NZ501423A - Receptors for TGF-beta-related neurotrophic factors and use for treating disease - Google Patents

Abstract

A growth factor receptor, TrnR2, is disclosed and the human and mouse amino acid sequences are identified. Human and mouse TrnR2 cDNA sequences have been cloned and sequenced. The TrnR2 polypeptide may be used for promoting growth and differentiation of cells in a culture medium, or for treating a disease condition mediated by the expression of a TrnR2 polypeptide.

Description

WO 98/46622 PCT/US98/07996 RECEPTORS FOR TGF-B-RELATED NEUROTROPHIC FACTORS Reference to Government Grants This invention was made with government support under National Institutes of Health Grant Numbers R01 AG13729 and R01 AG13730. The government has certain 5 rights in this invention.

Related Applications This application claims the benefit of U.S. Provisional Application entitled TrnR2, A Novel Receptor Which Mediates Neurturin And GDNF Signaling Through Ret 10 filed April 17, 1997.

Background of the - Invention (1) Field of the Invention" This invention relates gatierally to receptors for trophic or growth factors and', - more particularly, to a 15 novel receptor for TGF-p-related neurotrophic* factors. (2) Description of the Related Art The development and maintenance of ^issues in complex organisms requires precise control over,the processes of cell proliferation, differentiation, 20 survival and function. A major mechanism whereby these processes are controlled is through the actions of polypeptides known as "growth factors". These structurally diverse molecules act through specific cell surface receptors to produce these actions. 25 Of particular importance are those growth factors, termed "neurotrophic factors", that promote the differentiation, growth and survival of neurons and reside in the nervous system or in innervated tissues. Nerve growth factor (NGF) was the fijrst neurotrophic 30 factor to be identified and characterizte<3 ,( Levi- Montalcini et al., J. Exp. Zool. 116:321, 1951 which is Printed from Mimosa 2 incorporated by reference). NGF exists as a non-covalently bound homodiraer that promotes the survival and growth of sympathetic, neural crest-derived sensory, and basal forebrain cholinergic neurons.

In recent years it has become apparent that growth factors fall into classes, i.e. families or superfamilies based upon the similarities in their amino acid sequences. Examples of such families that have been identified include the fibroblast growth factor family, 10 the neurotrophin family and the transforming growth factor-beta (TGF-B) family. As an example of family member sequence similarities, TGF-p family members have 7 canonical framework cysteine residues which identify members of this superfamily.

NGF is the prototype member of the neurotrophin family. Brain-derived neurotrophic factor (BDNF), the second member of this family to be discovered, was shown to be related to NGF by virtue of the conservation of all six cysteines that form the three internal disulfides of 20 the NGF monomer (Barde, Progr Growth Factor Res 2:237-248, 1990 and Liebrock et al. Nature 342:149-152, 1989 which are incorporated by reference). By utilizing the information provided by BDNF of the highly conserved portions of two factors, additional members (NT-3, NT-25 4/5) of this neurotrophin family were rapidly found by several groups (Klein, FASEB J 8:738-44, 1994 which is incorporated by reference).

Signal transduction for neurotrophins is mediated by a family of closely related tyrosine kinase receptors 30 (Trk). The known members of the Trk family of receptors are: TrkA, identified as the NGF receptor; TrkB, which mediates signaling by BDNF and NT-4/5; and TrkC, which transduces the signals of NT-3 (for review, see (Tuszynski et al., Ann Neurol 35:S9-S12, 1994 which is 35 incorporated by reference). In addition to these preferential specificities, there is evidence of cross- Printed from Mimosa IhicLLECTUAL PROPERTY OFFICE OF N.Z. 2 8 NOV 2001 RECEIVED J talk between different members of the neurotrophin and Trk families, particularly in fibroblast-based model systems. For example, NT-3 stimulates phosphorylation of TrkB expressed in fibroblasts with a dose-response 5 relationship equivalent to that of BDNF and NT-4/5 (Ip et al, Neuron 10:137-149, 1993, incorporated herein by reference), while in PC12 cells, the TrkB receptor is 100-fold more sensitive to stimulation by BDNF and NT-4/5 than by NT-3. NT-3 may also signal through TrkA, 10 although with different specificities than NGF (Ip et aJ , supra and Cordon-Cardo et al, Cell 66:173-183, 1991, incorporated herein by reference).

Recently, a new family of neurotrophic factors has been identified whose members are not structurally 15 related to NGF and other neurotrophins but are structurally similar to TGF-p. As described in PCT/US96/14065 and PCT/US97/03461 , which are incorporated herein by reference, the known members of this new family, which has been named TRN (TGF-p Related 20 Neurotrophic factors), are glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), and persephin (PSP).

The placement of human GDNF and NTN into the same growth factor family is based on the similarities of 25 their physical structures and biological activities.

These two proteins have 42% identity in their amino acid sequences including seven cysteine residues whose positions are exactly conserved in neurturin and GDNF. The biological activities of GDNF and NTN include 30 supporting the survival of rat superior cervical, nodose, and dorsal root ganglion neurons In vitro, although NTN is more potent than GDNF in promoting SCG survival (Kotzbauer et al., supra). In addition, as disclosed in the copending international patent application, WO 35 97/08196, which is incorporated herein by reference, the accumulation of radiolabeled NTN in the sensory neurons following injection into partially crushed scxatic nerves of rats can be blocked by a 100 fold excess of unlabeled NTN or unlabeled GDNF, suggesting that NTN and GDNF compete for the same receptor.

Recently, it was reported that GDNF acts through a multicomponent receptor complex in which a transmembrane signal transducing component, the Ret protein-tyrosine kinase (Ret or Ret PTK), is activated upon the binding of GDNF with another protein, called GDNF Receptor a (GDNFR-10 a) which has no transmembrane domain and is attached to the cell surface via a glycosyl-phosphatidylinositol (GP1) linkage (Durbec et al., Nature 381:789-793, 1996; Jing et al.. Cell 85:1113-1124, 1996; Treanor et al., Nature 382:80-83, 1996; Truppet al.. Nature 381:785-789, 15 1996, which are incorporated herein by reference). GDNF also induces activation of the Ret PTK when a soluble form of GDNFR-a is added to the culture medium along with GDNF, demonstrating that GDNFR-a does not need to be anchored to the cell membrane to interact with Ret (Jing 20 et al., supra). The formation of a functional GDNF receptor complex by GDNFR-a and Ret is supported, in part, by the observations that mice deficient in either GDNF or Ret are phenotypically similar and that GDNFR-a and Ret are expressed together in the developing nephron, 25 midbrain, and motor neurons, all known targets of GDNF action.

Neuronal degeneration and death occur during development, during senescence, and as a consequence of pathological events throughout life. It is now generally 30 believed that neurotrophic factors regulate many aspects of neuronal function, including survival and development in fetal life, and structural integrity and plasticity in adulthood. Since both acute nervous system injuries as well as chronic neurodegenerative diseases are 35 characterized by structural damage and, possibly, by disease-induced apoptosis, it is likely that neurotrophic Printed from Mimosa factors play some role in these afflictions. Indeed, a considerable body of evidence suggests that neurotrophic factors may be valuable therapeutic agents for treatment of these neurodegenerative conditions, which are perhaps 5 the most socially and economically destructive diseases now afflicting our society. Nevertheless, because different neurotrophic factors can act preferentially through different receptors and on different neuronal cell types, there remains a continuing need for the 10 identification and characterization of these receptors of growth factors in the diagnosis and treatment of a variety of acute and chronic diseases of the nervous system.

Summary of the Invention: Briefly, therefore, the present invention i"S directed to the identification and isolation of substantially purified polypeptides that mediate the survival and growth promoting effects of neurotrophic factors on neurons. Accordingly, the inventors herein 20 have succeeded in discovering that members of the TRN growth factor family share receptors and signal transduction pathways. In particular, the inventors have discovered that signaling of NTN and GDNF through the Ret tyrosine kinase receptor is mediated by a novel family of 25 co-receptors, referenced herein as TrnR (TGF-p-related neurotrophic factor Receptors). The TrnR co-receptor family includes the known co-receptor protein GDNFR-a, referred to herein as TrnRl, and a novel protein, TrnR2, either of which can form a functional receptor complex 30 with Ret for both NTN and GDNF.

The existence of this co-receptor family was established by the isolation and characterization of a cDNA encoding TrnR2 which shows significant homology with TrnRl. In particular, a comparison of their respective 35 predicted amino acid sequences revealed that human TrnRl and human TrnR2 are 48% identical at the amino acid level Printed from Mimosa 6 and share 30 of 31 cysteine residues with nearly identical spacing, indicating a conserved cysteine backbone structure. Both co-receptors also contain a predicted N-terminal signal sequence, a putative C-5 terminal GPI linkage signal peptide (GPIsp), and three potential N-linked glycosylation sites.

Recently, new nomenclature for this family of GPI-linked co-receptors for GDNF and neurturin has been adopted by scientists in the field such that the official 10 name for the TrnR family is now GFRa, with individual members of the family being named GFRal (previously known as GDNFRa, TrnRl and RetLl), GFRa2 (previously TrnR2, NTNRa and RetL2) and GFRa3 (previously TrnR3). Nomenclature Committee, Neuron 19(3)i485, 1997. However, 15 the older TrnR nomenclature will be used herein.

