GFR ⁇ -X antibodies.
  • the invention also provides an antigenic peptide of GFR ⁇ -X which comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of GFR ⁇ -X such that an antibody raised against the peptide forms a specific immune complex with GFR ⁇ -X.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues and has a high antigenicity index as shown in Figure 2.
  • the invention further provides an antibody that specifically binds GFR ⁇ -X.
  • the antibody is monoclonal.
  • the antibody is coupled to a detectable substance.
  • the antibody is incorporated into a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable carrier.
  • Another aspect of the invention pertains to methods for modulating a GFR ⁇ -X mediated cell activity, e.g., function, proliferation or differentiation.
  • Such methods include contacting the cell with an agent which modulates a GFR ⁇ -X protein activity or GFRa-X nucleic acid expression such that a cell associated activity is altered relative to a cell associated activity (e.g., the same cell associated activity) of the cell in the absence of the agent.
  • the cell is capable of responding to a neurotrophic factor through a signaling pathway involving a GFR ⁇ -X protein.
  • the agent which modulates GFR ⁇ -X activity can be an agonist agent, an agent which stimulates GFR ⁇ -X protein activity or GFRa-X nucleic acid expression; or an antagonist agent, an agent which inhibits GFR ⁇ -X protein activity or GFRa-X nucleic acid expression.
  • agents which stimulate GFR ⁇ -X protein activity or GFRa-X nucleic acid expression include small molecules and nucleic acids encoding GFR ⁇ -X that have been introduced into the cell.
  • agents which inhibit GFR ⁇ -X activity or expression include small molecules, antisense GFRa-X nucleic acid molecules, and antibodies that specifically bind to GFR ⁇ -X.
  • the cell is present within a subject and the agent is administered to the subject.
  • the present invention also pertains to methods for treating subjects having disorders mediated by abnormal GFR ⁇ -X activity/expression.
  • the invention pertains to methods for treating a subject having a disorder characterized by aberrant GFR ⁇ -X protein activity or nucleic acid expression such as a neurological disorder, e.g., a central nervous system disorder, e.g., Parkinson's disease, or a disorder associated with abnormal or aberrant cell, e.g., neural cell, development.
  • a neurological disorder e.g., a central nervous system disorder, e.g., Parkinson's disease
  • a disorder associated with abnormal or aberrant cell e.g., neural cell, development.
  • GFR ⁇ -X modulator e.g., a small molecule
  • the invention pertains to methods for treating a subject having a neurological disorder, e.g., a central nervous system disorder, e.g., Parkinson's disease, or a disorder associated with abnormal or aberrant cell, e.g., neural cell, development, comprising administering to the subject a GFR ⁇ -X modulator such that treatment occurs.
  • a neurological disorder e.g., a central nervous system disorder, e.g., Parkinson's disease
  • a disorder associated with abnormal or aberrant cell e.g., neural cell, development
  • the invention pertains to methods for treating a subject having a neurological disorder, e.g., a central nervous system disorder, e.g., Parkinson's disease or a disorder associated with abnormal or aberrant cell, e.g., neural cell, development, comprising administering to the subject a GFR ⁇ -X protein or portion thereof such that treatment occurs.
  • a neurological disorder e.g., a central nervous system disorder, e.g., Parkinson's disease or a disorder associated with abnormal or aberrant cell, e.g., neural cell, development
  • Neurological disorders and disorders associated with abnormal or aberrant cell, e.g., neural cell, development can also be treated according to the invention by administering to the subject having the disorder a nucleic acid encoding a GFR ⁇ -X protein or portion thereof such that treatment occurs.
  • the invention also pertains to methods for detecting genetic mutations in a GFR a-X gene, thereby determining if a subject with the mutated gene is at risk for (or is predisposed to have) a disorder characterized by aberrant or abnormal GFRa-X nucleic acid expression or GFR ⁇ -X protein activity, e.g., a central nervous system disorder, e.g., Parkinson's disease or a disorder associated with abnormal or aberrant cell, e.g., neural cell, development.
  • a central nervous system disorder e.g., Parkinson's disease
  • a disorder associated with abnormal or aberrant cell e.g., neural cell, development.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic mutation characterized by an alteration affecting the integrity of a gene encoding a GFR ⁇ -X protein, or the misexpression of the GFRa-X gene.
  • Another aspect of the invention pertains to methods for detecting the presence of
  • the methods involve contacting a biological sample (e.g., a neural cell sample) with a compound or an agent capable of detecting GFR ⁇ -X protein or GFR ⁇ -X encoding mRNA such that the presence of GFR ⁇ -X is detected in the biological sample.
  • a biological sample e.g., a neural cell sample
  • the compound or agent can be, for example, a labeled or labelable nucleic acid probe capable of hybridizing to GFR ⁇ -X encoding mRNA or a labeled or labelable antibody capable of binding to GFR ⁇ -X protein.
  • the invention further provides methods for diagnosis of a subject with, for example, a central nervous system disorder, e.g., Parkinson's disease, or a disorder associated with abnormal or aberrant cell, e.g., neural cell, development, based on detection of GFR ⁇ -X protein or mRNA.
  • a central nervous system disorder e.g., Parkinson's disease
  • a disorder associated with abnormal or aberrant cell e.g., neural cell, development, based on detection of GFR ⁇ -X protein or mRNA.
  • the method involves contacting a cell, tissue, or fluid sample (e.g., a neural cell sample) from the subject with an agent capable of detecting GFR ⁇ -X protein or mRNA, determining the amount of GFR ⁇ -X protein or mRNA expressed in the sample, comparing the amount of GFR ⁇ -X protein or mRNA expressed in the sample to a control sample and forming a diagnosis based on the amount of GFR ⁇ -X protein or mRNA expressed in the sample as compared to the control sample.
  • the sample is a neural cell sample. Kits for detecting GFR ⁇ -X, or fragments thereof, in a biological sample are also within the scope of the invention.
  • Still another aspect of the invention pertains to methods, e.g., screening assays, for identifying a compound for treating a disorder characterized by aberrant GFRa-X nucleic acid expression or protein activity, e.g., a central nervous system disorder, e.g., Parkinson's disease or a disorder associated with abnormal or aberrant cell, i.e., neural cell, development.
  • These methods typically include assaying the ability of the compound or agent to modulate the expression of the GFRa-X gene or the activity of the GFR ⁇ -X protein thereby identifying a compound for treating a disorder characterized by aberrant GFRa-X nucleic acid expression or protein activity.
  • the method involves contacting a biological sample obtained from a subject having the disorder with the compound or agent, determining the amount of GFR ⁇ -X protein expressed and/or measuring the activity of the GFR ⁇ -X protein in the biological sample, comparing the amount of GFR ⁇ -X protein expressed in the biological sample and/or the measurable GFR ⁇ -X biological activity in the cell to that of a control sample.
  • An alteration in the amount of GFR ⁇ -X protein expression or GFR ⁇ -X activity in the cell exposed to the compound or agent in comparison to the control is indicative of a modulation of GFR ⁇ -X expression and/or GFR ⁇ -X activity.
  • the invention also pertains to methods for identifying a compound or agent which interacts with (e.g., binds to) a GFR ⁇ -X protein.
  • These methods can include the steps of contacting the GFR ⁇ -X protein, a fragment thereof, or a cell expressing GFR ⁇ - X, with the compound or agent under conditions which allow binding of the compound to the GFR ⁇ -X protein to form a complex and detecting the formation of a complex of the GFR ⁇ -X protein and the compound in which the ability of the compound to bind to the GFR ⁇ -X protein is indicated by the presence of the compound in the complex.
  • the invention further pertains to methods for identifying a compound or agent which modulates, e.g., stimulates or inhibits, the interaction of the GFR ⁇ -X protein with a target molecule, e.g., GDNF, NTN, a complex of GDNF and NTN, or the tyrosine kinase receptor RET.
  • a target molecule e.g., GDNF, NTN, a complex of GDNF and NTN, or the tyrosine kinase receptor RET.
  • the GFR ⁇ -X protein is contacted, in the presence of the compound or agent, with the target molecule under conditions which allow binding of the target molecule to the GFR ⁇ -X protein to form a complex.
  • An alteration, e.g., an increase or decrease, in complex formation between the GFR ⁇ -X protein and the target molecule as compared to the amount of complex formed in the absence of the compound or agent is indicative of the ability of the compound or agent to modulate the interaction of the GFR ⁇ -X protein with a target molecule.
