WO2002100349A2

WO2002100349A2 - Use of the parathroid hormone-2 (pth2) receptor to screen for agents to treat pain

Info

Publication number: WO2002100349A2
Application number: PCT/US2002/018771
Authority: WO
Inventors: Ted Usdin
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 2001-06-13
Filing date: 2002-06-12
Publication date: 2002-12-19
Anticipated expiration: 2003-12-13
Also published as: WO2002100349A3; AU2002316237A1

Abstract

The invention is related to use of parathyroid hormone-2 (PTH2) receptor to screen for agents to treat pain.

Description

Use Of The Parathyroid Hormone-2 (PTH2) Receptor To Screen For Agents To Treat

Pain

1. Field of the Invention

2. Background of the Invention The parathyroid hormone 2 (PTH2) receptor was originally identified in a screen for novel G-protein coupled receptors (Usdin, T.B. et al. 1995 J Biol Chem 270:15455-15458). It is distinguished both pharmacologically and anatomically from the PTH1 (or PTH/PTHrP) receptor (Jϋppner, H. et al. 1991 Science 254:1024-1026), which mediates the calcium regulating functions of parathyroid hormone (PTH), a classic endocrine hormone, and effects of the distinct peptide parathyroid hormone-related peptide, which regulates development, remodeling, and epithelial transport in several tissues (Brown, E.M. et al. 1996 Baillieres Clin Endocrinol Metab 10:123-61; Kronenberg, H.M. et al. 1998 Recent Prog Harm Res 53:283-301; Wysolmerski, J.J. & Stewart, A. F. 1998 Annu Rev Physiol 60:431-60).

The biological roles of the PTH2 receptor are not yet established. It is present at greatest levels in the nervous system, and unlike the PTH1 receptor it is found at low density and in only a few cells in kidney and bone (Usdin, T.B. et al. 1995 J Biol Chem 270:15455-15458; Usdin, T. B. et al. 1996 Endocrinology 137:4285-4297; Usdin, T.B. et al. 1999 Endocrinology 140:3363-3371). Parathyroid hormone-related peptide has low affinity for PTH2 receptors, and does not significantly activate them (Gardella, TJ. et al. 1996 J Biol Chem 271:19888-19893). Unlike the human PTH2 receptor, which was initially characterized and lead to the receptor's name, rat and zebrafish PTH2 receptors are poorly activated by PTH (Hoare, S.RJ. et al. 1999 Endocrinology 140:4419-4425; Hoare, S.R. et al. 2000 Endocrinology 141:3080-6). We recently used selective activation of the PTH2 receptor as an assay to purify a previously undescribed peptide from bovine hypothalarnus (Usdin, T.B. et al. 1999 Nat Neuroscience 2:941-943). This peptide, tuberoinfundibular peptide of 39 residues (TIP39), has a structure and functional architecture similar to PTH and parathyroid hormone-related peptide (Piserchio, A. et al. 2000 JBiol Chem 275:27284-90), but at most nine residues in common with PTH from any species (Usdin, T.B. 2000 Trends Pharmacol Sci 21:128-30). TIP39 potently activates human, rat, and zebrafish PTH2 receptors and has no effect on PTH1 receptors (Hoare, S.R. et al. 2000 Endocrinology 141:3080-6; Usdin, T.B. et al. 1999 Nat Neuroscience 2:941- 943). It may be the physiological ligand for PTH2 receptors.

Detailed mapping of the PTH2 receptor's anatomical distribution suggests a number of possible biological functions (Usdin, T. B. et al. 1996 Endocrinology 137:4285-4297; Usdin, T.B. et al. 1999 Endocrinology 140:3363-3371; Wang, T. et al. 2000 Neuroscience 100:629-49). The receptor is expressed at high levels in the hypothalamus (Wang, T. et al. 2000 Neuroscience 100:629-49) and recent experiments support a role for it in regulation of hypothalamic function (Ward, H.L. et al. 2001 Endocrinology 142:3451-6). It is also synthesized by a population of dorsal root ganglion (DRG) cells as well as by neurons within the dorsal horn of the spinal cord (Wang, T. et al. 2000 Neuroscience 100:629-49). A PTH2 receptor selective antibody intensely labels nerve fibers and terminals in the superficial dorsal horn of the spinal cord, and the corresponding caudal part of the spinal trigeminal nucleus. These areas contain most of the central terminals of nociceptive primary afferents. TIP39 increases cAMP in F-ll cells (Usdin, T.B. et al. 1999 Nat Neuroscience 2:941-943), which are a DRG-neuroblastoma hybrid cell line that possesses some of the properties of peptidergic nociceptors (Kusano, K. & Gainer, H. 1993 J Neuroscience Res 34: 158-69). Other agents that increase cAMP in DRG neurons potentiate nociception (Taiwo, Y.O. et al. 1989 Neuroscience 32:577-80).

3.1. Segue to the Invention We have now used local administration of TLP39, and a TIP39 sequestering antibody, to explore the involvement of the PTH2 receptor and TLP39 in nociception. We also employed double-label immunohistochemistry to define the distribution of the PTH2 receptor in the spinal cord more precisely, and determined the locations of TLP39 synthesis. Our data show that TIP39 causes or potentiates nocifensive responses, that sequestering TLP39 with an antibody decreases withdrawal responses, and that TLP39 is synthesized by neurons in areas that have projections to the sensory trigeminal and spinal cord regions rich in PTH2 receptors. Thus TIP39 is envisioned as being a novel modulator of nociception. 3.2. Summary of the invention The invention is related to use of parathyroid hormone-2 (PTH2) receptor to screen for agents to treat pain.

4.1. Brief Description of the Drawings Figure 1 shows detection of PTH2 receptor mRNA in rat spinal cord and DRG by in situ hybridization, (a) Low magnification darkfield micrograph. Part of the spinal cord gray matter at the thoracic level, and an attached dorsal root ganglion (DRG) are outlined. Note hybridization in outer layers of the spinal cord dorsal horn, in scattered cells in deeper spinal cord layers, and in the DRG. (b) Higher magnification brightfield image of a DRG. Note accumulation of silver grains (black) over some smaller neurons. Gray shows cellular counterstaining. * indicates central canal. Bars: 1 mm (a), 100 μm (b). Approximately 10-15 % of DRG neurons are distinctly labeled by in situ hybridization. Antibody labeling shows a continuum of intensity and numerical estimates are difficult to make.

Figure 2 shows laminar localization of the PTH2 receptor in the dorsal horn of mouse spinal cord. PTH2 receptor immunoreactivity was detected with a green (depicted as light gray) labeled secondary antibody and calcitonin gene-related peptide (CGRP) (a, b), isolectin B4 (c, d), protein kinase C gamma (e) and substance P (f), with red (depicted as dark gray) labeled secondary antibodies. High power confocal microscopy indicates that there is virtually no co-localization between PTH2 receptor and CGRP (b) immunoreactivity or isolectin B4 labeling (d). Bars: 100 μm (a, c, e, f), 20 μm (b, d).

Figure 3 shows responses of animals to TIP39 administration, (a) Force of the paw withdrawal response immediately after intraplantar administration of various doses of TIP39. (b) Time spent performing scratching, biting and licking (SBL) behaviors over the 20 min following intrathecal administration of TIP39. Figure 4 shows modulation of nociceptive responses by intrathecal administration of an antibody to TIP39 or TIP39 itself. Tail-flick (a, d), paw pressure (b, e), and Hargreaves (radiant heat directed to a paw; c, f) assays were performed as described herein. Responses following intrathecal delivery of vehicle (Veh), antibody to TIP39 (α-TLP), or pre-immune serum (Pre Irnm) are shown at 30 min following injection, the time at which the largest effect of the antibody was observed (a-c). Responses at various times after injection of vehicle or 100 fmol of TIP39 (TBP39) are shown (d-f). Figure 5 shows PCR amplification of TIP39 cDNA from rat neuronal and peripheral tissues. The predicted size of the product amplified from cDNA product is 297 bp; from genomic DNA it is 594 bp.