Accordingly, the invention provides a substantially purified TrnR2 polypeptide. It is believed that TrnR2 homologs of different mammalian species have at least 85% amino acid sequence identity while amino 20 acid sequence identity may be as low as 65% in TrnR2 homologs of non-mammalian species such as avian species. TrnR2 polypeptides identified herein include predicted precursor and mature forms of TrnR2 protein in which the predicted mature protein lacks the N-terminal signal 25 sequence and the C-terminal GPIsp but is otherwise identical to the precursor protein. Human precursor and mature proteins have the predicted amino acid sequences set forth in SEQ ID N0S:2 and 3, respectively. The corresponding predicted precursor and mature forms of 30 mouse TrnR2 protein have the amino acid sequences shown in SEQ ID N0S:5 and 6 (Figure 2). In addition, TrnR2 polypeptides of the invention include variants of human and mouse precursor proteins translated from alternatively spliced TrnR2 mRNA having the amino acid 35 sequences shown in SEQ ID N0S:7 and 8. Soluble TrnR2 polypeptides which lack a GPI anchor are also Printed from Mimosa 7 contemplated by the invention. Such soluble TrnR2 polypeptides include soluble forms of alternatively spliced variants of TrnR2. TrnR2 polypeptides also include biologically active fragments of the full-length 5 precursor or mature proteins which are capable of binding a TRN growth factor, or which are capable of activating the Ret PTK in the presence of the TRN growth factor, or which are capable of eliciting in a host animal antibodies specific for TrnR2.

The present invention also provides nucleotide sequences that encode a TrnR2 polypeptide. Human precursor and mature TrnR2 proteins are encoded by residues 36 to 1427 and residues 99 to 1331, respectively, of the nucleotide sequence set forth in SEQ 15 ID N0:1. Mouse precursor and mature TrnR2 proteins are encoded by residues 1 to 1389 and residues 64 to 1296, respectively, of the nucleotide sequence set forth in SEQ ID NO:4.

Expression vectors and stably transformed cells 20 are also provided. The transformed cells can be used in a method for producing a TrnR2 polypeptide.

In another embodiment, the present invention provides a method for preventing or treating neuronal degeneration comprising administering to a patient in 25 need thereof a therapeutically effective amount of a TrnR2 polypeptide, optionally along with a therapeutically effective amount of NTN or GDNF. A patient may also be treated by implanting transformed cells which express a TrnR2 polypeptide or a DNA sequence 30 which encodes TrnR2 into a patient's tissues which would benefit from increased sensitivity to a TRN such as NTN or GDNF. In another embodiment, a patient with neuronal degeneration is treated by implanting neuronal cells cultured and expanded by growth in the presence of TrnR2 35 and NTN or GDNF.

Printed from Mimosa 8 Another embodiment provides a method for treating tumor cells by administering a composition comprising an effective amount of TrnR2 and an effective amount of NTN or GDNF or a composition comprising DNA sequences 5 encoding TrnR2 and NTN or GDNF to produce a maturation and differentiation of the cells.

Yet another embodiment involves the use of a soluble TrnR2 polypeptide as an agonist of TRN growth factors.

In another embodiment the present invention provides isolated and purified TrnR2 antisense polynucleotides.

The present invention also provides compositions and methods for detecting TrnR2 expression. One method 15 detects TrnR2 protein using anti-TrnR2 antibodies and other methods are based upon detecting TrnR2 mRNA using recombinant DNA techniques.

Among the several advantages found to be achieved by the present invention, therefore, may be noted the 20 provision of a new co-receptor for neurturin and GDNF which mediates the ability of these growth factors to maintain and prevent the atrophy, degeneration or death of certain cells, in particular neurons; the provision of other members of the TrnR family of growth factor 25 receptors by making available new methods capable of obtaining said other family members; the provision of methods for obtaining TrnR2 by recombinant techniques; the provision of methods for preventing or treating diseases producing cellular degeneration and, 30 particularly, neuronal degeneration; the provision of methods for limiting the effects of TRN growth factors in a patient; and the provision of methods that can detect and monitor TrnR2 levels in a patient.

Printed from Mimosa Brief Description of the Drawings Figure 1 illustrates the homology of the amino acid sequences for the predicted precursor forms of TrnRl (human, SEQ ID N0:12; rat, SEQ ID N0:13) and TrnR2 5 (human, SEQ ID NO:2; mouse, SEQ ID NO:5) with identical amino acid residues enclosed in boxes and shared cysteine residues shaded; Figure 2 illustrates the nucleotide sequence (SEQ ID NO:4) and amino acid translation (SEQ ID NO:5) of the 10 long splice variant of precursor mouse TrnR2 with the predicted N-terminal signal sequence and C-terminal hydrophobic domain underlined, a potential GPI attachment site indicated by a asterisk, the potential N-linked glycosylation sites enclosed in boxes, and the amino acid 15 region missing in the short splice variant shaded; Figures 3A-C illustrate the effect of NTN and GDNF on Ret phosphorylation as detected by an immunoassay using antibodies specific for phosphotyrosine and Ret in (Fig. 3A) fibroblasts stably transfected with Ret alone 20 (Ret) or both Ret and the long splice variant of TrnR2 (Ret/TrnR2) and treated with GDNF or NTN or not treated (-), (Fig. 3B) fibroblasts expressing both Ret and TrnR2-LV which were pre-treated (+) or not treated (-) with phosphatidylinositol-specific phospholipase C (PIPLC) 25 before growth factor treatment, and (Fig. 3C) fibroblasts stably expressing both Ret and TrnR2-LV (TrnR2/Ret) or Ret and TrnRl (TrnRl/Ret) and treated with increasing amounts of NTN or GDNF; Figure 3D illustrates the effect of GDNF, NTN, and 30 persephin (PSP) on Ret tyrosine phosphorylation as detected by an immunoassay using antibodies specific for phosphotyrosine and Ret in fibroblasts coexpressing Ret and either the long splice variant of TrnR2 (TmR2-LV) or the short splice variant (TmR2-SV); Figure 3E illustrates the binding affinities of soluble TrnR2-LV fused with the Fc region of human IgGx Printed from Mimosa (R2-Ig) for GDNF, NTN and PSP as measured in an ELISA binding assay; Figure 4 illustrates the tissue distribution of TmR2 mRNA in adult mouse showing a Northern blot of 5 total RNA probed with a 32P-labeled TrnR2 cDNA fragment; Figures 5A-D illustrates the expression of TrnRl, TrnR2, and Ret in known sites of GDNF and/or NTN action showing in situ hybridization analysis using 33P-labeled RNA probes of tissue samples from (Fig. 5A) E14 mouse 10 (developing) ventral mesencephalon (vra), (Fig. 5B) adult mouse spinal cord, (Fig. 5C) E14 mouse (developing) kidney (k), gut (g) and dorsal root ganglia (drg), and (Fig. 5D) adult rat superior cervical ganglion (SCG); Figure 6 illustrates TrnRl, TrnR2, and Ret 15 expression in primary SCG cultures containing a contaminating population of non-neuronal cells showing the amount of different mRNAs at varying times after removal of nerve growth factor as measured by reverse transcription-polymerase chain reaction (RT-PCR) using 20 primers specific for Ret, TrnR2, neuron-specific enolase NSE, TrnRl, and a Schwann cell marker (S100); Figure 7 illustrates that expression of TrnRl, but not TrnR2, is up-regulated in the distal sciatic nerve after nerve injury as shown by Northern blot analysis of 25 total RNA isolated from normal sciatic nerve (N) and the distal segment of sciatic nerve seven days post-transection (7D) using 32P-labeled TrnRl and TrnR2 probes and brain RNA as a positive control for the detection of TrnR2 mRNA.