  • Figure 2 depicts a structural analysis of the mouse GFR ⁇ -X protein.
  • Figure 3 provides an alignment of the amino acid sequence of members of the GRF ⁇ family of protein.
  • a neurotrophic factor refers to a protein that modulates a biological activity of a cell, particularly a neuronal cell, through a neurotrophic factor signaling pathway, such as the RET signaling pathway.
  • biological activities include, but are not limited to, neural cell survival and/or neural cell function.
  • neurotrophic factors include GDNF and NTN.
  • RET signaling pathway includes a cell, e.g., neural cell, signaling pathway which involves the tyrosine kinase receptor RET.
  • An example of such a pathway includes the GDNF or NTN (neurotrophic factor) signaling pathway.
  • a neurotrophic factor responsive cell includes a cell which has a biological activity that can be modulated (e.g., stimulated or inhibited) by a neurotrophic factor. Examples of such functions include mobilization of intracellular molecules which participate in a signal transduction pathway, production or secretion of molecules, alteration in the structure of a cellular component, cell proliferation, cell migration, cell differentiation, and cell survival.
  • Cells responsive to neurotrophic factors preferably express a neurotrophic factor receptor, e.g., a GFR ⁇ receptor, such as GFR ⁇ - X, and/or a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET.
  • neurotrophic factor responsive cells include neural cells, e.g., cells of the central nervous system and peripheral nervous system cells (e.g., sympathetic and parasympathetic neurons), cells of the enteric nervous system, embryonic smooth and striated muscles around the enteric nervous system in the esophagus, gut and stomach, developing nephrons, and cells of the pancreatic primordium.
  • neural cells e.g., cells of the central nervous system and peripheral nervous system cells (e.g., sympathetic and parasympathetic neurons), cells of the enteric nervous system, embryonic smooth and striated muscles around the enteric nervous system in the esophagus, gut and stomach, developing nephrons, and cells of the pancreatic primordium.
  • neurotrophic factors regulate neural survival and neural function.
  • Abnormal or aberrant activity of proteins involved in the neurotrophic signaling pathway can lead to a variety of neurological disorders, e.g., central nervous system disorders.
  • neurological disorders e.g., central nervous system disorders.
  • abnormal or aberrant activity of GFR ⁇ -X in a neurotrophic factor signaling pathway in the thalamus e.g., the reticular thalamic nucleus, the zona certa, the anteromedial and dorsal thalamic nucleus, the lateral habenular nucleus, and the medial habenular nucleus
  • Sensory disorders are disorders which detrimentally affect normal sensory function. Examples of such sensory disorders include Dejerine-Roussy syndrome, contralateral anesthesia, and dense hypesthesia.
  • Abnormal or aberrant activity in cells of the peripheral or enteric nervous system can lead to eating disorders.
  • Abnormal or aberrant activity of a GFR ⁇ -X (or abnormal or aberrant nucleic acid expression of the nucleic acid encoding the protein)in a neurotrophic signaling factor pathway in the midbrain or mesencephalon e.g., the substantia nigra compacta and scattered cells of SN reticulata, the ventral segmental area, the interpenduncular nucleus, the supramammilary nucleus, the red nucleus, and the dorsal raphe nucleus
  • Motor disorders are disorders which detrimentally affect normal motor functions. Examples of such motor disorders include ataxia, facial infarction, tremors, tics, athetosis, amyotrophic lateral sclerosis (ALS), and Parkinson's disease.
  • Abnormal or aberrant activity of GFR ⁇ -X in a neurotrophic factor signaling pathway in the cerebellum can also lead to motor disorders. Examples of such motor disorders include loss of equilibrium and multiple sclerosis.
  • Abnormal or aberrant activity of GFR ⁇ -X in a neurotrophic factor signaling pathway in the pons can lead to motor disorders.
  • a neurotrophic factor signaling pathway in the pons e.g., the pontine reticular nucleus, the pontine nucleus, the motor trigeminal nucleus, the inferior olive nuclei, the locus coeruleus, the dorsal cochlear nucleus, the facial nucleus, the vestibular nucleus, and the hypoglossal nucleus
  • motor disorders include facial palsy, and limb ataxia.
  • Abnormal or aberrant activity of GFR ⁇ -X in a neurotrophic factor signaling pathway in the olfactory system can lead to sensory disorders.
  • Sensory disorders are disorders which detrimentally affect normal sensory function.
  • An example of such a sensory disorder includes the loss of olfaction functionality.
  • neocortex also known as the neopallium or isocortex
  • hippocampus a neurotrophic factor in a signaling pathway in the neocortex
  • cognitive disorders are disorders which detrimentally affect normal cognitive functions.
  • An example of such a cognitive disorder is Alzheimer's disease.
  • Abnormal or aberrant activity of GFR ⁇ -X in a neurotrophic factor signaling pathway in the amygdala can lead to motor disorders.
  • motor disorders include athetosis, dystoia, and tremors.
  • neurotrophic factors such as GDNF and NTN and the interaction with GFRs also promote proper development of a variety of cell types.
  • neurotrophic factor/receptor interactions promote development and function of certain peripheral organs and cells of their associated nervous system innervation.
  • Abnormal or aberrant activity of GFR ⁇ -X in a neurotrophic factor signaling pathway in these peripheral organs can lead to disorders associated with cellular development of cells of these organs.
  • An example of a disorder associated with development of the enteric nervous system is Hirschsprung's disease. Examples of disorders associated with development of the kidneys include kidney dysfunction, renal agenesis, and severe dysgenesis.
  • a murine GFRa-X nucleic acid molecule was identified from a positional cloning process in which the mouse mahogany locus was being sequenced to identify genes involved in obesity (described in detail in Example 1). During sequencing of a larger genomic region, an open reading frame was identified that encoded a protein that showed sequence homology to GFR ⁇ -1. Probes were generated based on portions of the genomic sequence and cDNA libraries were screened. Nucleotide sequences were determined and assembled and various methods such as RACE and genomic sequence analysis were used to extend the 5' sequence. The nucleotide sequence of the isolated mouse GFRa-X cDN A and the predicted amino acid sequence of the mouse GFR ⁇ -X protein are shown in Figure 1 and in SEQ ID NOs:l and 2, respectively. A plasmid containing the nucleotide sequence encoding mouse GFR ⁇ -X was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas,
  • the murine GFRa-X nucleic acid molecule is approximately 1019 nucleotides in length, and encodes a protein that is approximately 339 amino acid residues in length. This clone is likely to be missing several nucleotides (and amino acids) that are present at the 5' end of the naturally occurring cDNA.
  • the GFR ⁇ -X protein is expressed at least in brain cells, particularly in Lateral septal neurons, Septohypothalamic neurons, paraventricular thalamic neurons (anterior), superchiasmatic neurons, anterior cortical amygdaloid neurons, piriform cortex, paracentral thalamic neurons, lateral habenular neurons, paraventricular hypothalamic neurons (PVN), amygdaloid nucleus area, arcuate neurons, and ventromedial hypothalamic neurons (VMH).
  • nucleic acid molecules that encode GFR ⁇ -X proteins, particularly human or murine orthologues, biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify GFR ⁇ -X-encoding nucleic acid molecules (e.g., GFR ⁇ -X encoding mRNA).
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated GFRa-X nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g., a neural cell).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a homologue or orthologue or human or murine GFRa-X D K can be isolated from a cDNA library, such as a brain library, using all or portion of SEQ ID NO:l as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1.
  • mRNA can be isolated from neural cells (e.g., by the guanidinium- thiocyanate extraction procedure of Chirgwin et al.
  • cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Louis, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequences shown in SEQ ID NO:l.
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to a GFRa-X nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1.
  • the sequence of SEQ ID NO:l corresponds to the mouse GFRa-X cDNA.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l or a portion of this nucleotide sequence.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:l, thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence shown in SEQ ID NO:l.
  • an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to the nucleotide sequence shown in SEQ ID NO:l or a portion of either of these nucleotide sequences.
  • such nucleic acid molecules encode naturally occurring allelic variants of the mouse GFRa-X nucleic acid molecules disclosed herein or non-mouse orthologues, such as human GFR ⁇ -X.
  • the nucleic acid molecule of the invention can comprise only a portion of the coding region of SEQ ID NO:l, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of GFR ⁇ -X such as a ligand binding domain or signaling partner binding site of GFR ⁇ -X.