Figure 6 shows brain localization of TIP39-containing cells, (a-e) caudal paralemniscal nucleus at -8.7 mm from the bregma level, (f-j) subparafascicular area at - 4.4 mm from the bregma. Drawings from the atlas of Paxinos and Watson (Andrezik, J. A. & Beitz, AJ. 1986 in: The Rat Nervous System, ed. Paxinos, G. Academic Press, Orlando, pp. 1-28) indicate the location of the micrographs (a, f). The localization of TIP39 mRNA detected by in situ hybridization histochemistry is shown at low magnification in dark-field micrographs (b, g) and at greater magnification of the framed areas in brightfield (c, h). The localization of TIP39 protein is demonstrated by peroxidase-immunocytochemistry in colchicine treated animals, shown at low magnification (d, i) and at greater magnification of the framed areas (e, j). DR, dorsal raphe nucleus; fr, fasciculus retroflexus; 11, lateral lemniscus; PH, posterior hypothalamic area; PnO, pontine reticular nucleus, oral part; PrC, precomissural nucleus, posterior; pv, periventricular fiber system; PVP, paraventricular thalamic nucleus, posterior part; py, pyramidal tract; rs, rubrospinal tract; scp: superior cerebellar peduncle; VLL, ventral nucleus of lateral lemniscus; 3V, third ventricle. Bars: 1 mm (a, b, d), 100 μm (c, e, h, j), 500 μm (g, h, i).

Figure 7 shows TIP39-immunoreactive fibers in the spinal cord, (a) a horizontal section showing TLP39-immunoreactive fibers in cranio-caudal orientation in the lateral funiculus. (b) a coronal section showing TIP39-immunoreactive fibers in lamina X and at the dorsal column-gray matter border. DH, dorsal horn; CC, central canal. Bar: 200 μm (a), 50 μm (b).

4.2. Brief Description of the Sequences SEQ ID NO: 1 - the sequence of a cDNA clone for human TIP39:

AGCCCACTGCACGGTGATGGAGACCCGCCAGGTGTCCAGGAGCCCTCGGGTTC GGCTGCTGCTGCTGCTGCTGCTGCTGCTGGTGGTGCCCTGGGGCGTCCGCACTG CCTCGGGAGTCGCCCTGCCCCCGGTCGGGGTCCTCAGCCTCCGCCCCCCAGGAC GGGCCTGGGCGGATCCCGCCACCCCCAGGCCGCGGAGGAGCCTGGCGCTGGCG GACGACGCGGCCTTCCGGGAGCGCGCGCGGTTGCTGGCCGCCCTCGAGCGCCG CCACTGGCTGAACTCGTACATGCACAAGCTGCTGGTGTTGGATGCGCCCTGAGC

GCCTGCCCGTCCCCATCTTAATAAAGACCATGCCCTGCGCTCCGG SEQ ID NO: 2 - the human precursor peptide corresponds to the second open reading frame and has the sequence:

METRQVSRSPRVRLLLLLLLLLVVPWGVRTASGVALPPVGVLSLRPPGRAWADPA TPRPPJRSLATADDAAFRERARLLAALERRHWLNSYMHKLLVLDAP SEQ ID NO: 3 - deduced human TIP39:

SLALADDAAFRERARLLAALERRHWLNSYMHKLLVLDAP

5. Detailed Description of the Preferred Embodiment 5.1 General The parathyroid hormone 2 (PTH2) receptor's anatomical distribution suggests that, among other functions, it may be involved in modulation of nociception. We localized PTH2 receptor protein to spinal cord lamina II and showed that it is synthesized by subpopulations of primary sensory neurons and intrinsic spinal cord dorsal horn neurons. Tuberoinfundibular peptide of 39 residues (TIP39) selectively activates the PTH2 receptor. Intraplantar microinjection of TIP39 caused a paw- withdrawal response and intrathecal injection caused scratching, biting and licking, a nocifensive response. Intrathecal administration of a TIP39 antibody decreased sensitivity in tail-flick and paw-pressure assays. Intrathecal administration of TIP39 potentiated responses in these assays. We determined the sequence of TIP39's precursor and found that mRNA encoding TIP39 and TIP39-like immunoreactivity is concentrated in two brainstem areas, the subparafascicular area and the caudal paralemniscal nucleus. Cells in these areas project to the superficial dorsal horn of the spinal cord. Our data indicate that TLP39 released from supraspinal fibers potentiates aspects of nociception within the spinal cord.

5.2. PTH2 Receptor Gene

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide), having GenBank Accession number NM_005048, which encodes for the PTH2 receptor. The PTH2 receptor is structurally related to the G-protein-

PTH receptor family. It contains an open reading frame encoding a protein of 541 amino acid residues. The protein exhibits the highest degree of homology to a human PTH receptor with 48.237% identity and 65.863% similarity over the entire amino acid stretch. The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the polypeptide may be identical to the coding sequence shown in GenBank Accession number NM_005048 or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the sequence from GenBank Accession number NM_005048.

The polynucleotide which encodes the polypeptide of the present invention may include: only the coding sequence for the polypeptide; the coding sequence for the polypeptide and additional coding sequence; the coding sequence for the polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non- coding sequence 5' and/or 3' of the coding sequence for the polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence from GenBank Accession number NM_005048. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. It includes receptors from other species that have homologous sequences and similar pharmacological properties, such as the rat PTH2 receptor, GenBank Accession number NM_031089.

Thus, the present invention includes polynucleotides encoding the same polypeptide that can be deduced from GenBank Accession number NM_005048 as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the PTH2 receptor polypeptide. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in GenBank Accession number NM_005048. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide. The polynucleotides may also encode for a soluble form of the PTH2 receptor polypeptide which is the extracellular portion of the polypeptide which has been cleaved from the transmembrane and intracellular domains of the full-length polypeptide of the present invention. The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al. 1984 Cell 37:767).

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove- described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the polypeptide encoded by GenBank Accession number NM_005048, i.e. function as a soluble PTH2 receptor by retaining the ability to bind the ligands for the receptor even though the polypeptide does not function as a membrane bound PTH2 receptor, for example, by eliciting a second messenger response. Alternatively, the polynucleotides may have at least 20 bases, preferably at least 30 bases and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which have an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of GenBank Accession number NM_005048, or for variants thereof, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer. Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to the polynucleotide of GenBank Accession number NM_005048 as well as fragments thereof, which fragments have at least 20 or 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

Fragments of the genes may be employed as a hybridization probe for a cDNA library to isolate other genes which have a high sequence similarity to the genes of the present invention, or which have similar biological activity. Probes of this type are at least 20 bases, preferably at least 30 bases and most preferably at least 50 bases or more. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene of the present invention including regulatory and promoter regions, exons and introns. An example of a screen of this type comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the genes of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

5.3. PTH2 Receptor Polypeptide

The present invention further relates to a PTH2 receptor polypeptide which has the amino acid sequence deduced from GenBank Accession number NM_005048, as well as fragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide encoded by GenBank Accession number NM_005048, means a polypeptide which either retains substantially the same biological function or activity as such polypeptide, i.e. functions as a PTH2 receptor, or retains the ability to bind the ligand for the receptor even though the polypeptide does not function as a G-protein PTH2 receptor, for example, a soluble form of the receptor.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analog of the polypeptide encoded by GenBank