Figure 8 illustrates the expression of GF (TRN) receptors and neurturin in the adult mouse forebrain showing darkfield photographs of coronal sections analyzed by in situ hybridization using 33P-labeled riboprobes to detect expression of GFRa-1 (TrnRl) (Fig. 35 8A), GFRa-2 (TrnR2) (Fig. 8B), Ret (Fig. 8C) and NTN (Fig. 8D), in which the various regions are abbreviated Printed from Mimosa 11 as Cg—cingulate cortex, CI—claustrum, DBB-nucleus of the diagonal band of Broca, DEr>—dorsal endopiriform nucleus, LS—lateral septal nucleus, MS—medial septal nucleus, Pip—piriform cortex, Tu—olfactory tubercle, and 5 VP—ventral pallidum; Figure 9 illustrates the expression of GF (TRN) receptors and neurturin in in the neocortex, hippocampus, thalamus, and hypothalamus showing darkfield photographs of coronal sections of the adult mouse brain analyzed by 10 in situ hybridization using 33P-labeled riboprobes to detect expression of (Fig. 8) GFRa-1 (TrnRl), (Fig. 8B) GFRa-2 (TrnR2), (Fig. 8C) Ret, and (Fig. 8D) NTN, in which the various regions are abbreviated as Th-thalamic nuclei, A-emygdala, H—hypothalamus, LD—laterodorsal 15 nucleus of the thalamus, MD-mediodorsal nucleus of the thalamus, MHb-medial habenula, Rt—reticular thalamic nucleus, STh—subthalamic nucleus, and ZI—zona incerta; Figure 10 illustrates the expression of GF (TRN) receptor components in the adult mouse midbrain showing 20 darkfield photographs of coronal sections of adult mouse midbrain analyzed by in situ hybridization using 33P-labeled riboprobes to detect expression of (Fig. 10A) GFRa-1 (TrnRl) in the compacta region of the substantia nigra, the VTA, the oculomotor nucleus and the 25 superficial layers of the superior colliculus, (Fig. 10B) GFRa-2 in the compacta region of the substantia nigra, in the VTA, and the oculomotor nucleus, (Fig. 10C) Ret mRNA in the SNc and the VTA, in which the various regions are abbreviated as 3—oculomotor nucleus, SN-substantia nigra, 30 SuMM—supramammillary nucleus, MGN—medial geniculate nucleus, and VTA-ventral tegmental area; Figure 11 illustrates the expression of NTN mRNA in the supraoptic and paraventricular nuclei of the hypothalamus showing (Fig. 11A) a darkfield photograph 35 and (Fig. 11B-11C) brightfield photographs at higher magnification which show detection of NTN expression in Printed from Mimosa 12 magnocellular neurons in the supraoptic (Fig. 11B) and paraventricular (Fig. 11C) nuclei with the various regions abbreviated as PV-paraventricular nucleus, SO-supraoptic nucleus, and 3V-^third ventricle; 5 Figure 12 illustrates expression of GDNF and GF (TRN) receptor mRNA in adult mouse midbrain and brainstem showing darkfield photographs of coronal sections analyzed by in situ hybridization using 33P-labeled riboprobes to detect expression of (Fig. 12A) Ret mRNA in 10 cranial nerve nuclei 10 and 12, and in the gigantocellular reticular nucleus (Gi), (Fig. 12B) GFRa-2 (TrnR2) in cranial nerve nuclei Sp5, 6, 7 and ventral cochlear nucleus (VC). (Fig. 12C) GDNF in the VC and the facial motor nucleus, (Fig. 12D) GFRa-1 in the facial 15 motor nucleus and in the dorsal cochlear nucleus (DC), (Fig. 12E) GFRa-2 in the inferior colliculus the tegmental nuclei, and the locus coeruleus, (Fig. 12F) Ret mRNA in the trigeminal motor nucleus and the inferior colliculus, with the various regions being identified as 20 6—abducens nucleus, 7—facial nucleus, 10—vagal motor nucleus, 13—hypoglossal nucleus, Gi—gigantocellular reticular nucleus, IG—inferior colliculus, LG—locus coeruleus, Sp5—spinal trigeminal nucleus, Mo5—motor trigeminal nucleus, Tg-^tegmental nuclei, VG-ventral 25 cochlear nucleus; Figure 13 illustrates GF (TRN) receptor expression in adult mouse cervical spinal cord showing darkfield photographs of transverse sections analyzed by in situ hybridization using 33P-labeled riboprobes to detect 30 expression of (Fig. 13A) GFRa-1 (TrnRl), (Fig. 13B) GFRa-2 (TrnR2), and (Fig. 13C) Ret, with various regions identified as DH—dorsal horn, and VH-ventral horn; Figure 14 illustrates GF (TRN) receptor and NTN mRNA expression in adult cerebellum showing darkfield 35 photographs of sagittal sections analyzed by in situ hybridization using 33P-labeled riboprobes to detect Printed from Mimosa 13 expression of (Fig. 14A) GFRa-1 (TrnRl) in cells adjacent to Purkinje neurons in the Purkinje layer, (Fig. 14B) GFRa-2 (TrnR2) in the granule cell layer and in neurons that appear to be Purkinje cells in the Purkinje layer, 5 (Fig. 14C) Ret in the Purkinje layer in cells surrounding Purkinje neurons and in the molecular layer, and (Fig. 14D) NTN in the Purkinje and granule cell layers, with the various regions abbreviated as Gr—granule cell layer, P—Purkinje layer, Mot-wolecular layer.

Description of the Preferred Embodiments The present invention is based upon the surprising discovery that NTN, like GDNF, can stimulate Ret PTK through the known co-receptor GDNFR-a and thereby cause 15 the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI-3-K) intracellular signaling pathways. As the first co-receptor known to mediate signaling by at least two members of the TRN family of growth factors, GDNFR-a is 20 referred to herein as TrnRl. The unexpected discovery that members of the TRN family of growth factors share a receptor complex and signal transduction pathways led to the identification, isolation and sequencing of a cDNA encoding a novel second co-receptor for NTN and GDNF, 25 TrnR2. Prior to this invention, TrnR2 was unknown and had not been identified as a discrete biologically active substance, nor had it been isolated in pure form.

TrnR2 was identified by searching a database of Expressed Sequence Tags (dbEST database) using the Basic 30 local alignment search tool (BLAST, Altschul et al., J.Mol.Biol. 215:403-410, 1990 incorporated herein by reference) and the full length rat TrnRl protein sequence (GenBank Accession No. U59486) as a query. Three human ESTs (H12981, R02135, W73681) which showed only partial, 35 but significant, homology to rat TrnRl were identified by the BLAST search. The determination and alignment of the Printed from Mimosa 14 complete sequences of these three ESTs, obtained from the WashU-Merck EST project, indicated that they all encoded partial cDNAs of an identical transcript.

The 5' end of the cDNA was obtained by the rapid 5 amplification of cDNA ends (RACE) technique using as templates human brain and placenta cDNA libraries (Marathon RACE libraries, Clontech, Palo Alto, CA) and the Klentaq LA polymerase chain reaction (PCR) technique described by Barnes, Proc.Natl.Acad.Sci.U.S.A. 91:2216-10 2220, 1994, incorporated herein by reference. Two alternatively spliced forms of TrnR2 mRNA were identified in both brain and placenta, the short splice variant (TrnR2-SV) is missing 399 nucleotides of the coding sequence from the 5' end of the long splice variant 15 (TrnR2-LV) (residues 75 to 473 of SEQ ID NO:l). The predicted amino acid sequence of the long splice variant of human precursor TrnR2 contains 464 amino acids and is shown in SEQ ID NO:2, the predicted amino acid sequence for the mature protein is shown in SEQ ID NO:3. The 20 short splice variant has a predicted amino acid sequence of 331 amino acids as set forth in SEQ ID NO:7^, which would be encoded by nucleotides 36-74 and 474-1427 of SEQ ID NO:1. The corresponding mouse cDNAs for both the long and short splice variants were also obtained by PCR, 25 using a brain cDNA template. The full length precursor murine cDNA is set forth in SEQ ID NO:4 and contains a single long open reading frame (ORF) encoding a predicted protein of 463 amino acids (SEQ ID N0:5); the short splice variant identified has an ORP encoding a predicted 30 330 amino acid polypeptide (SEQ ID NO:8) which would be encoded by nucleotides 1-39 and 438-1389 of SEQ ID NO:4.

All physical features of TrnR2 indicate that it is closely related to TrnRl. As shown in Figure 1, the predicted amino acid sequence for TrnR2-LV shows 35 significant homology with TrnRl. (The human and rat TrnRl sequences (SEQ ID Nos. 12 and 13, respectively) are those Printed from Mimosa reported by Jing et al., supra.) The predicted protein for precursor TrnR2 contains a putative 21 amino acid signal sequence (residues 1-21 of SEQ ID NO:2 and SEQ ID NO:5 for human and mouse proteins, respectively) at the 5 amino terminus, three potential N-linked glycosylation sites, and has a stretch of 16 carboxyl-terminal hydrophobic amino acids (residues 449-464 of SEQ ID NO:2 and 448-463 of SEQ ID NO:5 for human and mouse proteins, respectively). The presence of the N- and C-terminal 10 hydrophobic regions indicates that mature TrnR2 is potentially a GPI-linked protein (Udenfriend and Kodukula, Ann. Rev. Biochem. 64:563-591, 1995, incorporated herein by reference), as has been demonstrated for TrnRl (Treanor et al. supra; Jing et 15 al., supra). A potential GPI attachment site for the human and mouse long splice variants is the glycine residue at position 411 of SEQ ID NO:2 and 3, respectively. Accordingly, the predicted GPI attachment site in the short splice variant is the glycine residue 20 at position 299 of SEQ ID NO:7 for the human protein and at position 299 of SEQ ID NO:8 for the mouse protein.

The inventors herein have found significant functional similarities and dissimilarities between TrnR2 and TrnRl. Experimental data which is discussed below 25 indicate that either NTN or GDNF can activate Ret in the presence of either TrnR2 or TrnRl. In addition. Ret activation in the presence of either co-receptor responds to stimulation with either NTN or GDNF in a dose-dependent manner. However, while the TrnRl/Ret receptor 30 complex responds equivalently to the same amounts of each ligand, the TrnR2/Ret complex is more sensitive to NTN than GDNF, indicating that, at least in the model system examined, the latter complex may function preferentially as a NTN receptor. Also, TrnRl and TrnR2 are expressed 35 in a partially overlapping manner. While both co- receptors are expressed in the dorsal root ganglia (DRG) Printed from Mimosa 16 and the brain, TrnR2 is not expressed or expressed at very low levels in several known targets of GDNF action in which both TrnRl and Ret are expressed, including embryonic and adult nigra, motor neurons, gut and kidney. 5 In contrast, TrnR2 and Ret appear to comprise the expressed receptor complex in SCG neurons.

Although the data indicate that a physiological pairing of ligand and receptor may exist for the TrnR family as it does for the Trk family, there is also in 10 vivo evidence of cross-talk between the TRN ligands and their receptors as observed in several ligand-receptor systems having multiple family members, including between members of the neurotrophin growth factor and Trk receptor families. For example, the inventors herein 15 have found that neuroblastoma cell lines may express either TrnRl, TrnR2 or both, but despite this heterogeneity, those cell lines which respond to GDNF or NTN always respond to both factors. Thus, it is believed that TrnR2, TrnRl or other as yet unidentified members of 20 the TrnR family can combine in vivo with Ret to form a functional receptor complex for NTN and GDNF, and possibly for persephin and other as yet unidentified members of the TRN growth factor family as well.