  • the nucleotide sequence determined from the cloning of the GFRa-X gene from a mouse allows for the generation of probes and primers designed for use in identifying and/or cloning GFRa-X homologues in other cell types, e.g., from other tissues, as well as GFRa-X orthologues from other mammals such as humans.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of SEQ ID NO:l sense, an anti-sense sequence of SEQ ID NO:l, or naturally occurring mutants thereof.
  • Primers based on the nucleotide sequence in SEQ ID NO:l can be used in PCR reactions to clone GFR ⁇ -X homologues. Probes based on the GFR ⁇ -X nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a GFR ⁇ -X protein, such as by measuring a level of a GFR ⁇ -X-encoding nucleic acid in a sample of cells from a subject, e.g., detecting GFR ⁇ -X encoding mRNA levels or determining whether a genomic GFRa-X gene has been mutated or deleted.
  • the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof maintains one or more of the activities possessed by GFR ⁇ -X.
  • homologous proteins include, but are not limited to, allelic variants of SEQ ID NO:2 and non-mouse orthologues (such as human GFR ⁇ -X) of SEQ ID NO:2.
  • the language "sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in SEQ ID NO:2) amino acid residues to an amino acid sequence of SEQ ID NO:2 such that the protein or portion thereof is able to bind a neurotrophic and modulate a response in a neurotrophic factor responsive cell.
  • the protein is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the entire amino acid sequence of SEQ ID NO:2.
  • portions of proteins encoded by the GFRa-X nucleic acid molecule of the invention preferably possess one or more activities of the GFR ⁇ -X protein.
  • biologically active portion of GFR ⁇ -X is intended to include a portion, e.g., a domain/motif, of GFR ⁇ -X that has one or more of the following activities: 1) it can interact with (e.g., bind to) a neurotrophic factor, e.g., GDNF and/or NTN; 2) it can interact with (e.g., bind to) a tyrosine kinase receptor or other signaling partner, e.g., the tyrosine kinase receptor RET; 3) it can modulate the activity of a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET; and 4) it can bind a neurotrophic factor and modulate a response in a neurotroph
  • Direct binding assays as described herein can be performed to determine the ability of a GFR ⁇ -X protein or biologically active portion thereof to interact with (e.g., bind to) a neurotrophic factor (e.g., GDNF and/or NTN or a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET).
  • a neurotrophic factor e.g., GDNF and/or NTN or a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET.
  • GFR ⁇ -X proteins of the present invention can be determined using the following assay. Plasmids including a nucleic acid molecule which encodes a fragment of the mouse GFR ⁇ -X protein either alone or as a chimeric fusion protein with, for example, an Ig constant region can be generated, as described in Sanicola et al. (1991) Proc. Natl Acad. Sci. 94:6238-6243, by ligating a DNA fragment encoding the GFR ⁇ -X fragment to suitable vector sequences. The plasmids can be transfected into 293-EBNA cells and stable lines obtained by using hygromycin selection. The GFR ⁇ -X fragment or fusion proteins can be purified and then exposed to rhGDNF (Promega, Madison, WI). Complexes of GFR ⁇ -X and rhGDNF can then be identified.
  • rhGDNF Promega, Madison, WI
  • GFR ⁇ -X protein or a biologically active portion thereof to interact with (e.g., bind to) a tyrosine kinase receptor can be determined using an assay similar to the assay described above for determining the ability of a GFR ⁇ -X protein or biologically active portion thereof to interact with (e.g., bind to) a neurotrophic factor (e.g., GDNF and/or NTN).
  • a neurotrophic factor e.g., GDNF and/or NTN
  • the mouse GFR ⁇ -X protein as described above can be exposed to proteins known to complex with members of the GFR ⁇ -X family of receptors. Complexes can be identified and detected using art known methods.
  • a fragment of a GFR ⁇ -X protein of the present invention to modulate the activity of a tyrosine kinase receptor e.g., the tyrosine kinase receptor RET
  • a tyrosine kinase receptor e.g., the tyrosine kinase receptor RET
  • RET tyrosine kinase receptor
  • Neuro-2a and SK-N-SH cells can be treated with PIPLC, and the response of RET to GDNF can be examined.
  • a change in tyrosine kinase RET phosphorylation in the cell lines treated with GDNF in combination with soluble GFR ⁇ -X compared to cell lines treated with GDNF alone indicates that the GFR ⁇ -X protein is capable of modulating the activity of a tyrosine kinase receptor, e.g., the tyrosine kinase receptor RET.
  • the biologically active portion of GFR ⁇ -X comprises the N- terminal domain of the GFR ⁇ -X protein.
  • Figure 2 provides a structural analysis of the mouse GFR ⁇ -X protein. Additional domains can e identified by analyzing conserved residue in the GFR ⁇ family of proteins ( Figure 3). Additional nucleic acid fragments encoding biologically active portions of GFR ⁇ -X can be prepared by isolating a portion of SEQ ID NO:l, expressing the encoded portion of GFR ⁇ -X protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of GFR ⁇ -X protein or peptide.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l (and portions thereof) due to degeneracy of the genetic code and thus encode the same GFR ⁇ -X protein as that encoded by the nucleotide sequence shown in SEQ ID NO: 1.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein comprising an amino acid sequence shown in SEQ ID NO:2.
  • GFR ⁇ -X nucleotide sequence shown in SEQ ID NO:l
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of GFR ⁇ -X may exist within a population.
  • Such genetic polymorphism in the GFRa-X gene may exist among individuals within a population due to natural allelic variation producing both active variants and inactive variants.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a GFR ⁇ -X protein, preferably a mammalian GFR ⁇ -X protein.
  • Such active natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the GFRa-X gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in GFR ⁇ -X that are the result of natural allelic variation are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding GFR ⁇ -X proteins from other species, and thus which have a nucleotide sequence which differs from the mouse sequence of SEQ ID NO:l, are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and human homologues of the mouse GFRa-X cDNA of the invention can be isolated based on their homology to the mouse GFRa-X nucleic acid disclosed herein using the mouse cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non- limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1 % SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein) and is encoded by a common genetic locus.
  • the nucleic acid encodes a natural human GFR ⁇ -X.
  • allelic variants of the GFR ⁇ -X sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by directed or random mutation into the nucleotide sequence of SEQ ID NO:l, thereby leading to changes in the amino acid sequence of the encoded GFR ⁇ -X protein, without altering the functional ability of the GFR ⁇ -X protein.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of GFR ⁇ -X (e.g., the sequence of SEQ ID NO:2) without altering the activity of GFR ⁇ -X, whereas an "essential" amino acid residue is required for GFR ⁇ -X activity.
  • conserved amino acid residues e.g., hydrophobic amino acids, in the N-terminal domain of GFR ⁇ -X are most likely important for binding to a neurotrophic factor and are thus essential residues of GFR ⁇ -X.
  • amino acid residues may not be essential for activity and thus are likely to be amenable to alteration without altering GFR ⁇ -X activity.
  • nucleic acid molecules encoding GFR ⁇ -X proteins that contain changes in amino acid residues that are not essential for GFR ⁇ -X activity.
  • GFR ⁇ -X proteins differ in amino acid sequence from SEQ ID NO:2 yet retain at least one of the GFR ⁇ -X activities described herein.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2 and is capable of binding a neurotrophic factor and modulating a response in a neurotrophic factor responsive cell.
  • the protein encoded by the nucleic acid molecule is at least about 70% homologous to SEQ ID NO:2, more preferably at least about 80-85% homologous to SEQ ID NO:2, even more preferably at least about 90% homologous to SEQ ID NO:2, and most preferably at least about 95-99% homologous to SEQ ID NO:2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the GFR ⁇ -X amino acid sequence of SEQ ID NO:2 having 177 amino acid residues, at least 80, preferably at least 100, more preferably at least 120, even more preferably at least 140, and even more preferably at least 150, 160 or 170 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent- identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J Mol. Biol.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • An isolated nucleic acid molecule encoding a GFR ⁇ -X protein homologous to the protein of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:l by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in GFR ⁇ -X is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a GFR ⁇ -X coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for a GFR ⁇ -X activity described herein to identify mutants that retain GFR ⁇ -X activity.