Accession number NM_005048 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the polypeptide which are employed for purification of the polypeptide, or (v) one in which a fragment of the polypeptide is soluble, i.e. not membrane bound, yet still binds ligands to the membrane bound receptor. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The polypeptides of the present invention include the polypeptide encoded by GenBank Accession number NM_005048 as well as polypeptides which have at least 70% similarity (preferably at least a 70% identity) to the polypeptide encoded by GenBank Accession number NM_005048 and more preferably at least a 90% similarity (more preferably at least a 90% identity) to the polypeptide encoded by GenBank Accession number NM_005048 and still more preferably at least a 95% similarity (still more preferably at least a 95% identity) to the polypeptide encoded by GenBank Accession number NM_005048 and also includes portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis, therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention. The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons). The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adeno virus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or S V40 promoter, the E. coli lac or trp, the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera frugiperda Sf9; animal cells such as CHO, COS or Bowes melanoma; adenovirus; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen); pbs, pDIO phagescript, psiX174, pbluescript SK, pbsks, PNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233 3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene); pSVK3, PBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include laci, lacZ, T3, T7, gpt, lambda PR, Pf, and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

5.4. PTH2 Receptor Polypeptide Expression

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell, introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L. et al. 1986 Basic Methods in Molecular Biology).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Full-length proteins can be expressed in vertebrate cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. 1989 Molecular Cloning: A Laboratory

Manual, Second Edition, Cold Spring Harbor, N.Y.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector.

Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within, the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman 1981 Cell 23:175, and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The G-protein PTH2 receptor polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

5.5. Screening Assays for Compounds that Modulate PTH2 Receptor Expression or

Activity The following assays are designed to identify compounds that interact with (e.g., bind to) PTH2 receptor (including, but not limited to the extracellular domain (ECD) or cytoplasmic domain (CD) or transmembrane domain (TM) of PTH2 receptor), compounds that interact with (e.g., bind to) intracellular proteins that interact with PTH2 receptor (including, but not limited to, TM and CD of PTH2 receptor), compounds that interfere with the interaction of PTH2 receptor with its natural ligand TIP39, or parathyroid hormone (PTH), as well as with transmembrane or intracellular proteins involved in PTH2 receptor- mediated signal transduction, and to compounds which modulate the activity of PTH2 receptor gene (i.e., modulate the level of PTH2 receptor gene expression) or modulate the level of PTH2 receptor. Assays may additionally be utilized which identify compounds which bind to PTH2 receptor gene regulatory sequences (e.g., promoter sequences) and which may modulate PTH2 receptor gene expression. See, e.g., Platt, K.A. 1994 J Biol Chem 269:28558-28562.

The compounds which may be screened in accordance with the invention include, but are not limited to peptides, antibodies and fragments thereof, and other organic compounds (e.g., peptidomimetics) that bind to the ECD or TM of the PTH2 receptor and inhibit the activity triggered by the natural ligand (i.e., antagonists); as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic the ECD of the PTH2 receptor (or a portion thereof) and bind to and "neutralize" natural ligand.

Such compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries (see, e.g., Lam, K.S. et al. 1991 Nature 354:82-84; Houghten, R. et al. 1991 Nature 354:84-86), and combinatorial chemistry-derived molecular library made of D- and or L- configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al. 1993 Cell 72:767-778), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Other compounds which can be screened in accordance with the invention include but are not limited to small organic molecules that are able to cross the blood-brain barrier, gain entry into an appropriate cell and affect the expression of the PTH2 receptor gene or some other gene involved in the PTH2 receptor signal fransduction pathway (e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or such compounds that affect the activity of the PTH2 receptor (e.g., by inhibiting or enhancing the enzymatic activity of the CD) or the activity of some other intracellular factor involved in the PTH2 receptor signal transduction pathway. Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate PTH2 receptor expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites, such as the interaction domains of TIP39 or PTH with PTH2 receptor itself. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found. Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain infra-molecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined. If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy. Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods. Finally, having determined the structure of the active site, either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential PTH2 receptor modulating compounds. Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity. Further experimental and computer modeling methods useful to identify modulating compounds based upon identification of the active sites of TIP39 or PTH, PTH2 receptor, and related fransduction and transcription factors will be apparent to those of skill in the art.

Examples of molecular modeling systems are the CHARMM and QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific- proteins, such as Rotivinen, et al. 1988 Acta Pharmaceutical Fennica 97:159-166; Ripka, 1988 New Scientist 54-57; McKinaly and Rossmann 1989 Annu Rev Pharmacol Toxicol 29:111-122; Perry and Davies, 1989 OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 Alan R. Liss, Inc.; Lewis and Dean 1989 Proc R Soc Lond 236:125-140 and 141-162; and, with respect to a model receptor for nucleic acid components, Askew, et al. 1989 J Am Chem Soc 111:1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, Calif), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA, once that region is identified.

Although described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators, preferably inhibitors.

Compounds identified via assays such as those described herein may be useful, for example, in modulating nociception. Assays for in vivo testing the effectiveness of compounds, identified by, for example, techniques such as those described in Section 5.5.1 through 5.5.5, are discussed below, in Section 5.6.

5.5.1. In Vitro Cell-Free Screening Assays for Compounds that Bind to PTH2 Receptor In vitro systems may be designed to identify compounds capable of interacting with (e.g., binding to) PTH2 receptor (including, but not limited to, the ECD or CD or TM of PTH2 receptor). Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant PTH2 receptor gene products; may be useful in elaborating the biological function of the PTH2 receptor; may be utilized in screens for identifying compounds that disrupt normal PTH2 receptor interactions; or may in themselves disrupt such interactions. The principle of the assays used to identify compounds that bind to the PTH2 receptor involves preparing a reaction mixture of the PTH2 receptor and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. The PTH2 receptor species used can vary depending upon the goal of the screening assay. For example, where antagonists of the natural ligand are sought, the full length PTH2 receptor, or a soluble truncated PTH2 receptor, e.g., in which the TM and/or CD is deleted from the molecule, a peptide corresponding to the ECD or a fusion protein containing the PTH2 receptor ECD fused to a protein or polypeptide that affords advantages in the assay system (e.g., labeling, isolation of the resulting complex, etc.) can be utilized. Where compounds that interact with the cytoplasmic domain are sought to be identified, peptides corresponding to the PTH2 receptor CD and fusion proteins containing the PTH2 receptor CD can be used.

The screening assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring the PTH2 receptor protein, polypeptide, peptide or fusion protein or the test substance onto a solid phase and detecting