Accordingly, the invention provides a 25 substantially purified TrnR2 polypeptide. A TrnR2 polypeptide of the invention includes growth factor receptors of any origin which are substantially homologous to and which are biologically equivalent to the human or mouse TrnR2 polypeptides characterized and 30 described herein. Such substantially homologous growth factor receptors may be native to any tissue or species and, similarly, biological activity can be characterized in any of a number of biological assay systems.

The term "biologically equivalent" is intended to 35 mean that the compositions of the present invention are capable of demonstrating some or all of the same signal Printed from Mimosa 17 mediating properties in a similar fashion, not necessarily to the same degree, as the recombinantly produced human or mouse TrnR2.

By "substantially homologous" it is meant that the 5 degree of amino acid homology of human or mouse TrnR2 to a TrnR2 from any species is greater than that between TrnR2 and TrnRl (GDNFR-a).

Sequence identity or percent identity is intended to mean the percentage of identical residues between two 10 sequences, referenced to human TrnR2 when determining percent identity with non-human TrnR2, referenced to TrnR2 when determining percent identity with non-TrnR2 growth factor receptors and referenced to human TrnRl when determining percent identity of non-TrnR2 growth 15 factor receptors with TrnRl, when-the two sequences are aligned using the Clustlal method (Higgins et al, Cabios 8:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, WI). In this method, multiple alignments are carried out 20 in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments. Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores 25 between the separate residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related proteins over a given evolutionary interval. Penalties for opening and lengthening gaps in the 30 alignment contribute to the score. The default parameters used with this program are as follows: gap penalty for multiple alignment = 10; gap length penalty for multiple alignment = 10; k-tuple value in pairwise alignment = 1; gap penalty in pairwise alignment = 3; 35 window value in pairwise alignment = 5; diagonals saved in pairwise alignment = 5. The residue weight table used Printed from Mimosa WO 98/46622 PCT/US98/07996 18 for the alignment program is PAM250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl. 3, p. 345, 1978).

Percent conservation is calculated from the above 5 alignment by adding the percentage of identical residues to the percentage of positions at which the two residues represent a conservative substitution (defined as having a log odds value of greater than or equal to 0.3 in the PAM250 residue weight table). Conservation is referenced 10 to human TrnR2 when determining percent conservation with non-human TrnR2, and referenced to TrnR2 when determining percent conservation with non-TrnR2 growth factor receptors. Conservative amino acid changes satisfying this requirement are: R-K; E-D, Y-F, L-M; V-I, Q-H. The 15 calculations of identity (I) and conservation (C) between human and mouse TrnR2 (hTrnR2 and mTrnR2, respectively) and between each of these and human and rat TrnRl (hTrnRl and rTrnRl, respectively) are shown in Table 1.

Table 1 COMPARISON % IDENTITY % CONSERVATION hTrnR2 v. mTrnR2 94 95 hTrnR2 v. hTrnRl 48 53 hTrnR2 v. rTrnRl 47 52 mTrnR2 v. hTrnRl 43 47 mTrnR2 v. rTrnRl 47 52 The degree of homology between the predicted \ precursor mouse and human TrnR2 proteins is about 94% sequence identity and all TrnR2 homologs of non-human mammalian species are believed to have at least about 85% sequence identity with human TrnR2. For non-mammalian 35 species such as avian species, it is believed that the degree of homology with TrnR2 is at least about 65% identity. By way of comparison, the variations between members of the TrnR family of receptors can be seen by comparing TrnRl and TrnR2 (Fig. 1). Human and mouse 40 precursor TrnR2 share about 94% identical amino acids and Printed from Mimosa have about 53% and 52% sequence conservation with human and rat precursor TrnRl, respectively. It is believed that the different TrnR family members similarly have a sequence identity of about 40% to that of TrnR2 and about 5 40% to that of TrnRl and within a range of about 30% to about 85% identity with TrnR2 and within a range of about 30% to about 85% sequence identity with TrnRl. Thus, a given non-TrnR2 and non-TrnRl family member from one species would be expected to show lesser sequence 10 identity with TrnR2 and with TrnRl from the same species than the sequence identity between human TrnR2 and TrnR2 from a non-human mammalian species, but greater sequence identity than that between human TrnR2 and any other known growth factor receptor except TrnRl. 15 A TrnR2 polypeptide of the invention can also include hybrid and modified forms of TrnR2 and fragments thereof in which certain amino acids have been deleted or replaced and modifications such as where one or more amino acids have been changed to a modified amino acid or 20 unusual amino acid and modifications such as glycosylations so long as the hybrid or modified form retains TrnR2 biological activity. By TrnR2 biological activity, it is meant that Ret PTK is activated in the presence of the hybrid or modified TrnR2 and NTN or GDNF 25 or other TRN growth factor, although not necessarily at the same level of potency as that of TrnR2 isolated from tissues or cells which naturally produce TrnR2 such as SCG neurons or that of the recombinantly produced human or mouse TrnR2.

Also included within the meaning of substantially homologous is any TrnR2 polypeptide which may be isolated by virtue of cross-reactivity with antibodies to the TrnR2 described herein or whose encoding nucleotide sequences including genomic DNA, mRNA or cDNA may be 35 isolated through degenerate PCR or by hybridization with the complementary sequence of genomic or subgenomic Printed from Mimosa nucleotide sequences or cDNA of the human or mouse TrnR2 described herein or fragments thereof. It will also be appreciated by one skilled in the art that allelic variants of TrnR2 are included within the present 5 invention.

Isolation of cDNAs corresponding to two alternatively spliced TrnR2 mRNAs of different lengths indicate a TrnR2 protein product for each spliced variant may exist. Thus, any and all TrnR2 proteins encoded by 10 alternatively spliced TrnR2 mRNAs are intended to be included within the term "TrnR2 polypeptide" as used herein.

The predicted amino acid sequence and biological function of TrnR2 indicate that it is an externally 15 disposed plasma membrane protein anchored to the extracellular surface of the cell membrane by a glycosyl-phosphatidyl (GPI) linkage. It is well-known in the art that GPI anchored membrane proteins are synthesized as a precursor protein with an N-terminal signal sequence and 20 a C-terminal GPIsp which are cleaved during the cellular processing events leading to the mature protein. Thus, all precursor TrnR2 proteins containing either or both of the signal sequence and GPIsp as well as mature TrnR2 proteins which contain neither signal peptide are 25 embraced by the term "TrnR2 polypeptide". It is also well-recognized in the art that GPI anchored proteins may exist in soluble form following cleavage of the GPI linkage with phospholipases. Therefore, the term "TrnR2 polypeptide also includes soluble TrnR2 polypeptides 30 generated by phospholipase cleavage of anchored TrnR2 polypeptides.

A preferred TrnR2 polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID N0:3, SEQ ID N0:5, and SEQ ID N0:6. A more 35 preferred TmR2 polypeptide is human mature TrnR2 protein which has the amino acid sequence set forth in SEQ ID Printed from Mimosa 21 NO: 3.

The term "TrnR2 polypeptide" is intended to include fragments which have one or more of the biological activities of precursor or mature TrnR2 protein. Such 5 activities include binding a member of the TRN growth factor family, particularly NTN or GDNF, and binding to Ret in the presence of NTN, GDNF, or other TRN family member, with such binding leading to Ret phosphorylation. It is believed that by using the nucleotide sequences 10 encoding precursor TrnR2, which are provided herein, those skilled in the art can readily construct multiple TrnR2 fragments and screen them for the desired biological activity.

A preferred TrnR2 fragment is one which lacks the 15 hydrophobic domain for the GPI-attachment site which are referred to herein as soluble TrnR2 fragments. For example, soluble TrnR2 fragments include, but are not limited to, polypeptides having an amino acid sequence encoded by nucleotide residues 99 to 1331 of SEQ ID N0:1 20 (human long splice variant), nucleotides 36-74 and 474-1331 of SEQ ID N0:1 (human short splice variant), nucleotide residues 64-1296 of SEQ ID NO:4 (mouse long splice varaint), nucleotides 1-39 and 438-1296 (mouse short splice variant), and fragments thereof.

* TrnR2 fragments also included in the scope of the invention are antigenic fragments which are capable of eliciting TrnR2 specific antibodies when administered to a host animal as conjugated to a carrier molecule or in nonconjugated form.

A TrnR2 protein of the present invention may be Isolated in purified form from tissues or cells which naturally produce TrnR2. Such tissues or cells may originate from any eukaryotic organism that naturally produce TrnR2. Alternatively, a substantially pure TrnR2 35 polypeptide may be prepared by recombinant DNA technology. By "pure form" or "purified form" or Printed from Mimosa 22 "substantially purified form" it is meant that a TrnR2 composition is substantially free of other proteins which are not TrnR2.

One skilled in the art can readily follow known 5 methods for isolating proteins in order to obtain the TrnR2 polypeptide substantially free of other proteins, including immunochromatography, size-exclusion chromatography, HPLC, ion-exchange chromatography, and ligand affinity chromatography. As readily appreciated 10 by those skilled in the art, an example of one way to obtain TmR2 protein naturally produced by cells in culture would be to treat the cells with PI-PLC to cleave the GPI-linked TrnR2 protein from the cell surface, and then purifying the soluble TrnR2 protein from the media 15 by ligand affinity chromatography using NTN or GDNF as the ligand or by immunochromatography using an antibody raised against TrnR2 protein or an antigenic TrnR2 peptide.