  • the encoded protein can be expressed recombinantly (e.g., as described in Examples 4 and 5) and the activity of the protein can be determined using, for example, assays described herein.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense-nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire GFR ⁇ -X coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding GFR ⁇ -X.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of GFR ⁇ -X encoding mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of GFR ⁇ -X encoding mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GFR ⁇ -X encoding mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a GFR ⁇ -X protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementary to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of an antisense nucleic acid molecule of the invention includes direct injection at a tissue site.
  • an antisense nucleic acid molecule can be modified to target selected cells and then administered systemically.
  • an antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen.
  • the antisense nucleic acid molecule can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also- comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analog (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) N ⁇ twre 334:585-591)) can be used to catalytically cleave GFR ⁇ -X encoding mR ⁇ A transcripts to thereby inhibit translation of GFR ⁇ -X encoding mR ⁇ A.
  • a ribozyme having specificity for a GFR ⁇ -X-encoding nucleic acid can be designed based upon the nucleotide sequence of a GFRa-X cDN A disclosed herein (i.e., SEQ ID ⁇ O:l).
  • a derivative of a Tetr ⁇ hymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a GFR ⁇ -X-encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071 and Cech et al. U.S. Patent No.
  • GFR ⁇ -X encoding mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., BarteL D. and Szostak, J.W. (1993) Science 261 :1411-1418.
  • GFRa-X gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the GFRa-X gene (e.g., the GFR ⁇ - X promoter and/or enhancers) to form triple helical structures that prevent transcription of the GFRa-X gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the GFRa-X gene e.g., the GFR ⁇ - X promoter and/or enhancers
  • vectors preferably expression vectors, containing a nucleic acid encoding GFR ⁇ -X (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GFR ⁇ -X proteins, mutant forms of GFR ⁇ -X, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of GFR ⁇ -X in prokaryotic or eukaryotic cells.
  • GFR ⁇ -X can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • the coding sequence of the GFR ⁇ -X is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N- terminus to the C-terminus, GST-thrombin cleavage site-GFR ⁇ -X.
  • the fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant GFR ⁇ -X unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the GFR ⁇ -X expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • GFR ⁇ -X can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ.
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to GFR ⁇ -X encoding mRNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • GFR ⁇ -X protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation or transfection techniques As used herein, the terms
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding GFR ⁇ -X or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) GFR ⁇ -X protein.
  • the invention further provides methods for producing GFR ⁇ -X protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding GFR ⁇ -X has been introduced) in a suitable medium until GFR ⁇ -X is produced.
  • the method further comprises isolating GFR ⁇ -X from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • the non-human transgenic animals can be used in screening assays designed to identify agents or compounds, e.g., drugs, pharmaceuticals, etc., which are capable of ameliorating detrimental symptoms of selected disorders such as neurological disorders and morphological disorders.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GFR ⁇ -X-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous GFR ⁇ -X sequences have been introduced into their genome or homologous recombinant animals in which endogenous GFR ⁇ -X sequences have been altered.
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • rodent such as a rat or mouse
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non- human animal, preferably a mammal, more preferably a mouse, in which an endogenous GFRa-X gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing GFR ⁇ -X- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the human homologue of the mouse GFRa-X nucleic acid molecule of SEQ ID NO:l can be isolated based on hybridization to the mouse GFRa-X cDNA (described further in subsection I above) and used as a transgene, e.g., introduced as a transgene into the genome of a non-human animal.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the GFR ⁇ -X transgene to direct expression of GFR ⁇ -X protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of the GFR ⁇ -X transgene in its genome and/or expression of GFR ⁇ -X encoding mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding GFR ⁇ -X can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a GFRa-X gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GFRa-X gene.
  • the GFR a-Xgene can be a human gene (e.g., from a human genomic clone isolated from a human genomic library screened with the cDNA of SEQ ID NO:l), but more preferably, is a non-human homologue of a human GFRa-X gene.
  • the mouse GFRa- gene can be used to construct a homologous recombination vector suitable for altering an endogenous GFRa-X gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous GFRa-X gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous GFRa-X gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GFR ⁇ -X protein).
  • the altered portion of the GFRa-X gene is flanked at its 5' and 3' ends by additional nucleic acid of the GFRa-X gene to allow for homologous recombination to occur between the exogenous GFRa-X gene carried by the vector and an endogenous GFRa-X gene in an embryonic stem cell.
  • flanking GFRa-X nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GFRa-X gene has homologously recombined with the endogenous GFRa-X gene are selected (see e.g., Li, E. et al.
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI.
  • cre/loxP recombinase system of bacteriophage PI.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • Another aspect of the invention pertains to isolated GFR ⁇ -X proteins, and biologically active portions thereof, as well as peptide fragments suitable for use as immunogens to raise anti-GFR ⁇ -X antibodies.
  • An "isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of GFR ⁇ -X protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of GFR ⁇ -X protein having less than about 30% (by dry weight) of non-GFR ⁇ -X protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-GFR ⁇ -X protein, still more preferably less than about 10% of non-GFR ⁇ -X protein, and most preferably less than about 5% non-GFR ⁇ -X protein.
  • GFR ⁇ -X protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of GFR ⁇ -X protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of GFR ⁇ -X protein having less than about 30% (by dry weight) of chemical precursors or non-GFR ⁇ -X chemicals, more preferably less than about 20% chemical precursors or non-GFR ⁇ -X chemicals, still more preferably less than about 10% chemical precursors or non-GFR ⁇ - X chemicals, and most preferably less than about 5% chemical precursors or non-GFR ⁇ - X chemicals.
  • isolated proteins or biologically active portions thereof lack contaminating proteins from the same animal from which the GFR ⁇ -X protein is derived.
  • such proteins are produced by recombinant expression of, for example, a human GFR ⁇ -X protein in a non-human cell.
  • an isolated GFR ⁇ -X protein or a portion thereof of the invention can bind a neurotrophic factor and modulate a response in a neurotrophic factor responsive cell.
  • the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of SEQ ID NO: 2 such that the protein or portion thereof maintains the ability to bind a neurotrophic factor and modulate a response in a neurotrophic factor responsive cell.
  • the portion of the protein is preferably a biologically active portion as described herein.
  • the GFR ⁇ -X protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence of the DNA SEQ ID NO:l.
  • the preferred GFR ⁇ -X proteins of the present invention also preferably possess at least one of the GFR ⁇ -X activities described herein.
  • the GFR ⁇ -X protein is substantially homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the protein of SEQ ID NO: 2 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the GFR ⁇ -X protein is a protein which comprises an amino acid sequence which is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the entire amino acid sequence of SEQ ID NO:2 and which has at least one of the GFR ⁇ -X activities described herein.
  • the invention pertains to a protein which is substantially homologous to the entire amino acid sequence of SEQ ID NO:2.
  • Biologically active portions of the GFR ⁇ -X protein include peptides comprising amino acid sequences derived from the amino acid sequence of the GFR ⁇ -X protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence of a protein homologous to the GFR ⁇ -X protein, which include less amino acids than the GFR ⁇ -X protein or the full length protein which is homologous to the GFR ⁇ -X protein, and exhibit at least one activity of the GFR ⁇ -X protein.
  • biologically active portions comprise a domain or motif, e.g., an N-terminal hydrophobic domain, with at least one activity of the GFR ⁇ -X protein.
  • the domain is an N-terminal hydrophobic domain derived from a human and is at least about 55-60%, preferably at least about 65-70%, even more preferably at least about 75-80%, and most preferably at least about 85-90% or more homologous to SEQ ID NO:2.
  • GFR ⁇ -X proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the GFR ⁇ -X protein is expressed in the host cell. The GFR ⁇ -X protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a GFR ⁇ -X protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native GFR ⁇ -X protein can be isolated from cells (e.g., neural cells), for example using an anti-GFR ⁇ -X antibody (described further below).
  • the invention also provides GFR ⁇ -X chimeric or fusion proteins.
  • a GFR ⁇ -X "chimeric protein” or “fusion protein” comprises a GFR ⁇ -X polypeptide operatively linked to a non-GFR ⁇ -X polypeptide.
  • GFR ⁇ -X polypeptide refers to a polypeptide having an amino acid sequence corresponding to GFR ⁇ -X, or a fragment thereof
  • a non-GFR ⁇ -X polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the GFR ⁇ -X protein, e.g., a protein which is different from the GFR ⁇ -X protein and which is derived from the same or a different organism.