PTH2 receptor/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the PTH2 receptor reactant may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nommmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nommmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nommmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nommmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for PTH2 receptor protein, polypeptide, peptide or fusion protein or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. Alternatively, cell-based assays, membrane vesicle-based assays and membrane fraction-based assays can be used to identify compounds that interact with PTH2 receptor. To this end, cell lines that express PTH2 receptor, or cell lines (e.g., COS cells, CHO cells, fibroblasts, etc.) that have been genetically engineered to express PTH2 receptor (e.g., by transfection or fransduction of PTH2 receptor DNA) can be used. Interaction of the test compound with, for example, the ECD of PTH2 receptor expressed by the host cell can be determined by comparison or competition with native TIP39 or PTH. 5.5.2. Assays for Intracellular Proteins that Interact with the PTH2 Receptor Any method suitable for detecting protein-protein interactions may be employed for identifying transmembrane proteins or intracellular proteins that interact with PTH2 receptor. Among the traditional methods which may be employed are co- immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns of cell lysates or proteins obtained from cell lysates and the PTH2 receptor to identify proteins in the lysate that interact with the PTH2 receptor. For these assays, the PTH2 receptor component used can be a full length PTH2 receptor, a soluble derivative lacking the membrane-anchoring region (e.g., a truncated PTH2 receptor in which the TM is deleted resulting in a truncated molecule containing the ECD fused to the CD), a peptide corresponding to the CD or a fusion protein containing the CD of PTH2 receptor. Once isolated, such an intracellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify proteins with which it interacts. For example, at least a portion of the amino acid sequence of an intracellular protein which interacts with the PTH2 receptor can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique. (See, e.g., Creighton 1983 Proteins: Structures and Molecular Principles W.H. Freeman & Co. N.Y. pp. 34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known (see, e.g., Ausubel et al. eds. 1994 Current Protocols in Molecular Biology N.Y. John Wiley & Sons; and Innis, M. et al. eds. 1990 PCR Protocols: A Guide to Methods and Applications Academic Press, Inc. New York). Additionally, methods may be employed which result in the simultaneous identification of genes which encode the transmembrane or intracellular proteins interacting with PTH2 receptor. These methods include, for example, probing expression libraries, in a manner similar to the well known technique of antibody probing of λgtl 1 libraries, using labeled PTH2 receptor protein, or a PTH2 receptor polypeptide, peptide or fusion protein, e.g., a PTH2 receptor polypeptide or PTH2 receptor domain fused to a marker (e.g., an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc domain. One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al. 1991 PNAS USA 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif). Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to a PTH2 receptor nucleotide sequence encoding PTH2 receptor, a PTH2 receptor polypeptide, peptide or fusion protein, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot because it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, PTH2 receptor may be used as the bait gene product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait PTH2 receptor gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait PTH2 receptor gene sequence, such as the open reading frame of PTH2 receptor (or a domain of PTH2 receptor), can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids. A cDNA library of the cell line from which proteins that interact with bait PTH2 receptor gene product are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait PTH2 receptor gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait PTH2 receptor gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which express HIS3 can be detected by their growth on Petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait PTH2 receptor gene-interacting protein using techniques routinely practiced in the art.

5.5.3. Assays for Compounds that Interfere with PTH2 Receptor/Intracellular or PTH2 Receptor/Transmembrane Macromolecule Interaction

The macromolecules that interact with the PTH2 receptor are referred to, for purposes of this discussion, as "binding partners". These binding partners are likely to be involved in the PTH2 receptor signal fransduction pathway, and therefore, in the role of PTH2 receptor in modulation of nociception. Therefore, it is desirable to identify compounds that interfere with or disrupt the interaction of such binding partners with TIP39 or PTH which may be useful in regulating the activity of the PTH2 receptor and control nociception associated with PTH2 receptor activity.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the PTH2 receptor and its binding partner or partners involves preparing a reaction mixture containing PTH2 receptor protein, polypeptide, peptide or fusion protein as described in Sections 5.5.1 and 5.5.2 above, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the PTH2 receptor moiety and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the PTH2 receptor moiety and the binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the PTH2 receptor and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal PTH2 receptor protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant PTH2 receptor. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal PTH2 receptors. The assay for compounds that interfere with the interaction of the PTH2 receptor and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the PTH2 receptor moiety product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction by competition can be identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the PTH2 receptor moiety and interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the PTH2 receptor moiety or the interactive binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished simply by coating the solid surface with a solution of the PTH2 receptor gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified. In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the PTH2 receptor moiety and the interactive binding partner is prepared in which either the PTH2 receptor or its binding partners is labeled, but the signal generated by the label is quenched due to formation of the complex (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt PTH2 receptor/intracellular binding partner interaction can be identified.

In a particular embodiment, a PTH2 receptor fusion protein can be prepared for immobilization. For example, the PTH2 receptor or a peptide fragment, e.g., corresponding to the CD, can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art. This antibody can be labeled with the radioactive isotope, for example, ¹²⁵I, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-PTH2 receptor fusion protein can be anchored to glutathione-agarose beads. The interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the PTH2 receptor gene product and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-PTH2 receptor fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. ^' This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the PTH2 receptor/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads. In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the PTH2 receptor and/or the interactive or binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized. For example, and not by way of limitation, a PTH2 receptor gene product can be anchored to a solid material as described above, by making a GST-PTH2 receptor fusion protein and allowing it to bind to glutathione agarose beads. The interactive binding partner can be labeled with a radioactive isotope, such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-PTH2 receptor fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the intracellular binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology. 5.5.4. Cell- and Membrane-based Screening Assays for PTH2 Receptor Inhibitors

Compounds, including but not limited to binding compounds identified via assay techniques such as those described in the preceding sections above can be tested for the ability to treat or prevent pain. The assays described above can identify compounds which affect PTH2 receptor activity, e.g., compounds that bind to PTH2 receptor, inhibit binding of the natural ligand, and either activate signal transduction (agonists) or block activation (antagonists), and compounds that bind to a natural ligand of PTH2 receptor and neutralize ligand activity; or compounds that affect PTH2 receptor gene activity (by affecting PTH2 receptor gene expression, including molecules, e.g., proteins or small organic molecules, that affect or interfere with splicing events so that expression of the full length PTH2 receptor can be modulated). However, it should be noted that the assays described can also identify compounds that modulate PTH2 receptor signal transduction (e.g., compounds which affect upstream or downstream signaling events). The identification and use of such compounds which affect another step in the PTH2 receptor signal transduction pathway in which the PTH2 receptor gene product is involved and, by affecting this same pathway may modulate the effect of PTH2 receptor on the development of pain are within the scope of the invention. Such compounds can be used as part of a therapeutic method for the modulation of pain. Cell-based systems, membrane vesicle-based systems, and membrane fraction- based systems can be used to identify compounds which may act to treat or prevent pain. Such systems can include, for example, recombinant or non-recombinant cells, such as cell lines, which express the PTH2 receptor gene. In addition, expression host cells (e.g., COS cells, CHO cells, fibroblasts) genetically engineered to express a functional PTH2 receptor and to respond to activation by the natural PTH2 receptor ligand (such as TIP39 or PTH), e.g., as measured by a chemical or phenotypic change, induction of another host cell gene, change in ion flux, phosphorylation of host cell proteins, etc., can be used as an end point in the assay. In utilizing such cell-based systems, cells may be exposed to a compound suspected of exhibiting an ability to treat or prevent pain, at a sufficient concentration and for a time sufficient to elicit chemical or phenotypic change, induction of another host cell gene, change in ion flux, phosphorylation of host cell proteins, etc., in the exposed cells. After exposure, the cells can be assayed to measure alterations in the expression of the PTH2 receptor gene, e.g., by assaying cell lysates for PTH2 receptor mRNA transcripts (e.g., by Northern analysis) or for PTH2 receptor protein expressed in the cell; compounds which regulate or modulate expression of the PTH2 receptor gene are good candidates as therapeutics. Alternatively, the cells are examined to determine whether one or more body pain-like cellular phenotypes has been altered to resemble a more normal or more wild type, pain-free cellular phenotype, or a cellular phenotype more likely to produce a lower incidence or severity of pain. Still further, the expression and/or activity of components of the signal transduction pathway of which PTH2 receptor is a part, or the activity of the PTH2 receptor signal transduction pathway itself can be assayed.