A recombinant TrnR2 polypeptide may be made by 20 expressing the DNA sequences encoding TrnR2 in a suitable transformed host cell. Using methods well known in the art, the DNA encoding the TmR2 polypeptide may be linked to an expression vector, transformed into a host cell and conditions established that are suitable for expression 25 of the TrnR2 polypeptide by the transformed cell.

Any suitable expression vector may be employed to produce recombinant TrnR2 such as, for example, the mammalian expression vector pCMV-neo (Brewer, Meth.Cell Biol. 43i233-245, 1994, incorporated herein by reference) 30 which was used herein or the E. coll pET expression vectors, in particular, pET-30a (Studier et al.. Methods Enzymol. 185:60-89, 1990 which is incorporated by reference). Other suitable expression vectors for expression in mammalian and bacterial cells are known in 35 the art as are expression vectors for use in yeast or insect cells. Baculovirus expression systems can also be Printed from Mimosa 23 employed.

In another embodiment, the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence that encodes a TrnR2 5 polypeptide. Nucleotide sequences included in the invention are those encoding human or mouse precursor and mature TrnR2 proteins. Preferred nucleotide sequences encoding human proteins are as set forth in SEQ ID N0:1: nucleotides 36-1427 encode a precursor TrnR2 and 10 nucleotides 99-1331 encode a human mature TrnR2.

Similarly, preferred nucleotide sequences which encode a mouse precursor and a mouse mature protein are nucleotides 1-1389 and nucleotides 64-1296 of SEQ ID NO:4, respectively. Nucleotide sequences encoding TrnR2 15 fragments are also contemplated, particularly soluble TmR2 fragments. Preferred nucleotide sequences encoding a soluble TrnR2 fragment are residues 99 to 1331 of SEQ ID N0:1 (human long splice variant), nucleotides 36-74 and 474-1427 of SEQ ID N0:1 (human short splice variant), 20 residues 64-1296 of SEQ ID N0:4 (mouse long splice variant) and nucleotides 1-39 and 438-1389 of SEQ ID NO:4 (mouse short splice variant). It is understood by the skilled artisan that degenerate DNA sequences can encode the TrnR2 polypeptides described herein and these are 25 also intended to be included within the present invention.

Based upon the high sequence conservation between the human and mouse TrnR2 coding sequences, it is believed that DNA probes and primers can be made and used 30 to readily obtain TrnR2-encoding cDNA clones from different species. Thus, a cDNA encoding a TrnR2 from a species other than human or mouse is embraced by the invention.

Also included within the scope of this invention are 35 nucleotide sequences that are substantially the same as a nucleic acid sequence encoding TrnR2. Substantially the Printed from Mimosa 24 same sequences may, for example, be substituted with codons more readily expressed in a given host cell such as E. coll according to well known and standard procedures. Such modified nucleic acid sequences would 5 be included within the scope of this invention.

Specific nucleic acid sequences can be modified by those skilled in the art and, thus, all nucleic acid sequences which encode for the amino acid sequences of TrnR2 or biologically active fragments thereof can 10 likewise be so modified. The present invention thus also includes polynucleotides containing a nucleic acid sequence which will hybridize with all such nucleic acid sequences — or complements of the nucleic acid sequences where appropriate — and encode for a polypeptide having 15 biological activity as a coreceptor for NTN or GDNF. The present invention also includes nucleic acid sequences which encode for polypeptides that have one or more of the biological activities of TrnR2 and those that are recognized by antibodies that bind to TrnR2. 20 The cDNA sequences provided herein allow genomic clones for the TrnR2 gene to be readily isolated. One use for genomic clones is for chromosome localization studies. For example, human and mouse genomic clones for the TmR2 gene were obtained by screening PI (mouse) and 25 PAC (human) genomic libraries (Genome Systems, St. Louis, MO) with a PCR assay using primers derived from the TrnR2 coding region. A human PAC genomic clone containing the TrnR2 gene in a 120 kb genomic fragment was used to localize the TrnR2 gene to the short arm of human 30 chromosome 8 in region pl2-21 by fluorescence in situ hybridization analysis (FISH). The mouse chromosomal location can readily be determined in a similar fashion using a mouse genomic clone.

A search of the database for neurological diseases 35 genetically mapped to the human locus revealed only one such disease, SPG5A, an autosomal recessive form of Printed from Mimosa spastic paraplegia, localized to the paracentric region of chromosome 8 (Hentati et al.. Hum.Molec.Genet. 3:1263-1267, 1994, incorporated herein by reference). Also, an amplification event on 8pl2 has been observed in some 5 cases of breast and ovarian cancer (Imbert et al., Genomics 32:29-38, 1996, incorporated herein by reference). Genomic clones for the TrnR2 gene are also useful for surveying for possible gene or chromosome rearrangements in patients suffering from a neurological 10 disease with no identified cause.

The present invention also encompasses vectors comprising expression regulatory elements operably linked to any of the nucleic acid, sequences included within the scope of the invention. This invention also includes 15 host cells, of any variety, that have been transformed with vectors comprising expression regulatory elements operably linked to any of the nucleic acid sequences included within the scope of the present invention.

In one embodiment, recombinant cells expressing both 20 Ret and TrnR2 are provided which are useful for screening compounds for TRN growth factor agonistic or antagonistic activity. The recombinant cells may be produced by transforming a suitable host cell such as fibroblasts with nucleotide sequences encoding for expression Ret and 25 TrnR2 proteins. The protein-encoding nucleotide sequences may be on the same or on different vectors. Ret-encoding nucleotide sequences may be readily isolated by screening a suitable cDNA library using an oligonucleotide probe corresponding to a region of the 30 known human and/or mouse amino acid sequences (Iwamoto, et al.. Oncogene 8, 1087-1091, 1993 incorporated herein by reference). Suitable cDNA libraries would be those prepared from tissues known to express Ret, including but not limited to placental tissue.

Agonistic or antagonistic activity of a test compound would be determined by incubating the target Printed from Mimosa Ret/TrnR2-expressing cells with the test compound in the absence or presence of a TRN growth factor such as NTN, GDNF, or persephin and assaying for Ret protein tyrosine kinase activity. Compounds which increase Ret PTK 5 activity in the absence of a TRN have agonistic activity, while compounds which reduce or block Ret PTK activity in the presence of a TRN are TRN antagonists.

The Ret PTK activity may be assayed by looking for tyrosine phosphorylation of Ret as described herein. 10 Alternatively, or additionally, the target cell may be engineered to include a reporter gene whose expression is under the control of a TRN-responsive enhancer/promoter region. NTN and GDNF are known to cause an increase in mitogen-activated protein kinase (MAPK) activation in SCG 15 neurons (Kotzbauer, et al., Nature 384:467-470, 1996 incorporated herein by reference) and the inventors herein have also discovered that phosphatidylinositol 3-kinase (PI-3-K) is also activated by NTN and GDNF. Thus, expression of a reporter gene operably linked to the 20 enhancer/promoter regions of genes downstream in the MAPK or PI-3-K intracellular signalling pathways would be expected to be increased in the presence of a TRN agonist and decreased in the presence of a TRN antagonist. It is believed that such enhancer/promoter regions are known to 25 those skilled in the art and can be readily isolated.

Known reporter genes which encode for readily detectable products include, but are not limited to, p-galactosidase, chloramphenicol acetyl transferase, luciferase and p-glucuronidase. Detection of the 30 expression of known reporter genes, which is well known to those skilled in the art, may serve as a sensitive indicator for any NTN or GDNF agonistic activity of test compounds.

Methods are also provided herein for producing TrnR2 35 polypeptides. Preparation can be by isolation from a variety of cell types so long as the cell type expresses Printed from Mimosa TrnR2 protein. Examples of biological material suitable for TrnR2 isolation include, but are not limited to, brain tissue, human neuroblastoma cell lines, and superior cervical ganglion cells. A second and preferred 5 method involves utilization of recombinant methods by isolating a nucleic acid sequence encoding a TrnR2 polypeptide, cloning the sequence along with appropriate regulatory sequences into suitable vectors and cell types, and expressing the sequence to produce TrnR2. In 10 one embodiment, the nucleotide sequence does not encode the C-terminal hydrophobic domain containing the GPI-attachment site, thus producing a soluble TrnR2 fragment that is secreted into the growth medium.

The present invention also provides probes which may 15 be used to identify cells and tissues which may be responsive to NTN or GDNF in normal or disease conditions by detecting TrnR2 expression in such cells. Detection of TrnR2 expression may also be useful to determine if a patient suffering from a NTN- or GDNF-related disorder 20 has aberrant TrnR2 expression or expresses a biologically inactive TrnR2 mutant. TrnR2 expression may be detected with probes which react with TrnR2 mRNA or TrnR2 protein.

For example, to detect the presence of mRNA encoding a TrnR2 polypeptide or biologically inactive mutant 25 thereof, a sample is obtained from a patient. The sample may be from blood or a tissue biopsy. The sample may be treated to extract the nucleic acids contained therein which may then be subjected to gel electrophoresis or other size separation techniques.

The mRNA of the sample is contacted with a polynucleotide probe comprising a nucleic acid sequence complementary to TrnR2 mRNA. The polynucleotide probe may be an oligonucleotide containing a minimum of about 8 to 12, preferably at least about 20, contiguous 35 nucleotides which are complementary to the TrnR2 target sequence, oligonucleotide probes may be prepared by any Printed from Mimosa 28 method known in the art such as, for example, excision, transcription or chemical synthesis. Alternatively, the polynucleotide probe may comprise a cDNA encoding TrnR2 or a fragment thereof as a probe.