  • the term "operatively linked" is intended to indicate that the GFR ⁇ -X polypeptide and the non-GFR ⁇ -X polypeptide are fused in-frame to each other.
  • the non-GFR ⁇ -X polypeptide can be fused to the N-terminus or C-terminus of the GFR ⁇ -X polypeptide.
  • the fusion protein is a GST- GFR ⁇ -X fusion protein in which the GFR ⁇ -X sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant GFR ⁇ -X.
  • a GFR ⁇ -X chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a GFR ⁇ -X- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GFR ⁇ -X protein.
  • the present invention also pertains to homologues of the GFR ⁇ -X proteins which function as either a GFR ⁇ -X agonist (mimetic) or a GFR ⁇ -X antagonist.
  • the GFR ⁇ -X agonists and antagonists stimulate or inhibit, respectively, a subset of the biological activities of the naturally occurring form of the GFR ⁇ -X protein.
  • specific biological effects can be elicited by treatment with a homologue of limited function.
  • treatment of a- subject with a homologue having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the GFR ⁇ -X protein.
  • Homologues of the GFR ⁇ -X protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the GFR ⁇ -X protein.
  • the term "homologue” refers to a variant form of the GFR ⁇ -X protein which acts as an agonist or antagonist of the activity of the GFR ⁇ -X protein.
  • An agonist of the GFR ⁇ -X protein can retain substantially the same, or a subset, of the biological activities of the GFR ⁇ -X protein.
  • an antagonist of the GFR ⁇ -X protein can inhibit one or more of the activities of the naturally occurring form of the GFR ⁇ -X protein, by, for example, competitively binding to a downstream or upstream member of the GFR ⁇ -X cascade which includes the GFR ⁇ -X protein.
  • the mammalian GFR ⁇ -X protein and homologues thereof of the present invention can be either positive or negative regulators of neurotrophic factor responses in cells responsive to a neurotrophic factor.
  • homologues of the GFR ⁇ -X protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the GFR ⁇ -X protein for GFR ⁇ -X protein agonist or antagonist activity.
  • a variegated library of GFR ⁇ -X variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of GFR ⁇ -X variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GFR ⁇ -X sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GFR ⁇ -X sequences therein.
  • fusion proteins e.g., for phage display
  • Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential GFR ⁇ -X sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.
  • libraries of fragments of the GFR ⁇ -X protein coding can be used to generate a variegated population of GFR ⁇ -X fragments for screening and subsequent selection of homologues of a GFR ⁇ -X protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a GFR ⁇ -X coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the GFR ⁇ -X protein.
  • Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GFR ⁇ -X homologues (Arkin and Yourvan (1992) PNAS 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
  • cell based assays can be exploited to analyze a variegated GFR ⁇ -X library.
  • a library of expression vectors can be transfected into a cell line ordinarily responsive to a particular neurotrophic factor.
  • the transfected cells are then contacted with the neurotrophic factor and the effect of the GFR ⁇ -X mutant on signaling by the neurotrophic factor can be detected, e.g., by measuring 3[H]thymidine incorporation. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of neurotrophic factor induction, and the individual clones further characterized.
  • GFR ⁇ -X protein or a portion or fragment thereof (particularly fragments comprising residues displaying high antigenicity scores, Fig. 3), can be used as an immunogen to generate antibodies that bind GFR ⁇ -X using standard techniques for polyclonal and monoclonal antibody preparation.
  • the GFR ⁇ -X protein of SEQ ID NO:2 can be used or, alternatively, the invention provides antigenic peptide fragments of GFR ⁇ -X for use as immunogens.
  • the antigenic peptide of GFR ⁇ -X comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of GFR ⁇ -X such that an antibody raised against the peptide forms a specific immune complex with GFR ⁇ -X.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of GFR ⁇ -X that are located on the surface of the protein, e.g., hydrophilic regions.
  • a GFR ⁇ -X immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed GFR ⁇ -X protein or a chemically synthesized GFR ⁇ -X peptide.
  • Prefered fragments of GFR ⁇ -X for use as an immunogen are fragments comprising high antigenicity scores shown in Figue 2 and conserved regions of high homology shown in Figure 3..
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic GFR ⁇ -X preparation induces a polyclonal anti-GFR ⁇ -X antibody response.
  • an aspect of the invention pertains to anti-GFR ⁇ -X antibodies.
  • the antibodies of the present invention will bind GFR ⁇ -X but will not bind GFR ⁇ -1 , GFR ⁇ -2, or GFR ⁇ -3.
  • the term "antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as GFR ⁇ -X.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind GFR ⁇ -X.
  • the term "monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GFR ⁇ -X.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular GFR ⁇ -X protein with which it immunoreacts.
  • Polyclonal anti-GFR ⁇ -X antibodies can be prepared as described above by immunizing a suitable subject with a GFR ⁇ -X immunogen.
  • the anti-GFR ⁇ -X antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized GFR ⁇ -X.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against GFR ⁇ -X can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) PNAS 76:2927-31 ; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds GFR ⁇ -X.
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind GFR ⁇ -X, e.g., using a standard ELISA assay.
  • a monoclonal anti-GFR ⁇ -X antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with GFR ⁇ -X to thereby isolate immunoglobulin library members that bind GFR ⁇ -X.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO
  • recombinant anti-GFR ⁇ -X antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No.
  • An anti-GFR ⁇ -X antibody (e.g., monoclonal antibody) can be used to isolate GFR ⁇ -X by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-GFR ⁇ -X antibody can facilitate the purification of natural GFR ⁇ -X from cells and of recombinantly produced GFR ⁇ -X expressed in host cells.
  • an anti-GFR ⁇ -X antibody can be used to detect GFR ⁇ -X protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the GFR ⁇ -X protein.
  • Anti-GFR ⁇ -X antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I, S or H.
  • compositions suitable for administration to a subject, e.g., a human.
  • Such compositions typically comprise the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a GFR ⁇ -X protein, fragment, or anti-GFR ⁇ -X antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a GFR ⁇ -X protein, fragment, or anti-GFR ⁇ -X antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • a GFR ⁇ -X protein of the invention has one or more of the activities described herein and can thus be used to, for example, bind a neurotrophic factor and modulate response in a neurotrophic factor responsive cell.
  • the isolated nucleic acid molecules of the invention can be used to express GFR ⁇ -X protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GFR ⁇ -X encoding mRNA (e.g., in a biological sample) or a genetic mutation in a GFRa-X gene, and to modulate GFR ⁇ -X activity, as described further below.
  • the GFR ⁇ -X proteins can be used to screen drugs or compounds which modulate GFR ⁇ -X protein activity as well as to treat disorders characterized by insufficient production of GFR ⁇ -X protein or production of GFR ⁇ -X protein forms which have decreased activity compared to wild type GFR ⁇ -X.
  • the anti-GFR ⁇ -X antibodies of the invention can be used to detect and isolate GFR ⁇ -X protein and modulate GFR ⁇ -X protein activity.
  • the invention provides methods for identifying compounds or agents that can be used to treat disorders characterized by (or associated with) aberrant or abnormal GFRa- X nucleic acid expression and/or GFR ⁇ -X protein activity. These methods are also referred to herein as drug screening assays and typically include the step of screening a candidate/test compound or agent for the ability to interact with (e.g., bind to) a GFR ⁇ - X protein, to modulate the interaction of a GFR ⁇ -X protein and a target molecule, and/or to modulate GFRa-X nucleic acid expression and/or GFR ⁇ -X protein activity.
  • Candidate/test compounds or agents which have one or more of these abilities can be used as drugs to treat disorders characterized by aberrant or abnormal GFRa-X nucleic acid expression and/or GFR ⁇ -X protein activity.
  • Candidate/test compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam, K.S. et al. (1991) Nature 354:82- 84; Houghten, R. et al.
  • the invention provides assays for screening candidate/test compounds which interact with (e.g., bind to) GFR ⁇ -X protein.
  • the assays are cell-based assays which include the steps of combining a GFR ⁇ -X protein, a biologically active portion thereof, or a cell expressing GFR ⁇ -X protein or fragment thereof, and a candidate/test compound, e.g., under conditions which allow for interaction of (e.g., binding of) the candidate/test compound to the GFR ⁇ -X protein or portion thereof to form a complex, and detecting the formation of a complex, in which the ability of the candidate compound to interact with (e.g., bind to) the GFR ⁇ -X protein or portion thereof is indicated by the presence of the candidate compound in the complex. Formation of complexes between the GFR ⁇ -X protein and the candidate compound can be quantitated, for example, using standard immunoassays.