For example, after exposure, the cell lysates can be assayed for the presence of phosphorylation of host cell proteins, as compared to lysates derived from unexposed control cells. The ability of a test compound to inhibit phosphorylation of host cell proteins in these assay systems indicates that the test compound inhibits signal transduction initiated by PTH2 receptor activation. The cell lysates can be readily assayed using a Western blot format; i.e., the host cell proteins are resolved by gel electrophoresis, transferred and probed using an antibody against a phosphorylated peptide residue (e.g., an antibody labeled with a signal generating compound, such as radiolabel, fluor, enzyme, etc.). See, e.g., Glenney et al. 1988 J Immunol Methods 109:277-285; Frackelton et al. 1983 Mol Cell Biol 3: 1343-1352. Alternatively, an ELISA format could be used in which a particular host cell protein involved in the PTH2 receptor signal transduction pathway is immobilized using an anchoring antibody specific for the target host cell protein, and the presence or absence of a phosphorylated peptide residue on the immobilized host cell protein is detected using a labeled antibody. See, King et al. 1993 Life Sciences 53:1465-1472. In yet another approach, ion flux, such as calcium, potassium, sodium, bicarbonate, chloride ion flux, can be measured as an end point for PTH2 receptor stimulated signal transduction.

In general, other cell-based screening procedures of the invention involve providing appropriate cells which express a PTH2 receptor polypeptide on the surface thereof. Such cells include cells from mammals, yeast, Drosophila or E. coli. In particular, a polynucleotide encoding the receptor of the present invention is employed to transfect cells to thereby express a PTH2 receptor. The expressed receptor is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which are fransfected to express a PTH2 receptor polypeptide. Such a screening techmque is described in PCT WO 92/01810, published Feb. 6, 1992. Such an assay may be employed to screen for a compound which inhibits activation of a receptor of the present invention by contacting the melanophore cells which encode the receptor with both a receptor ligand, such as TIP39 or PTH, and a compound to be screened. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor, i.e., inhibits activation of the receptor.

The technique may also be employed for screening of compounds which activate a receptor of the present invention by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the receptor. Other screening techniques include the use of cells which express a PTH2 receptor

(for example, fransfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation. In this technique, compounds may be contacted with cells expressing a receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction or pH changes, is then measured to determine whether the potential compound activates or inhibits the receptor.

Another screening techmque involves expressing a PTH2 receptor polypeptide in which the receptor is linked to phospholipase C or D. Representative examples of such cells include, but are not limited to, endothelial cells, smooth muscle cells, and embryonic kidney cells. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal. Another method involves screening for compounds which are antagonists, and thus inhibit activation of a receptor polypeptide of the present invention by determining inhibition of binding of labeled ligand, such as TIP39 or PTH, to cells which have the receptor on the surface thereof, or cell membranes containing the receptor. Such a method involves transfecting a eukaryotic cell with a DNA encoding a PTH2 receptor polypeptide such that the cell expresses the receptor on its surface (or using a eukaryotic cell that expresses the PTH2 receptor on its surface). The cell is then contacted with a potential antagonist in the presence of a labeled form of a ligand, such as TIP39 or PTH. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity associated with fransfected cells or membrane from these cells. If the compound binds to the receptor, the binding of labeled ligand to the receptor is inhibited as determined by a reduction of labeled ligand which binds to the receptors. This method is called a binding assay.

Another such screening procedure involves the use of eukaryotic cells which are fransfected to express a receptor of the present invention (or use of eukaryotic cells that express the PTH2 receptor on their surface). The cells are loaded with an indicator dye that produces a fluorescent signal when bound to calcium, and the cells are contacted with a test substance and a receptor agonist, such as TIP39 or PTH. Any change in fluorescent signal is measured over a defined period of time using, for example, a fluorescence specfrophotometer or a fluorescence imaging plate reader. A change in the fluorescence, signal pattern generated by the ligand indicates that a compound is a potential antagonist (or agonist) for the receptor.

Another such screening procedure involves use of eukaryotic cells which are fransfected to express the PTH2 receptor of the present invention (or use of eukaryotic cells that express the PTH2 receptor on their surface), and which are also fransfected with a reporter gene construct that is coupled to activation of the receptor (for example, luciferase or beta-galactosidase behind an appropriate promoter). The cells are contacted with a test substance and a receptor agonist, such as TIP39 or PTH, and the signal produced by the reporter gene is measured after a defined period of time. The signal can be measured using a luminometer, specfrophotometer, fluorimeter, or other such instrument appropriate for the specific reporter construct used. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor. Another such screening technique for antagonists or agonists involves introducing

RNA encoding a PTH2 receptor polypeptide into Xenopus oocytes to transiently or stably express the receptor. The receptor oocytes are then contacted with a receptor ligand, such as TIP39 or PTH, and a compound to be screened. Inhibition or activation of the receptor is then determined by detection of a signal, such as, cAMP, calcium, proton, or other ions. Another method involves screening for PTH2 receptor polypeptide inhibitors by determining inhibition or stimulation of PTH2 receptor polypeptide-mediated cAMP and/or adenylate cyclase accumulation or- diminution. Such a method involves transiently or stably transfecting a eukaryotic cell with a PTH2 receptor polypeptide to express the receptor on the cell surface (or using a eukaryotic cell that expresses the PTH2 receptor on its surface). The cell is then exposed to potential antagonists in the presence of PTH2 receptor polypeptide ligand, such as TIP39 or PTH. The amount of cAMP accumulation is then measured, for example, by radio-immuno or protein binding assays (for example using Flashplates or a scintillation proximity assay). Changes in cAMP levels can also be determined by directly measuring the activity of the enzyme, adenylyl cyclase, in broken cell preparations. If the potential antagonist binds the receptor, and thus inhibits PTH2 receptor polypeptide binding, the levels of PTH2 receptor polypeptide-mediated cAMP, or adenylate cyclase activity, will be reduced or increased.

The present invention also provides a method for determining whether a ligand not known to be capable of binding to PTH2 receptor can bind to such receptor which comprises contacting a eukaryotic cell which expresses a PTH2 receptor with the ligand, such as TIP39 or PTH, under conditions permitting binding of candidate ligands to a PTH2 receptor, and detecting the presence of a candidate ligand which binds to the receptor thereby determining whether the ligand binds to the PTH2 receptor. The systems hereinabove described for determining agonists and/or antagonists may also be employed for determining ligands which bind to the receptor. 5.5.5. Potential PTH2 Receptor Antagonists Examples of potential PTH2 receptor polypeptide antagonists include antibodies or, in some cases, oligonucleotides, which bind to the receptor but do not elicit a second messenger response such that the activity of the receptor is prevented. Potential antagonists also include proteins which are closely related to a ligand of the PTH2 receptor polypeptide, i.e., a fragment of the ligand, which have lost biological function and when binding to the PTH2 receptor polypeptide, elicit no response.

A potential antagonist also includes an antisense construct prepared through the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both methods of which are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the PTH2 receptor polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix— see Lee, et al. 1979 Nucl Acids Res 6:3073; Cooney, et al 1988 Science 241:456; and Dervan, et al. 1991 Science 251:1360), thereby preventing transcription and production of a PTH2 receptor polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule to a PTH2 receptor polypeptide (antisense—Okano, J. 1991 Neurochem 56:560; Oligonucleotides as antisense inhibitors of gene expression 1988 CRC Press, Boca Raton, Fla.). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of a PTH2 receptor polypeptide.

Another potential antagonist is a small molecule which binds to a PTH2 receptor polypeptide, making it inaccessible to ligands such that normal biological activity is prevented. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules.

Potential antagonists also include soluble forms of a PTH2 receptor polypeptide, e.g., fragments of the polypeptide, which bind to the ligand, TIP39 or PTH, and prevent the ligand from interacting with membrane bound PTH2 receptor polypeptides. 5.6. Assays for in vivo Identification of Compounds that Treat Pain

Animal-based systems may be used to identify compounds capable of treating or preventing pain. Such animal models may be used as test substrates for the identification of pharmaceuticals, therapies and interventions which may be effective in treating or preventing pain. For example, animal models may be exposed to a compound, suspected of exl ibiting an ability to treat or prevent pain, at a sufficient concentration and for a time sufficient to elicit such a treatment or prevention of pain in the exposed animals. The response of the animals to the exposure may be monitored by assessing the reversal of pain.