To enable detection of hybridization between the polynucleotide probe and the target sequence, the probe may be labelled with any detectable label known in the art such as, for example, radioactive or fluorescent labels or enzymatic markers. Labeling of the probe can 10 be accomplished by any method known in the art such as by PCR, random priming, end labelling, nick translation or the like. One skilled in the art will also recognize that other methods not employing a labelled probe can be used to determine the hybridization. Examples of methods 15 that can be used for detecting hybridization include Southern blotting, fluorescence in situ hybridization, and single-strand conformation polymorphism with PCR amplification.

Hybridization conditions for the type of probe used 20 may be readily determined by those skilled in the art. High stringency conditions are preferred in order to prevent false positives. The stringency of hybridization is determined by a number of factors xn the hybridization and washing steps. Such factors are well known to those 25 skilled in the art and outlined in, for example, Sambrook et al. (Sambrook, et al., 1989, supra).

The sensitivity of detection in a sample of TrnR2 mRNA may be increased using the technique of reverse transcription/polymerization chain reaction (RT/PCR) to 30 amplify cDNA transcribed from TrnR2 mRNA using primers specific for a TrnR2-encoding nucleotide sequence (see example 4 and Fig. 6 below). The method of RT/PCR is well known and routinely performed by those skilled in the art.

The present invention further provides for methods to detect the presence of the TrnR2 protein or Printed from Mimosa 29 biologically inactive mutants thereof in a sample obtained from a patient. Any method known in the art for detecting proteins can be used. Such methods include, but are not limited to immunodiffusion, 5 Immunoelectrophoresis, immunochemical methods, binder-ligand assays, immunohistochemical techniques, agglutination and complement assays. (For example, see Basic and Clinical Immunology, Sites and Terr, eds., Appleton & Lange, Norwalk, Conn, pp 217-262, 1991 which 10 is incorporated by reference). Preferred are binder-ligand immunoassay methods which involve reacting antibodies with an epitope or epitopes of a TrnR2 protein or derivative thereof to competitively displace a labeled TrnR2 polypeptide.

As used herein, a derivative.of the TrnR2 protein is intended to include a polypeptide in which certain amino acids have been deleted or replaced or changed to modified or unusual amino acids wherein the derivative is biologically equivalent to TrnR2 and wherein the 20 polypeptide derivative cross-reacts with antibodies raised against the TrnR2 protein. By cross-reaction it is meant that an antibody reacts with an antigen other than the one that induced its formation.

Numerous competitive and non-competitive protein 25 binding immunoassays are well known in the art.

Antibodies as used herein are intended to include full-length anti-TrnR2 antibody molecules and TrnR2 binding fragments of such antibody molecules. The anti-TrnR2 antibody may be unlabeled, for example as used in 30 agglutination tests, or labeled for use in a wide variety of assay methods. Labels that can be used include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes and the like for use in radioimmunoassay 35 (RIA), enzyme immunoassays, e.g., enzyme-linked immunosorbent assay (EL1SA), fluorescent immunoassays and Printed from Mimosa the like.

Polyclonal or monoclonal antibodies to the TrnR2 protein or an epitope thereof can be made for use in immunoassays by any of a number of methods known in the 5 art. By epitope reference is made to an antigenic determinant of a polypeptide. An epitope could comprise 3 amino acids in a spacial conformation which is unique to the epitope. Generally an epitope consists of at least 5 such amino acids. Methods of determining the 10 spatial conformation of amino acids are known in the art, and include, for example, x-ray crystallography and 2 dimensional nuclear magnetic resonance.

One approach for preparing antibodies to a protein is the selection and preparation of an amino acid 15 sequence of all or part of the protein, chemically synthesizing the sequence and injecting it into an appropriate animal, usually a rabbit or a mouse (See Example 11).

Oligopeptides can be selected as candidates for the 20 production of an antibody to the TrnR2 protein based upon the oligopeptides lying in hydrophilic regions, which are thus likely to be exposed in the mature protein.

Antibodies to TrnR2 can also be raised against oligopeptides that include one or more of the conserved 25 regions identified herein such that the antibody can cross-react with other family members. Such antibodies can be used to identify and isolate the other family members.

Methods for preparation of the TrnR2 protein or an 30 epitope thereof include, but are not limited to chemical synthesis, recombinant DNA techniques or isolation from biological samples. Chemical synthesis of a peptide can be performed, for example, by the classical Merrifeld method of solid phase peptide synthesis (Merrifeld, J Am 35 Chem Soc 85:2149, 1963 which is incorporated by reference) or the FMOC strategy on a Rapid Automated Printed from Mimosa 31 Multiple peptide Synthesis system (DuPont Company, Wilmington, DE) (Caprino and Han, J Org Chem 37:3404, 1972 which is incorporated by reference).

Polyclonal antibodies can be prepared by immunizing 5 rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate intervals. The animals are bled and sera assayed against purified TrnR2 protein, usually by ELISA or by bioassay based upon the ability to block one or more of the biological activities of TrnR2. 10 When using avian species, e.g. chicken, turkey and the like, the antibody can be isolated from the yolk of the egg. Monoclonal antibodies can be prepared after the method of Milstein and Kohler by fusing splenocytes from immunized mice with continuously replicating tumor cells 15 such as myeloma or lymphoma cells. (Milstein and Kohler Nature 256:495-497, 1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone and Banatis eds.. Academic Press, 1981 which are incorporated by reference). The hybridoma cells so 20 formed are then cloned by limiting dilution methods and supernates assayed for antibody production by ELISA, RIA or bioassay.

If a patient suffering from a GDNF or NTN-related disorder expresses apparently normal levels of TrnR2, it 25 may be possible that the expressed TrnR2 may be a biologically inactive mutant. To verify this, cDNA obtained from mRNA isolated from a sample of a relevant target tissue may be sequenced using methods known in the art.

The present invention also includes therapeutic or pharmaceutical compositions comprising an effective amount of a TrnR2 polypeptide for treating patients with cellular degeneration and a method for promoting cell survival which comprises administering to a patient in 35 need thereof a therapeutically effective amount of a TrnR2 polypeptide.

Printed from Mimosa 32 Certain degeneration disorders may be related to a lack of or reduced expression of biologically active TrnR2, while NTN or GDNF expression is normal. In addition, it may be desirable under certain circumstances 5 to increase TrnR2 levels even where TrnR2 expression is not decreased. It has been shown that soluble TrnRl added to Ret-expressing cells, i.e., the TrnRl is not bound to the membrane, can activate Ret in the presence of GDNF (Jing et al, supra). Thus, it is believed that 10 administering a TrnR2 polypeptide will increase the number of cells which have a functional TrnR2/Ret receptor complex and thus capable of responding to endogenously produced NTN and/or GDNF.

Additional survival or growth promoting effects may 15 be achieved by administering NTN and/or GDNF along with the TmR2 polypeptide. It is believed that treatment with one or both of these growth factors together with a TrnR co-receptor would increase the sensitivity of cells normally responsive to the growth factor(s). In 20 addition, such treatment would be expected to promote the survival or growth of other cell types that express Ret but that are not normally responsive to NTN or GDNF.

Alternatively, expression of TrnR2 could be increased in tissues defective in such expression by gene 25 therapy. Patients may be implanted with vectors or cells capable of producing a biologically-active TrnR2 polypeptide. In one approach, cells that secrete soluble TrnR2 may be encapsulated into semipermeable membranes for implantation into a patient. The cells can be those 30 that normally express a TrnR2 protein or the cells can be transformed to express a TrnR2 polypeptide. When the patient is human, it is preferred that the TrnR2 be human TrnR2. However, the formulations and methods herein can be used for veterinary as well as human applications and 35 the term "patient" as used herein is intended to include human and veterinary patients.

Printed from Mimosa 33 Cells can be grown ex vivo, for example, for use in transplantation or engraftment into patients (Muench et al., Leuk & Lymph 16:1-11, 1994 which is incorporated by reference). TrnR2 in combination with NTN or GDNF can be 5 administered to such cells to elicit growth and differentiation, provided the cells express Ret. Ret expression has been observed during embryogenesis in many cell lineages of the developing central and peripheral nervous systems. Ret has also been detected outside the 10 nervous system as well, including gut and kidney. Thus, in another embodiment of the present invention, a composition comprising TrnR2 and NTN or GDNF is used to promote the ex vivo expansion of Ret-expressing cells for transplantation or engraftment.

These compositions and methods are useful for treating a number of degenerative diseases. Where the cellular degeneration involves neuronal degeneration, the diseases include, but are not limited to peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's 20 disease, Parkinson's disease, Huntington's disease, ischemic stroke, acute brain Injury, acute spinal chord injury, nervous system tumors, multiple sclerosis, peripheral nerve trauma or injury, exposure to neurotoxins, metabolic diseases such as diabetes or renal 25 dysfunctions and damage caused by infectious agents.

Where the cellular degeneration involves bone marrow cell degeneration, the diseases include, but are not limited to disorders of insufficient blood cells such as, for example, leukopenias including eosinopenia and/or 30 basopenia, lymphopenia, monocytopenia, neutropenia, anemias, thrombocytopenia as well as an insufficiency of stem cells for any of the above. The above cells and tissues can also be treated for depressed function.