  • the invention provides screening assays to identify candidate/test compounds which modulate (e.g., stimulate or inhibit) the interaction (and most likely GFR ⁇ -X activity as well) between a GFR ⁇ -X protein and a molecule (target molecule) with which the GFR ⁇ -X protein normally interacts.
  • target molecules includes proteins in the same signaling path as the GFR ⁇ -X protein, e.g., proteins which may function upstream (including both stimulators and inhibitors of activity) e.g., GDNF and/or NTN or downstream of the GFR ⁇ -X protein in the neurotrophic factor signaling pathway, e.g., the tyrosine kinase RET receptor.
  • the assays are cell-based assays which include the steps of combining a GFR ⁇ -X protein, a biologically active portion thereof, or a cell expressing GFR ⁇ -X protein or fragment thereof, a GFR ⁇ -X target molecule (e.g., a GFR ⁇ -X ligand) and a candidate/test compound, e.g., under conditions wherein but for the presence of the candidate compound e.g., GDNF or NTN, the GFR ⁇ -X protein or biologically active portion thereof interacts with (e.g., binds to) the target molecule, and detecting the formation of a complex which includes the GFR ⁇ -X protein and the target molecule or detecting the interaction/reaction of the GFR ⁇ -X protein and the target molecule.
  • a GFR ⁇ -X target molecule e.g., a GFR ⁇ -X ligand
  • a candidate/test compound e.g., under conditions wherein but for the presence of the candidate compound e.g.
  • Detection of complex formation can include direct quantitation of the complex by, for example, measuring inductive effects of the GFR ⁇ -X protein.
  • a statistically significant change, such as a decrease, in the interaction of the GFR ⁇ -X and target molecule (e.g., in the formation of a complex between the GFR ⁇ -X and the target molecule) in the presence of a candidate compound (relative to what is detected in the absence of the candidate compound) is indicative of a modulation (e.g., stimulation or inhibition) of the interaction between the GFR ⁇ -X protein and the target molecule.
  • Modulation of the formation of complexes between the GFR ⁇ -X protein and the target molecule can be quantitated using, for example, an immunoassay.
  • GFR ⁇ -X GFR ⁇ -X
  • its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Interaction e.g., binding of
  • GFR ⁇ -X to a target molecule, in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/ GFR ⁇ -X fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g. 35s-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated.
  • glutathione sepharose beads Sigma Chemical, St. Louis, MO
  • glutathione derivatized microtitre plates which are then combined with the cell lysates (e.g. 35s-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following in
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of GFR ⁇ -X-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • Other techniques for immobilizing proteins on matrices can also be used in the drug screening assays of the invention.
  • GFR ⁇ -X or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated GFR ⁇ -X molecules can be prepared from biotin-NHS (N-hydroxy- succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, IL
  • streptavidin-coated 96 well plates Piereptavidin-coated 96 well plates
  • antibodies reactive with GFR ⁇ -X but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and GFR ⁇ -X trapped in the wells by antibody conjugation.
  • preparations of a GFR ⁇ -X-binding protein and a candidate compound are incubated in the GFR ⁇ -X-presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the GFR ⁇ -X target molecule, or which are reactive with GFR ⁇ -X protein and compete with the target molecule; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
  • the invention provides a method for identifying a compound (e.g., a screening assay) capable of use in the treatment of a disorder characterized by (or associated with) aberrant or abnormal GFRa-X nucleic acid expression or GFR ⁇ -X protein activity.
  • This method typically includes the step of assaying the ability of the compound or agent to modulate the expression of the GFRa-X nucleic acid or the activity of the GFR ⁇ -X protein thereby identifying a compound for treating a disorder characterized by aberrant or abnormal GFRa-X nucleic acid expression or GFR ⁇ -X protein activity.
  • disorders characterized by aberrant or abnormal GFRa-X nucleic acid expression or GFR ⁇ -X protein activity are described herein.
  • Methods for assaying the ability of the compound or agent to modulate the expression of the GFRa-X nucleic acid or activity of the GFR ⁇ -X protein are typically cell-based assays.
  • cells which are sensitive to ligands, e.g., GDNF, which transduce signals via a pathway involving GFR ⁇ -X can be induced to overexpress a GFR ⁇ -X protein in the presence and absence of a candidate compound.
  • ligands e.g., GDNF
  • Candidate compounds which produce a statistically significant change in GFR ⁇ -X-dependent responses can be identified.
  • expression of the GFR a-X nucleic acid or activity of a GFR ⁇ -X protein is modulated in cells and the effects of candidate compounds on the readout of interest (such as rate of cell proliferation or differentiation) are measured.
  • the expression of genes which are up- or down-regulated in response to a GFR ⁇ -X-dependent signal cascade can be assayed.
  • the regulatory regions of such genes e.g., the 5' flanking promoter and enhancer regions, are operably linked to a detectable marker (such as luciferase) which encodes a gene product that can be readily detected.
  • Phosphorylation of GFR ⁇ -X or GFR ⁇ -X target molecules can also be measured, for example, by immunoblotting.
  • modulators of GFR ⁇ -X expression e.g., compounds which can be used to treat a disorder characterized by aberrant or abnormal GFRa-X nucleic acid expression or GFR ⁇ -X protein activity
  • a cell is contacted with a candidate compound and the expression of GFR ⁇ -X encoding mRNA or protein in the cell is determined.
  • the level of expression of GFR ⁇ -X encoding mRNA or protein in the presence of the candidate compound is compared to the level of expression of GFR ⁇ -X encoding mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of GFRa-X nucleic acid expression based on this comparison and be used to treat a disorder characterized by aberrant GFRa-X nucleic acid expression. For example, when expression of GFR ⁇ -X encoding mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GFR ⁇ -X encoding mRNA or protein expression. Alternatively, when expression of GFR ⁇ -X encoding mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GFR ⁇ -X encoding mRNA or protein expression.
  • the level of GFR ⁇ -X encoding mRNA or protein expression in the cells can be determined by methods described herein for detecting GFR ⁇ -X encoding mRNA or protein.
  • the GFR ⁇ -X proteins can be used as "bait proteins" in a two-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • GFR ⁇ -X-binding proteins or "GFR ⁇ -X-bp"
  • GFR ⁇ -X-binding proteins are also likely to be involved in the propagation of signals by the GFR ⁇ -X proteins as, for example, upstream or downstream elements of the GFR ⁇ -X pathway.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for GFR ⁇ -X is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcription regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with GFR ⁇ -X.
  • a reporter gene e.g., LacZ
  • Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with GFR ⁇ -X.
  • Modulators of GFR ⁇ -X protein activity and/or GFRa-X nucleic acid expression identified according to these drug screening assays can be to treat, for example, neurological diseases or disorders described herein.
  • These methods of treatment include the steps of administering the modulators of GFR ⁇ -X protein activity and/or nucleic acid expression, e.g., in a pharmaceutical composition as described in subsection IV above, to a subject in need of such treatment, e.g., a subject with a neurological disease.
  • the invention further provides a method for detecting the presence of GFR ⁇ -X in a biological sample.
  • the method involves contacting the biological sample with a compound or an agent capable of detecting GFR ⁇ -X protein or mRNA such that the presence of GFR ⁇ -X is detected in the biological sample.
  • a preferred agent for detecting GFR ⁇ -X encoding mRNA is a labeled or labelable nucleic acid probe capable of hybridizing to GFR ⁇ -X encoding mRNA.
  • the nucleic acid probe can be, for example, the GFRa-X cDNA of SEQ ID NO: 1 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GFR ⁇ -X encoding mRNA.
  • a preferred agent for detecting GFR ⁇ -X protein is a labeled or labelable antibody capable of binding to GFR ⁇ -X protein.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • labeled or labelable with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
  • the detection method of the invention can be used to detect GFR ⁇ -X encoding mRNA or protein in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of GFR ⁇ -X encoding mRNA include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of GFR ⁇ -X protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • GFR ⁇ -X protein can be detected in vivo in a subject by introducing into the subject a labeled anti-GFR ⁇ -X antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample is a neural cell sample.
  • the neural cell sample can comprise neural tissue or a suspension of neural cells.
  • a tissue section for example, a freeze-dried or fresh frozen section of neural tissue removed from a patient, can be used as the neural cell sample.