With regard to intervention, any treatments which reverse any aspect of pain should be considered as candidates for therapeutic or preventive intervention.

Tests for evaluation of nociceptive responses include tail-flick test (Ueda, H. et al. 1986 Neurosci Lett 65:247-52); capsaicin test (Tan-No, K. et al. 1998 Neuropeptides 32:411-5); von Frey fiber test; Hargreaves test (Hargreaves, K. et al. 1988 Pain 32:77-88); as well as evaluation of nocifensive responses (Hylden, J.L. & Wilcox, G.L. 1980 Eur J Pharmacol 67:313-6); and evaluation of flexor responses as described by Inoue, M. et al. 1998 PNAS USA 95:10949-53; Ueda, H. 1999 Jpn J Pharmacol 79:263-8.

5.7. Pharmaceutical Preparations and Methods of Administration

The antagonist compounds that are identified by the screening methods as described above can be administered to a patient at therapeutically effective doses to treat or prevent pain disorders, including chronic pain syndromes, hypersensitivity, windup and allodynia.

A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms of pain.

5.7.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concenfration range that includes the IC₅₀ (i.e., the concenfration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

5.7.2. Formulations and Use The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.

The compounds of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application, which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., vitamins. For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Ampoules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules. A syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.

Sustained or directed release compositions can be formulated, e.g., by inclusion in liposomes or those wherein the active compound is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc. It is also possible to freeze- dry these compounds and use the lyophilizates obtained, for example, for the preparation of products for injection.

For topical application, there are employed as non-sprayable forms, viscous to semi- solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a freon.

It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the compositions formulated, the mode of application, and the particular situs and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Effects of TIP39 Administration.

Both dorsal root ganglion (DRG) and spinal cord dorsal horn neurons synthesize PTH2 receptors. The PTH2 receptor synthesizing DRG neurons seem to be of the smaller size class, and the spinal cord neurons are within the superficial layers of the dorsal horn, (Fig. 1). We previously observed that a PTH2 receptor selective antibody labels nerve fibers and terminals much more strongly than cell bodies, suggesting that the receptor accumulates to much greater levels in the terminals and dendrites than the soma of neurons (Wang, T. et al. 2000 Neuroscience 100:629-49). Fibers in the superficial layers of the dorsal horn of the spinal cord were particularly intensely labeled. We now performed double-labeling experiments, comparing the distribution of the PTH2 receptor with well studied markers, to define more precisely the PTH2 receptor's localization in the spinal cord (Fig. 2). Most PTH2 receptor labeling was ventral to the area of greatest substance P and calcitonin gene-related peptide (CGRP) labeling (marginal layers), and dorsal to the layer of labeling by an antibody to protein kinase C gamma (lamina 111). The labeling was largely co-laminar with a band of labeling by isolectin B4. On the basis of extensive studies of these markers (Molliver, D.C. et al. 1995 JComp Neurol 361:404-16; Malmberg, A.B. et al. 1997 Science 278:279-83; Polgar, E. et al. 1999 Brain Res 833:71-80), this labeling places the PTH2 receptor within lamina II. Partial overlap occurred in labeling by the PTH2 receptor and substance P and CGRP antibodies, but high magnification confocal microscopy showed no colocalization of substance P and the PTH2 receptor, and little colocalization of the PTH2 receptor with CGRP (Fig. 2b). There was also no apparent colocalization with isolectin B4 despite extensive laminar overlap (Fig. 2d). CGRP and isolectin B4 together label nearly all small-diameter primary afferent fibers (Snider, W.D. & McMahon, S.B. 1998 Neuron 20:629-32). Relatively little PTH2 receptor synthesis occurs in supraspinal areas that project to the superficial dorsal horn (Wang, T. et al. 2000 Neuroscience 100:629-49). Thus, most PTH2 receptors present in the spinal cord seem to be on the processes of intrinsic dorsal horn neurons, which is consistent with the in situ hybridization pattern seen with the PTH2 receptor probe.

The localization of PTH2 receptor immunoreactivity and mRNA synthesis within the spinal cord and the small size of the PTH2 receptor expressing DRG cells indicate its involvement in nociception. Because many proteins synthesized by DRG neurons are transported in both their peripheral and central processes, we initially tested the effect of activating peripheral PTH2 receptors. Microinjection of TIP39 into the plantar surface of a mouse paw elicited a dose-dependent withdrawal response (Fig. 3a). The maximal effect occurred with approximately 10 fmol of peptide. By comparison, 1-10 fmol of nociceptin/orphanin FQ, 2 pmol of bradykinin, and 10 pmol of substance P cause maximal responses in this pain assay (Inoue, M. et al. 1998 PNAS USA 95:10949-53; Ueda, H. et al. . 2000 Peptides 21:1215-21). In situ hybridization histochemistry studies have shown that the PTH2 receptor is present at low levels on vascular endothelium (Usdin, T. B. et al. 1996 Endocrinology 137:4285-4297; Usdin, T.B. et al. 1999 Endocrinology 140:3363- 3371), so we cannot be absolutely certain that the effect of TIP39 results from activation of PTH2 receptors on the peripheral processes of sensory neurons. However, TIP39 has no effect on vascular tone in a perfused mesentery preparation and the labeling of DRG neurons, is stronger than that of vasculature. We next examined the effects of central administration of TIP39. Intrathecal injection of TIP39 stimulated a dose-dependent nocifensive response, caudally directed scratching, biting, and licking (Fig. 3b). The responses to peripheral and central TIP39 administration show that it can activate nociceptive circuits. However, these observations do not address the effects of endogenous TIP39. Because no TIP39 antagonists were available, we sequestered TJJP39 with an antibody, and asked what nociceptive responses were affected. Intrathecal injection of the TIP39 antibody increased the response latency in the thermal tail-flick assay (Fig. 4a). In the paw pressure test, intrathecal administration of the TIP39 antibody increased the latency to paw withdrawal, corresponding to decreased pressure sensitivity (Fig. 4b). In contrast, intrathecal delivery of the TIP39 antibody did not significantly affect paw withdrawal latency in response to radiant heat (Fig. 4c; Hargreaves assay; Hargreaves, K. et al. 1988 Pain 32:77-88), and it did not affect the response to intraplantar injection of the chemoirritant capsaicin. Inhibition by the TIP39 antibody of responses in the thermal tail- flick and paw pressure assays suggest that TIP39 may have a facilitatory role in the underlying circuits. We tested this possibility by performing the nociceptive assays after intrathecal administration of TIP39 (Fig. 4 d-f). Infrathecal TIP39 administration decreased the tail-flick and paw-pressure withdrawal latencies, but did not have a significant effect on withdrawal in response to paw heating. These effects are consistent with those predicted from the antibody sequestration experiments and with a facilitatory role of TIP39 in nociception. The tail-flick response is thought to be primarily a spinal reflex, whereas paw withdrawal from heat requires supraspinal mediation. The different effects of TIP39 and TIP39 sequestration on these responses may reflect differences in PTH2 receptor expression on neurons in the underlying circuits. However, the intrathecally applied reagents may also reach relevant sites with different efficiency. We next wanted to identify the source(s) of TIP39. We originally determined the amino acid sequence of TIP39 purified from bovine hypothalamus. We searched genomic databases using this sequence and identified homologous human and mouse sequences. From these we predicted sequences for TIP39's precursor: METC(R)QM(V)SRSPRE(V)RLLLLLLLLLL(V)VPWGT(V)G(R)P(T)*ASGVALPL(P) A(V)GVF(L)—