The compositions and methods herein can also be 35 useful to prevent degeneration and/or promote survival in other non-neuronal tissues as well. One skilled in the Printed from Mimosa 34 art can readily determine using a variety of assays known in the art whether a particular cell type expresses Ret and would thus likely be activated in the presence of TrnR2 and a TRN such as NTN or GDNF.

In certain circumstances, it may be desirable to modulate or decrease the trophic effect of endogenously synthesized TRN growth factors, including NTN and/or GDNF. This may be achieved by blocking binding of the growth factor to its receptor in the target tissue or by 10 decreasing TrnR2 expression in the target tissue. Thus, appropriate treatments, for example, may involve administration of TrnR2 antibodies or other compounds having TRN antagonist properties, or the use of antisense polynucleotides to modulate TrnR2 expression. 15 Specific antibodies, either polyclonal or monoclonal, may be capable of preventing binding of NTN an/or GDNF to TrnR2 or, alternatively, may prevent the formation of a functional TrnR2/Ret receptor complex.

Such antibodies can be produced by any suitable method 20 known in the art. For example, murine or human monoclonal antibodies can be produced by hybridoma technology or by combinatorial antibody library technology, including panning a phage display library. The antibody may be engineered using recombinant 25 techniques to produce an antibody with desirable characteristics such as being "humanized" to be better tolerated by the patient or having specificities for both TrnR2 and Ret or both TrnR2 and a TRN growth factor.

Such antibody engineering techniques are known in the 30 art. See for example, Hayden et al., Curr. Opin.

Immunol. 9(2):201-212, 1997, incorporated herein by reference. Alternatively, the TrnR2 protein, or an immunologically active fragment thereof, or an anti-idiotypic antibody, or fragment thereof can be 35 administered to an animal to elicit the production of antibodies capable of recognizing and binding to the Printed from Mimosa TrnR2 protein. Such antibodies can be from any class of antibodies including, but not limited to IgG, IgA, IgM, IgD, and IgE or in the case of avian species, IgY and from any subclass of antibodies.

It is also envisioned that soluble TrnR2 polypeptides and fragments can also serve as TRN antagonists. For example, it is believed that a soluble TrnR2 administered in excess would GDNF, NTN, and possibly other TRN growth factors, thereby sequestering 10 the TRN growth factor from the anchored TrnR2 receptors on target cells. Similarly, if excessive levels of a TRN growth factor, particularly GDNF or NTN, was circulating, administration of soluble TrnR2 may act to sequester the growth factor in the plasma and possibly facilitate their 15 excretion, thereby limiting the effects of the growth factor in the body.

In another aspect of the present invention, TrnR2 antisense oligonucleotides can be made and a method utilized for diminishing the level of expression of TrnR2 20 protein by a cell comprising administering one or more TrnR2 antisense oligonucleotides. By TrnR2 antisense oligonucleotides reference is made to oligonucleotides that have a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid 25 sequence involved in the expression of TrnR2 such that the expression of TrnR2 is reduced. Preferably, the specific nucleic acid sequence involved in the expression of TrnR2 is contained within a genomic DNA molecule or mRNA molecule that encodes TnrR2. A genomic DNA molecule 30 may comprise regulatory regions of the TrnR2 gene and/or coding sequences for precursor or mature TrnR2 protein. The term complementary to a nucleotide sequence in the context of TrnR2 antisense oligonucleotides and methods therefor means sufficiently complementary to such a 35 sequence as to allow hybridization to that sequence in a cell, i.e., under physiological conditions. The TrnR2 Printed from Mimosa 36 antisense oligonucleotides preferably comprise a sequence containing from about 8 to about 100 nucleotides and more preferably the TrnR2 antisense oligonucleotides comprise from about 15 to about 30 nucleotides.

The TrnR2 antisense oligonucleotides can also include derivatives which contain a variety of modifications that confer resistance to nucleolytic degradation such as, for example, modified internucleoside linkages modified nucleic acid bases 10 and/or sugars and the like (Uhlmann and Peyman, Chemical Reviews 90:543-584, 1990; Schneider and Banner, Tetrahedron Lett 31:335, 1990; Milligan et al., J Med Chem 36:1923-1937, 1993; Tseng et al., Cancer Gene Therap 1:65-71, 1994; Miller et al.. Parasitology 10:92-97, 1994 15 which are incorporated by reference). Such derivatives include but are not limited to backbone modifications such as phosphotriester, phosphorothioate, methylphosphonate, phosphoramidate, phosphorodithioate and formacetal as well as morpholino, peptide nucleic 20 acid analogue and dithioate repeating units.

The TrnR2 antisense polynucleotides of the present invention can be used in treating overexpression of TrnR2 or reduce sensitivity of cells to inappropriate expression of NTN or GDNF. Such treatment can also 25 include the ex vivo treatment of cells.

The therapeutic or pharmaceutical compositions of the present Invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or 30 intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, 35 administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that Printed from Mimosa TrnR2 be administered to cells in the central nervous system, administration can be by intravenous injection with one or more agents capable of promoting penetration of TrnR2 across the blood-brain barrier such as an 5 antibody to the transferrin receptor. Co-administration may comprise physically coupling any known blood-brain penetrating agent to TrnR2. (See for example, Friden et al., Science 259:373-377, 1993 which is incorporated by reference).

A TrnR2 polypeptide can also be linked or conjugated with agents that provide other desirable pharmaceutical or pharmacodynamic properties. For example, a TrnR2 Jpolypeptide can be stably linked to a polymer such as * polyethylene glycol to obtain desirable properties of 15 solubility, stability, half-life and other pharmaceutically advantageous properties. (See for example Davis et al. Enzyme Eng 4:169-73, 1978; Burnham, Am J Hosp Pharm 51:210-218, 1994 which are incorporated by reference).

The compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other 25 pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition. Such 30 solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection. The primary solvent can be aqueous or alternatively non-aqueous.

TrnR2 can also be incorporated into a solid or semi-solid 35 biologically compatible matrix which can be implanted into tissues requiring treatment.

Printed from Mimosa 38 The carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the 5 formulation. Similarly, the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier. Such excipients are those substances usually and customarily 10 employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or periodic infusion.

Dose administration can be repeated depending upon 15 the pharmacokinetic parameters of the dosage formulation and the route of administration used.

It is also contemplated that certain formulations containing TrnR2 are to be administered orally. Such formulations are preferably encapsulated and formulated 20 with suitable carriers in solid dosage forms. Some examples of suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, 25 polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending 30 agents, preserving agents, sweetening agents or flavoring agents. The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The 35 formulations can also contain substances that diminish proteolytic degradation and promote absorption such as.

Printed from Mimosa 39 for example, surface active agents.

The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The 5 dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. It is believed that 10 such calculations can be readily made by one skilled in the art in light of the dose-response curves disclosed herein for NTN- or GDNF-induced Ret activation in TrnR2/Ret-expressing cells. Exact dosages are determined in conjunction with standard dose-response studies. It 15 will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, 20 weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.

The invention also provides the identification of a novel receptor gene family for TRN neurotrophic factors. 25 The known members of this family, Trnl and TrnR2, share approximately 48% percent amino acid sequence identity and about 53% sequence homology. The inventors herein believe that other unidentified genes may exist that encode proteins that have substantial amino acid sequence 30 homology to TrnRl and TrnR2 and which function as receptors for growth factors selective for the same or different tissues having the same or different biological activities. A different spectrum of activity with respect to tissues affected and/or response elicited 35 could result from preferential activation of different receptors by different family members as is known to Printed from Mimosa 40 occur with members of the NGF family of neurotrophic factors and Trk receptors (Tuszynski and Gage, supra).

As a consequence of members of a particular gene family showing substantial conservation of amino acid 5 sequence among the protein products of the family members, there is considerable conservation of sequences at the DNA level. This forms the basis for a new approach for identifying other members of the receptor gene family to which TrnRl and TrnR2 belong. The method 10 used for such identification is cross-hybridization using nucleic acid probes derived from one family member to form stable hybrid duplex molecules with homologous sequences from different members of the gene family or to amplify nucleic acid sequences from different family 15 members. (See for example, Kaisho et al. FEBS Letters 266:187-191, 1990 which is incorporated by reference). The sequence from the different family member may not be identical to the probe, but will, nevertheless be sufficiently related to the probe sequence to hybridize 20 with the probe. Alternatively, PCR using primers from one family member can be used to amplify homologous sequences in additional family members.

The above approaches would not have heretofore been successful in identifying other gene family members 25 because only one family member, TrnRl (GDNFR-a) was known. With the identification of TrnR2 herein, however, unique new probes and primers can be made that contain sequences from the longer conserved regions of this gene family (see boxed regions in Figure 1). The new probes 30 and primers made available from the present work make possible this powerful new approach which can now successfully identify other gene family members. Using this new approach, one may screen for genes related to TrnRl and TrnR2 in amino acid sequence homology by 35 preparing DNA or RNA probes based upon the conserved regions in the TrnRl and TrnR2 proteins.

Printed from Mimosa 41 Therefore, one embodiment of the present invention comprises probes and primers that are unique to or derived from a nucleotide sequence encoding such conserved regions and a method for identifying further 5 members of the TrnR gene family. Examples of such conserved region amino acid sequences include but are not limited to Cys-Arg-Cys-Lys-Arg-Gly-Met-Lys-Lys-Glu (SEQ ID N0:9); Cys-Asn-Arg-Arg-Lys-Cys-His-Lys-Ala-Lys-Arg (SEQ ID NO:10), and Cys-Leu-Xaa-Asn-Ala-Ile-Glu-Ala-Phe-10 Gly-Asn-Gly (SEQ ID NO:11) where Xaa is Lys or Arg.