  • the biological sample can comprise a biological fluid (e.g., cerebrospinal fluid) obtained from a subject having a neurological disorder.
  • the biological sample is a neural cell sample (e.g., a sample which includes motomeuron cells).
  • kits for detecting the presence of GFR ⁇ -X in a biological sample can comprise a labeled or labelable compound or agent capable of detecting GFR ⁇ -X protein or mRNA in a biological sample; means for determining the amount of GFR ⁇ -X in the sample; and means for comparing the amount of GFR ⁇ -X in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect GFR ⁇ -X encoding mRNA or protein.
  • the methods of the invention can also be used to detect genetic mutations in a GFRa-X gene, or the allelic form of GFRa-X found in a subject, thereby determining if a subject with the mutated gene is at risk for a disorder characterized by aberrant or abnormal GFRa-X nucleic acid expression or GFR ⁇ -X protein activity as defined herein.
  • the methods include detecting, in a sample from the subject, the presence or absence of a genetic mutation characterized by at least one of an alteration affecting the integrity of a gene encoding a GFR ⁇ -X protein, or the misexpression of the GFRa-X gene.
  • such genetic mutations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a GEi? ⁇ r-A'gene; 2) an addition of one or more nucleotides to a GFRa- gene; 3) a substitution of one or more nucleotides of a GFRa-X gene, 4) a chromosomal rearrangement of a GFRa-X gene; 5) an alteration in the level of a messenger RNA transcript of a GFR a-X gene, 6) aberrant modification of a GFR a-X gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a GFRa-X gene, 8) a non- wild type level of a GFR ⁇ -X-protein, 9) allelic loss of a GFRa-X gene, and 10) inappropriate post-translational modification of a GFR ⁇
  • detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the sample, contacting the nucleic acid with one or more primers which specifically hybridize to a GFRa-X gene under conditions such that hybridization and amplification of the GFRa- -gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • primers which specifically hybridize to a GFRa-X gene under conditions such that hybridization and amplification of the GFRa- -gene (if present) occurs
  • detecting the presence or absence of an amplification product or detecting the size of the amplification product and comparing the length to a control sample.
  • mutations in a GFRa-X gene from a sample cell can be identified by alterations in restriction enzyme
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the GFRa-X gene and detect mutations by comparing the sequence of the sample GFRa-X with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463).
  • a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol 38:147-159).
  • RNA/RNA or RNA/DNA duplexes Other methods for detecting mutations in the GFRa-X gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al. (1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397; Saleeba et al. (1992) Meth. Enzymol 217:286-295), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al.
  • Another aspect of the invention pertains to methods for treating a subject, e.g., a human, having a disease or disorder characterized by (or associated with) aberrant or abnormal GFRa-X nucleic acid expression and/or GFR ⁇ -X protein activity. These methods include the step of administering a GFR ⁇ -X modulator to the subject such that treatment occurs.
  • the language "aberrant or abnormal GFRa-X expression” refers to expression of a non- wild-type GFR ⁇ -X protein or a non- wild-type level of expression of a GFR ⁇ -X protein.
  • Aberrant or abnormal GFR ⁇ -X activity refers to a non- wild-type GFR ⁇ -X activity or a non- wild-type level of GFR ⁇ -X activity.
  • GFR ⁇ -X protein As the GFR ⁇ -X protein is involved in a pathway involving neurological and developmental functions, aberrant or abnormal GFR ⁇ -X protein activity or nucleic acid expression interferes with normal neurological functions and/or developmental functions.
  • neurological disorders or diseases characterized by or associated with abnormal or aberrant GFR ⁇ -X protein activity or nucleic acid expression in neural cells include sensory, disorders, e.g., Dejerine-Roussy Syndrome, motor disorders, e.g., Parkinson's disease, ALS, and cognitive disorders, e.g., Alzheimer's disease.
  • disorders or diseases characterized by or associated with abnormal or aberrant GFR ⁇ -X protein activity or nucleic acid expression in cells associated with developmental function include disorders of the enteric nervous system, e.g., Hirschsprung's disease and eating disorders.
  • treating refers to reduction or alleviation of at least one adverse effect or symptom of a disorder or disease, e.g., a disorder or disease characterized by or associated with abnormal or aberrant GFR ⁇ -X protein activity or GFRa-X nucleic acid expression.
  • a GFR ⁇ -X modulator is a molecule which can modulate GFRa-
  • a GFR ⁇ -X modulator can modulate, e.g., upregulate (activate) or downregulate (suppress), GFRa-X nucleic acid expression.
  • a GFR ⁇ -X modulator can modulate (e.g., stimulate or inhibit) GFR ⁇ -X protein activity.
  • a GFR ⁇ -X modulator can be an antisense molecule, e.g., a ribozyme, as described herein.
  • antisense molecules which can be used to inhibit GFR a-X nucleic acid expression include antisense molecules which are complementary to a portion of the 5' untranslated region of the GFR ⁇ -X encoding sequence which also includes the start codon and antisense molecules which are complementary to a portion of the 3' untranslated region.
  • a GFR ⁇ -X modulator which inhibits GFRa-X nucleic acid expression can also be a small molecule or other drag, e.g., a small molecule or drag identified using the screening assays described herein, which inhibits GFRa-X nucleic acid expression.
  • a GFR ⁇ -X modulator can be, for example, a nucleic acid molecule encoding GFR ⁇ -X (e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO:l) or a small molecule or other drug, e.g., a small molecule (peptide) or drag identified using the screening assays described herein, which stimulates GFRa-X nucleic acid expression.
  • a nucleic acid molecule encoding GFR ⁇ -X e.g., a nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO:l
  • a small molecule or other drug e.g., a small molecule (peptide) or drag identified using the screening assays described herein, which stimulates GFRa-X nucleic acid expression.
  • a GFR ⁇ -X modulator can be an anti-GFR ⁇ -X antibody or a small molecule or other drag, e.g., a small molecule or drug identified using the screening assays described herein, which inhibits GFR ⁇ -X protein activity.
  • a GFR ⁇ -X modulator can be an active GFR ⁇ -X protein or portion thereof (e.g., a GFR ⁇ -X protein or portion thereof having an amino acid sequence which is homologous to the amino acid sequence of SEQ ID NO: 2 or a portion thereof) or a small molecule or other drag, e.g., a small molecule or drug identified using the screening assays described herein, which stimulates GFR ⁇ -X protein activity.
  • a subject having a neurological disorder can be treated according to the present invention by administering to the subject a GFR ⁇ -X protein, preferably a portion thereof, or a nucleic acid encoding a GFR ⁇ -X protein or portion thereof such that treatment occurs.
  • a subject having a developmental disorder can be treated according to the present invention by administering to the subject a GFR ⁇ -X protein or portion thereof or a nucleic acid encoding a GFR ⁇ -X protein or portion thereof such that treatment occurs.
  • a cell associated activity refers to a normal or abnormal activity or function of a cell. Examples of cell associated activities include proliferation, migration, differentiation, production or secretion of molecules, such as proteins, and cell survival.
  • the cell is neural cell of the CNS, e.g., motomeuron of the spinal cord.
  • the agent stimulates GFR ⁇ -X protein activity or GFRa-X nucleic acid expression.
  • stimulatory agents include an active GFR ⁇ -X protein, a nucleic acid molecule encoding GFR ⁇ -X that has been introduced into the cell, and a modulatory agent which stimulates GFR-X ⁇ protein activity or GFRa-X nucleic acid expression and which is identified using the drug screening assays described herein.
  • the agent inhibits GFR ⁇ -X protein activity or GFRa-X nucleic acid expression.
  • inhibitory agents include an antisense GFRa-X nucleic acid molecule, an anti-GFR-X ⁇ antibody, and a modulatory agent which inhibits GFR ⁇ -X protein activity or GFR a-X nucleic acid expression and which is identified using the drug screening assays described herein.
  • modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the modulatory methods are performed in vivo, i.e., the cell is present within a subject, e.g., a mammal, e.g., a human, and the subject has a disorder or disease characterized by or associated with abnormal or aberrant GFR ⁇ -X protein activity or GFRa-X nucleic acid expression.
  • a nucleic acid molecule, a protein, a GFR ⁇ -X modulator, a compound etc. used in the methods of treatment can be incorporated into an appropriate pharmaceutical composition described herein and administered to the subject through a route which allows the molecule, protein, modulator, or compound etc. to perform its intended function. Examples of routes of administration are also described herein under subsection IV.