SLRA(P)PGRAWAG(D^L(P)G(A S(T)PL(R S(P RRSLALADDAAFRERARLLAALER RR(H)WLD(N)SYMO(H)KLLL LDAP. Bold letters indicate residues that differ between the mouse (SEQ ID NO: 4) and human (SEQ ID NO: 2) sequences, with the human residue in parentheses following the mouse residue. An asterisk indicates a predicted signal peptide cleavage site (http://www.cbs.dtu.dk/services/SignalP/; Nielsen, H. et al. 1997 hit J Neural Syst 8:581-99; Nielsen, H. et al. 1997 Protein Eng 10:1-6), and the dash indicates the location of an intron in the genomic sequences. Residues corresponding to the purified peptide are underlined. We amplified cDNAs corresponding to this mRNA from mouse and rat hypothalamus, and isolated a clone containing this sequence from a human hypothalamic cDNA library. The deduced human TIP39 peptide sequence (SEQ ID NO: 3) (underlined sequence above) is identical to purified bovine TIP39, while the predicted mouse peptide differs at 4 out of the 39 residues. Sequences corresponding to the purified peptide were followed by a stop codon and preceded by nucleotides encoding two arginine residues. The entire predicted TIP39 precursor is 103 residues and includes a predicted signal peptide of 33 residues.

With reverse transcription-PCR we found TIP39 mRNA in rat brain, dorsal root ganglion (DRG), eye, and testis but not in the spinal cord or other tissues (Fig. 5). We determined the central nervous system distribution of TIP39 mRNA by in situ hybridization histochemistry (Fig. 6). No clear signal accured after film autoradiography. After emulsion autoradiography two groups of intensely labeled cells were present in each of 9 animals examined. One was the subparafascicular area (Fig. 6 g, h; see, Peschanski, M. & Mantyh, P. W. 1983 Brain Res 263:181-90 for a detailed anatomical description of this area), in which TIP39 synthesizing cells were present both in the most rostral part of the central gray matter, medial to the fasciculus retroflexus at the diencephalic-midbrain junction, and in the parvicellular part of the subparafascicular nucleus. The dopaminergic Al 1 cell group is present in this area, but using adjacent sections it was clear that the TIP39 cells were tyrosine hydroxylase negative. The other group of intensely labeled cells was present in a well-defined area of the lateral pons bordered by the oral reticular pontine, ventral lateral lemniscal, and Kδlliker-Fuse nuclei (Fig. 6 b, c). We identified this latter area as the caudal paralemniscal nucleus on the basis of its topographical relation to the ventral nucleus of the lateral lemniscus and the rubrospinal tract (Zemlan, F.P. et al. 1979 J Anat 128:489-512; Andrezik, LA. & Beitz, A.J. 1986 in: The Rat Nervous System, ed. Paxinos, G. Academic Press, Orlando, pp. 1-28; Martin, G.F. et al. 1990 in: The Human Nervous System, ed. Paxinos, G. Academic Press, San Diego, pp. 203-220). Significantly weaker hybridization signals were seen in cells scattered in the olfactory bulb, amygdala, hypothalamus, cerebellum and the pineal gland. To test the highly discrete localization revealed by in situ hybridization histochemistry we performed reverse franscription-PCR on micro- and macrodissected tissue samples (Fig. 5b). Micropunches of the subparafascicular area and the caudal paralemniscal nucleus contained TIP39 mRNA. TIP39 mRNA was detectable in the hypothalamus, thalamus, pons, midbrain, olfactory bulb, amygdala, pineal gland, and the cerebellum but not in the cerebral cortex, hippocampus, striatum, pituitary, medulla oblongata (including microdissected spinal trigeminal nucleus), or spinal cord.

An antibody to TIP39 labels the same cell populations in rat (Fig. 6 d, e, i, j) and mouse brain as detected by in situ hybridization. In the spinal cord, fine scattered fibers are found in the lateral funiculus, the lateral intermediate gray matter, central gray matter, and lamina II and III (Fig. 7). Most of TIP39 fibers are in the lateral funiculus adjacent to the gray matter, an area that contains both descending and primary afferent fibers. We did not observe labeling of DRG neurons significantly over background using either in situ hybridization or immunohistochemistry. The reverse franscription-PCR signal was consistently positive but 35 cycles of amplification were used, suggesting that TIP39 may be present in DRG cells at a very low level. Since we have no evidence for TIP39 within spinal cord interneurons, the TJJP39 in deep layers of the spinal cord is most likely within descending fibers. Thus, most of the TIP39 normally present in the spinal cord may be in descending fibers.

Cells within the areas of TIP39 concenfration, the caudal paralemniscal nucleus (Andrezik, J.A. &. Beitz, AJ. 1986 in: The Rat Nervous System, ed. Paxinos, G. Academic Press, Orlando, pp. 1-28; Martin, G.F. et al. 1990 in: The Human Nervous System, ed. Paxinos, G. Academic Press, San Diego, pp. 203-220) and subparafascicular area (Peschanski, M. & Mantyh, P. W. 1983 Brain Res 263:181-90) project to the dorsal horn, and to the caudal part of the sensory trigeminal nucleus (Peschanski, M. & Mantyh, P. W. 1983 Brain Res 263:181-90; Zemlan, F.P. et al. 1979 JAnat 128:489-512; Martin, G.F. et al. 1990 in: The Human Nervous System, ed. Paxinos, G. Academic Press, San Diego, pp. 203-220), the medullary equivalent of the spinal dorsal horn. These areas have a high level of PTH2 receptor expression. Studies have implicated the caudal paralemniscal nucleus in nociception (Martin, G.F. et al. 1990 in: The Human Nervous System, ed. Paxinos, G. Academic Press, San Diego, pp. 203-220). These matches support the suggestion that TIP39 is a physiological ligand for the PTH2 receptor. DRG neurons also project to these areas. Thus TIP39 is envisioned to contribute to modulation of spinal nociceptive information. Blockade of the PTH2 receptor is envisioned as being a useful strategy for pain management.

6. EXAMPLE 1 Animals. Male ddY mice weighing 20 - 22 g were used in physiological experiments. Procedures were approved by Nagasaki University Animal Care Committee and complied with the recommendations of the International Association for the Study of Pain (Zimmermann, M. 1983 Pain 16:109-10). Anatomical experiments were approved by the National Institute of Mental Health Animal Care Committee. Materials. TIP39 was synthesized by Midwest Biomolecules (Waterloo, Illinois).

Other reagents were from standard commercial suppliers. Intrathecal antibody injections were performed with IgG, purified from a rabbit immunized with bovine (b) TIP39 coupled via glutaraldehyde to keyhole limpet hemocyanin, or from pre-immune serum. Iπrmunohistochemistry used affinity purified antibody from a rabbit immunized with mouse (m) TIP39 coupled via l-ethyl-3-(3-dimethylaminopropyl)carbodiimide to keyhole limpet hemocyanin. Titers (50% maximum binding to immobilized peptide) of anti- bTIP39 IgG and affinity purified anti-mTιP39 were 800 ng/ml and 6 ng/ml against bTIP39 and 900 ng/ml and 3 ng/ml against mTIP39. Both antibodies exhibited approximately 1% cross reactivity with PTH and no detectable cross reactivity with other peptides tested including parathyroid hormone-related peptide, calcitonin, substance P, vasoactive intestinal peptide, glucagon, and calcitonin gene-related peptide (CGRP). In vivo testing.