  • Test/candidate compounds, or modulators which have a stimulatory or inhibitory effect on GFR ⁇ -X activity can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., neural disorders, e.g., central and peripheral nervous system disorders) associated with aberrant GFR ⁇ -X activity.
  • disorders e.g., neural disorders, e.g., central and peripheral nervous system disorders
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drag.
  • the pharmacogenomics of the individual permit the selection of effective compounds (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of GFR ⁇ -X polypeptide, expression of GFRa-X nucleic acid, or mutation content of GFRa-X genes in an individual can be determined to thereby select appropriate compound(s) for therapeutic or prophylactic treatment of the individual. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drag disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (1996) Clin. Exp. Pharmacol.
  • pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drags act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drags (altered drag metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymo ⁇ hisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drag action.
  • the discovery of genetic polymo ⁇ hisms of drag metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymo ⁇ hisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drag response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its C YP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses.
  • GFR ⁇ -X polypeptide activity of GFR ⁇ -X polypeptide, expression of GFRa-X nucleic acid, or mutation content of GFRa-X genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of a subject.
  • pharmacogenetic studies can be used to apply genotyping of polymo ⁇ hic alleles encoding drag-metabolizing enzymes to the identification of a subject's drag responsiveness phenotype.
  • GFR ⁇ -X e.g., the ability to modulate the effects of neurotrophic factors on neurotrophic factor responsive cells
  • the effectiveness of an agent determined by a screening assay, as described herein, to increase GFRa-X gene expression, polypeptide levels, or up-regulate GFR ⁇ -X activity can be monitored in clinical trials of subjects exhibiting decreased GFRa-X gene expression, polypeptide levels, or down-regulated GFR ⁇ -X activity.
  • the effectiveness of an agent, determined by a screening assay, to decrease GFRa-X gene expression, polypeptide levels, or down- regulate GFR ⁇ -X activity can be monitored in clinical trials of subjects exhibiting increased GFRa-X gene expression, polypeptide levels, or up-regulated GFR ⁇ -X activity.
  • the expression or activity of GFR ⁇ -X and, preferably, other genes which have been implicated in, for example, a neural disorder, e.g., a central nervous system disorder can be used as a "read out" or markers of the neurotrophic factor responsiveness of a particular cell.
  • genes including GFR ⁇ -X, which are modulated in cells by treatment with a compound (e.g., drug or small molecule) which modulates GFR ⁇ -X activity (e.g., identified in a screening assay as described herein) can be identified.
  • a compound e.g., drug or small molecule
  • GFR ⁇ -X activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of GFR ⁇ -X and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of polypeptide produced, by one of the methods described herein, or by measuring the levels of activity of GFR ⁇ -X or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the compound. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the compound.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with a compound (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the compound; (ii) detecting the level of expression of an GFR ⁇ -X polypeptide, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting the level of expression or activity of the GFR ⁇ -X polypeptide, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the GFR ⁇ -X polypeptide, mRNA, or genomic DNA in the pre-administration sample with the GFR ⁇ -X polypeptide, mRNA, or genomic DNA in the post administration sample or samples;
  • a compound
  • increased administration of the compound may be desirable to increase the expression or activity of GFR ⁇ -X to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of GFR ⁇ -X to lower levels than detected, i.e., to decrease the effectiveness of the compound.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (a) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (b) identify an individual from a minute biological sample (tissue typing); and (c) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the GFR ⁇ -X sequence, described herein, can be used to map the location of the GFRa-X gene, respectively, on a chromosome. The mapping of the GFRa-X sequence to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • the GFRa-X gene can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GFR ⁇ -X sequences.
  • GFR ⁇ -X sequence can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GFR ⁇ -X sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the GFR ⁇ -X sequence to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a GFR ⁇ -X sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) PNAS, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1 ,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GFRa-X gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymo ⁇ hisms.
  • the GFR ⁇ -X sequence of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the GFR ⁇ -X sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the GFR ⁇ -X sequence of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses.
  • the noncoding sequences of SEQ ID NO:l can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If the predicted coding sequence, such as the one in SEQ ID NO:2 is used, a more appropriate number of primers for positive individual identification would be 500- 2,000.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a pe ⁇ etrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e., another DNA sequence that is unique to a particular individual).
  • another "identification marker” i.e., another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO: 1 are particularly appropriate for this use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the GFR ⁇ -X sequence or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO: 1 having a length of at least 20 bases, preferably at least 30 bases.
  • the GFR ⁇ -X sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such GFR ⁇ -X probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., GFR ⁇ -X primers or probes can be used to screen tissue culture for contamination (i.e., screen for the presence of a mixture of different types of cells in a culture).
  • GFR ⁇ -X primers or probes can be used to screen tissue culture for contamination (i.e., screen for the presence of a mixture of different types of cells in a culture).
  • the GFR ⁇ -X mouse cDNA was identified in a positional cloning process in which the mouse mahogany locus was being sequenced.
  • a cDNA library from a human brain cell library (available from Stratagene, LaJolla, CA, or Clontech, Palo Alto, CA) is screened under low stringency conditions (e.g., as described in Sambrook, J.,
  • Lateral septal neurons Septohypothalamic neurons, paraventricular thalamic neurons (anterior), superchiasmatic neurons, anterior cortical amygdaloid neurons, piriform cortex, paracentral thalamic neurons, lateral habenular neurons, paraventricular hypothalamic neurons (PVN),
  • the pcDNA/Amp vector by Invitrogen Co ⁇ oration (San Diego, CA) is used.
  • This vector contains an S V40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire GFR ⁇ -X protein and a HA tag (Wilson et al. (1984) Cell 31:161) fused in-frame to its 3' end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.
  • the GFR ⁇ -X DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the GFR ⁇ -X coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag and the last 20 nucleotides of the GFR ⁇ -X coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the GFRa-X gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E coli cells (strains HB101, DH5a, SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.
  • COS cells are subsequently transfected with the GFR ⁇ -X-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE- dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression of the GFR ⁇ -X protein is detected by radiolabelling (3 s-methionine or 35s-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 3 s-methionine (or 35s-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated proteins are then analyzed by SDS-PAGE.
  • DNA containing the GFR ⁇ -X coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the GFR ⁇ -X protein is detected by radiolabelling and immunoprecipitation using a GFR ⁇ -X specific monoclonal antibody
  • the amino acid sequence of the GFR ⁇ -X protein was compared to amino acid sequences of known proteins and various motifs were identified.
  • the GFR ⁇ -X protein the amino acid sequence of which is shown in Figure 1 (SEQ ID NO:2), is a novel protein which includes 340 amino acid residues.
  • a comparison of GFR ⁇ -X with other members of the GFR family of proteins is provided in Figure 3.

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Abstract

Molécules isolées d'acide nucléique qui codent des nouveaux membres de la famille GRFα de protéines, désignés par GFRα-X qui est une abréviation pour des molécules d'acide nucléique de la famille de récepteurs α-X de GDNF. Cette famille de protéines se lie à des facteurs neurotrophiques et transmet des signaux impliqués dans la régulation des fonctions cellulaires neuronales. La présente invention concerne également des molécules d'acide nucléique antisens, des vecteurs d'expression contenant des molécules d'acide nucléique GFRα-X, des cellules hôtes dans lesquelles ont été introduits des vecteurs d'expression et des animaux transgéniques dans lesquels un gène GFRα-X a été introduit ou disloqué. La présente invention concerne en outre des polypeptides GFRα-X isolés, des protéines de fusion, des peptides antigéniques, ainsi que des anticorps anti-GFRα-X. Des méthodes de diagnostic, de criblage et thérapeutiques reposant sur les compositions selon la présente invention sont également décrites.
PCT/US1999/006631 1998-03-31 1999-03-25 GFRα-X, NOUVEAU RECEPTEUR DE FACTEUR NEUROTROPHIQUE DERIVE DE CELLULES GLIALES, ET SON UTILISATION Ceased WO1999050298A1 (fr)

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US7576185B2 (en) 2002-03-05 2009-08-18 Genentech, Inc. PRO34128 antibodies
US7642242B2 (en) 2002-03-05 2010-01-05 Genentech, Inc. PRO34128 polypeptides

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WO1999050298A9 (fr) 1999-11-25

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