Evaluation of flexor responses. Experiments were performed as previously described (Inoue, M. et al. 1998 PNAS USA 95:10949-53; Ueda, H. 1999 Jpn J Pharmacol 79:263-8). Briefly, mice were held in a suspended cloth sling. Test agents were delivered through two polyethylene cannulae inserted into the plantar surface of the right hindlimb, which was connected to an isotonic transducer/recorder. For normalization the largest spontaneous response occurring immediately after cannulation was considered the maximal withdrawal force for each animal. Nociceptive activity, measured after complete recovery (20-30 min) from light ether anesthesia, was expressed as the ratio of the test elicited force to the maximal force in each mouse. Intraplantar TIP39 injections were made at 5 min intervals through one camiula. In the dose-response experiments increasing doses were given at 5 min intervals with each dose administered twice.

Evaluation of nocifensive responses. Animals were adapted to individual plastic cages for one hour. Five μl of test agent was injected between lumbar disk 5 and 6 (Hylden, J.L. & Wilcox, G.L. 1980 Eur JPharm 67:313-6), each mouse was returned to its fransparent cage, and the time spent exhibiting characteristic nociceptive behaviors, such as reciprocal hind limb scratching, and caudally directed scratching, biting and licking over 20 min of observation was measured (Hylden, J.L. & Wilcox, G. L. 1981 Brain Res 217:212- 5; Hylden, J.L. & Wilcox, G. L. 1983 J Pharmacol Exp Ther 226:398-404; Inoue, M. et al. 1999 J Pharmacol Exp Theτ 291:308-13). Tail-flick test. Animals were gently restrained by hand, and a light beam adjusted for 10-12 sec latency in naive mice was focused onto the blackened dorsal surface of the tail. Latency up to a cut-off time of 30 sec was measured (Ueda, H. et al. 1986 Neuroscience Lett 65:247-52).

Paw pressure test. Mice were placed in a Plexiglas chamber on a wire mesh grid and allowed to accommodate for a period of one hour. A mechanical stimulus was then delivered onto the middle of the plantar surface of the right hindpaw using a 0.8-0.9 mm diameter filament connected to an automatic Transducer Indicator (Model 1601, IITC Inc., Woodland Hills, CA). The filament used produces 10 g of force at 5 sec, when paw withdrawal is elicited in naive mice. A 20 sec cut-off time was used to avoid tissue damage.

Hargreaves test. A thermal beam was focussed on a hind limb footpad of mice placed on a glass surface and the withdrawal response latency measured, with a 20 sec cutoff time, as described by Hargreaves et al. (Hargreaves, K. et al. 1988 Pain 32:77-88).

Sequence identification, reverse transcription-PCR, in situ hybridization and immunohistochemistry. Genomic sequences of mouse (GenBank #AC073740) and human TIP39 (GenBank #AC068670) were identified in public high throughput gene sequence by tBLASTn (Altschul, S.F. et al. 1990 JMol Biol 215:403-410) search with the amino acid sequence of bovine TIP39. cDNA sequences were predicted using GENE (Salamov, A.A. & Solovyev, V.V. 2000 Genome Res 10:516-22). Fragments corresponding to amino acids -55 to 37 and -18 to 37 were amplified from total mouse brain cDNA with primers incorporating RNA polymerase recognition sites, [³⁵S]-labeled antisense and sense riboprobes were synthesized, and hybridized to cryostat sections as described on the World Wide Web

(http://inframural.nimh.nih.gov/lcmr/snge/Protocols/ISITH/ISHH.html). The two probes produced equivalent hybridization patterns. For the tissue distribution survey approximately 1 μg of DNase treated RNA prepared from rat tissues was reverse- transcribed and then PCR amplified (35 cycles) using primers: 5'- GGAGACCTGCCAGATGTCCA (SEQ ID NO: 5) and 5'-GTCCAGTAGCAACAGCTT (SEQ ID NO: 6) in different TJJP39 exons. Immunolabeling with a PTH2 receptor selective antibody was performed as previously described (Usdin, T.B. et al. 1999 Endocrinology 140:3363-3371; Wang, T. et al. 2000 Neuroscience 100:629-49). Labeling with a TIP39 selective antibody, performed using free floating 50 μm vibratome sections from paraformaldehyde perfused animals, was detected using Vectastain ABC reagents (Vector Laboratories, Burlingame CA) or by tyramide mediated amplification (Hunyady, B. et al. 1996 J Histochem Cytochem 44:1353-62). Incubation with TJP39 eliminated immunostaining. Some animals received an infraventricular injection of 80 μg of colchicine 48 hours before euthanasia.

Statistics. All data represent the mean ± SEM from a minimum of 5 separate experiments. While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of ameliorating pain comprising administering a compound that modulates a PTH2 receptor to a patient in need thereof.

2. Use of a compound that modulates a PTH2 receptor for the preparation of a medicament for the amelioration of pain.

3. A method of identifying a compound that can be used to modulate pain comprising: incubating a cell that expresses a PTH2 receptor, or membrane vesicle or membrane fraction thereof, in a presence and absence of a test compound; determining an amount of phosphorylation present on said PTH2 receptor in the presence and absence of said test compound; selecting said test compound that alters the amount of said phosphorylation; and identifying said selected compound as being a candidate compound useful for the modulation of pain.

4. A method of identifying a compound that can be used to modulate pain comprising: incubating a cell that expresses a PTH2 receptor, or membrane vesicle or membrane fraction thereof, in a presence and absence of a test compound; determining activity of said PTH2 receptor in the presence and absence of said test compound; selecting said test compound that alters the activity of said receptor; and identifying said selected compound as being a candidate compound useful for the modulation of pain.

5. A method of identifying a compound that can be used to modulate pain comprising: incubating a cell that expresses a PTH2 receptor, or membrane vesicle or membrane fraction thereof, in a presence and absence of a test compound; determining whether said test compound binds to said PTH2 receptor; selecting said test compound that binds to said PTH2 receptor; and identifying said selected compound as being a candidate compound useful for the modulation of pain.

6. A method of identifying a compound that can be used to modulate pain comprising: incubating a cell that expresses a PTH2 receptor, or membrane vesicle or membrane fraction thereof, in a presence and absence of a test compound; detecting a change in expression of a PTH2 receptor gene or a change in activity of a PTH2 receptor gene product expressed by the cell; selecting a test compound that alters said expression or activity; and identifying said selected compound as being a candidate compound useful for the modulation of pain.

7. A method of identifying a compound that can be used to modulate pain comprising: incubating a cell that expresses a PTH2 receptor, and said receptor being associated with a second component that provides a detectable signal in response to binding of a compound to said receptor, in a presence and absence of a test compound; determining whether said test compound binds to said PTH2 receptor by measuring a level of a signal generated from interaction of the test compound with said receptor; selecting said test compound that binds to said PTH2 receptor; and identifying said selected compound as being a candidate compound useful for the modulation of pain.

8. The method of Claim 7 which further comprises conducting the identification of the compound in a presence of labeled or unlabeled known agonist.

9. A method of identifying a compound that can be used to modulate pain comprising: incubating a cell that expresses a PTH2 receptor, or membrane vesicle or membrane fraction thereof, in a presence of a known ligand, and additionally in a presence and absence of a test compound; determining whether said test compound inhibits binding of said ligand to said PTH2 receptor by measuring an amount of said ligand bound to said receptor; selecting said test compound that causes reduction of binding of said ligand; and identifying said selected compound as being a candidate compound useful for the modulation of pain.

10. The method of Claim 9, wherein the ligand is labeled or unlabeled known agonist.

11. A method of making a pharmaceutical composition comprising combining the compound identified by any of Claims 3-10 with a pharmaceutically acceptable carrier.

12. A method of ameliorating pain comprising administering the pharmaceutical composition of Claim 11 to a patient in need thereof.