Degenerate oligonucleotides containing all of the possible nucleotide sequences which code for one or more of the TrnR2 conserved amino acid sequences can be synthesized for use as hybridization probes or 15 amplification primers. The nucleotide sequence may be based on the above listed conserved sequences or chosen from the other boxed conserved regions shown in Figure 1. To reduce the number of different oligonucleotides in a degenerate mix, an inosine base, or another "universal" 20 base, can be incorporated in the synthesis at positions where all four nucleotides are possible. Univeral bases such as inosine form base pairs with each of the four normal DNA bases which are less stabilizing than AT and GC base pairs but which are also less destabilizing than 25 mismatches between the normal bases (i.e. AG, AC, TG, TC).

Sources of nucleic acid for screening would include mammalian genomic DNA, cDNA reversed transcribed from mRNA obtained from mammalian cells, or genomic or cDNA 30 libraries prepared from mammalian species cloned into any suitable vector.

Hybridization using the new probes to conserved regions of the nucleic acid sequences would be performed under reduced stringency conditions. Factors involved in 35 determining stringency conditions are well known in the art (for example, see Sambrook et al., Molecular Cloning, Printed from Mimosa 42 2nd Ed., 1989 which is incorporated by reference).

Sources of nucleic acid for screening would include genomic DNA libraries from mammalian species or cDNA libraries constructed using RNA obtained from mammalian 5 cells cloned into any suitable vector.

PCR primers would be utilized under PCR conditions of reduced annealing temperature which would allow amplification of sequences from gene family members other than TrnRl and TrnR2. To identify the sequences of these 10 products, they can be gel purified and ligated into any suitable cloning vector and transformed into bacteria. The resulting clones can be screened with an oligonucleotide probe for either a unique TrnRl or a unique TrnR2 sequence in the amplified region. Clones 15 not hybridizing to either unique probe can be sequenced and if found to encode previously unisolated family members, the sequence of that clone can be used to Isolate full length cDNA clones and genomic clones. A similar method was used to isolate new gene members 20 (GDF-3 and GDF-9) of the TGF-B superfamily based on homology between previously identified genes (McPherron J Biol Chem 268: 3444-3449, 1993 which is incorporated by reference).

Alternatively, other TrnR family members may be 25 identified and/or obtained by screening a cDNA expression library for the presence of proteins cross-reacting with an antibody capable of reacting with a polypeptide containing a TrnR conserved region, e.g., an amino acid sequence selected from the group consisting of SEQ ID 30 NO:9, SEQ ID NO:10 and SEQ ID NO:11. Preparation of cDNA libraries in mammalian expression systems is known in the art (see e.g., Jing et al., supra, Treanor et al., supra, Gearing et al., EMBO J. 8:3667-3676, 1989, and Takebe, et al., Mol. Cell. Biol. 8:466-472, 1988, incorporated 35 herein by reference). A clone expressing a polypeptide that binds to an anti-TrnR conserved region would be Printed from Mimosa 43 isolated and its cDNAs sequenced to determine whether it encoded a TrnR family member based on comparing its predicted amino acid sequences with those of TrnRl and TrnR2.

Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein.

It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples.

' Example 1 This example illustrates that either TrnRl or TrnR2 can mediate the signaling of NTN or GDNF through the Ret protein tyrosine kinase.

Generation of fibroblasts expressing Ret and TrnRl or TrnR2 To examine the possibility that TrnRl or TrnR2 can form a functional receptor complex with Ret for NTN and/or GDNF, NIH3T3 fibroblasts which stably express Ret alone, both Ret and TrnRl, or both Ret and TrnR2 were generated.

Briefly, full length human Ret cDNA (gift of Dr. H.

Donnis-Keller, Washington University, St. Louis, MO) was subcloned into the pCMV-Neo vector (Brewer, C.B., Meth. Cell Biol. 431233-245, 1994, incorporated herein by reference). NIH3T3 cells (subclone MG87; Zhan et al., 1987) were transfected with the Ret-CMV-neo plasmid, grown in DMEM plus 10% fetal bovine serum (Hyclone), and stable transfectants expressing Ret were selected with 1 mg/ml G418. Positive clones were screened for Ret expression on immunoblots probed with an anti-Ret antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Printed from Mimosa 44 A clonal Ret-expressing cell line was used as the parent to generate TrnRl/Ret and TrnR2/Ret expressing cells by transfection with TrnRl or TrnR2 expression constructs. To prepare the TrnRl expression construct, a 5 TrnRl cDNA was obtained from a rat postnatal-day-1 library by Klentaq LA PCR with the primers: 5' -GCGGTACCATGTTCCTAGCCACTCTGTACTTCGC-3' (SEQ ID NO:14) and 5'-GCTCTAGACTACGACGTTTCTGCCAACGATACAG-3' (SEQ ID NO:15). The amplified product was cloned into the EcoRV site of 10 pBluescript KS (Stratagene, La Jolla, CA, sequenced and subcloned into the Hindlll and BamHI sites of pCMV-Neo (Brewer, 1994). For the TrnR2 expression construct, the coding region of the long form of human TrnR2 cDNA was amplified from the same Marathon RACE human brain cDNA 15 library used to clone and sequence TrnR2. Amplification primers were 5'-GCGGTACCATGATCTTGGCAAACGTCTGC-3' (SEQ ID NO:16) and 5'-GCTCTAGAGTCAGGCGGCTGTTCTTGTCTGCG-3' (SEQ ID NO:17). The product was cloned into pCMV-neo and the insert confirmed by sequencing.

The TrnRl-CMV-Neo plasmid or the TrnR2 CMV-Neo plasmid was co-transfected with SV2-HisD (gift of Dr. Richard Mulligan, Massachusetts Institute of Technology) into the Ret-expressing 3T3 cells and double transfectants selected in 2 mM L-histidinol (Sigma, St. 25 Louis, MO). TrnRl- and TrnR2-expressing clones were confirmed by western (Ret) and northern (TrnRl or TrnR2) blotting.

Preparation of recombinant NTN and GDNF A synthetic gene for the mature mouse NTN coding 30 sequence was prepared from four partially overlapping oligonucleotides containing the Eschericia coli (E. coll) codon preferences: 5'-GCA TAT GCC GGG TGC TCG TCC GTG CGG CCT GCG TGC AAC TGG AAG TTC GTG TTT CTG AAC TGG GTC TGG GTT ACA CTT CTG ACG AAA CTG T-3'(SEQ ID NO:18); 5'-GCT 35 GAC GCA GAC GAC GCA GAC CCA GGT CGT AGA TAC GGA TAG CAG CTT CGC ATG CAC CAG CGC AGT AAC GGA ACA GAA CAG TTT CGT- Printed from Mimosa 45 3' (SEQ ID NO:19); 5'-CTG CGT CAG CGT CGT CGT GTT CGT CGT GAA CGT GCT CGT GCT CAC CCG TGC TGC CGT CCG ACT GCT TAC GAA GAC GAA GTT TCT TTC-3' (SEQ ID NO:20); 5'-CGG ATC CTT AAA CGC AAG CGC ATT CAC GAG CAG ACA GTT CCT GCA GAG TGT 5 GGT AAC GAG AGT GAA CGT CCA GGA AAG AAA CTT CG-3' (SEQ ID NO:21). These oligos were gel purified and then annealed for 10 min at 68" C followed by 30 min at 22° C to form a linear sequence. The annealed oligos were extended with Klenow fragment, kinased and ligated into the Bluescript 10 KS plasmid. After verifying the authenticity of the cloned NTN fragment by DNA sequencing, the fragment was transferred to the Ndel and BamHI site of the expression ,1 vector pET30a(+) (Novagen, Madison, WI). A histidine tag followed by a enterokinase site was placed at the amino 15 terminus of the NTN sequence by inserting oligonucleotide linkers A (5'-TAT GCA CCA TCA TCA TCA TCA TGA CGA CGA CGA CAA GGC-3')(SEQ ID NO:22) and B (5'-TAG CCT TGT CGT CGT CGT CAT GAT GAT GAT GAT GGT GCA-3')(SEQ ID NO:23) into the Ndel site.

The mature rat GDNF coding sequence was obtained from an embryonic-day-21 rat kidney cDNA library by PCR using primers 5'-CAG CAT ATG TCA CCA GAT AAA CAA GCG GCG GCA CT-3' (SEQ ID NO:24) and 5'-CAG GGA TCC GGG TCA GAT ACA TCC ACA CCG TTT AGC-3' (SEQ ID NO:25). The amplified 25 cDNA fragment was subcloned into the Ndel and Sail sites of pET30a(+). A six-His tag and enterokinase site were added to the amino terminus of the NTN sequence at the Ndel site using linkers A and B as above.

The NTN and GDNF pET30a(+) constructs were sequenced 30 to confirm their authenticity and then transformed into E. coll strain BL21/DE3. The transformed bacteria were grown at 37* C in 2XYT medium (30 /*g/ml kanamycin) with vigorous shaking. For GDNF production, IPTG was added to a final concentration of 1.0 mM to induce expression of 35 the protein after the culture reached an optical density of A600 = 0.7. Incubation was continued for an additional Printed from Mimosa 46 2 h. Bacteria containing the NTN expression construct were grown for 24 h without IPTG.

Cells were harvested by centrifugation at 4000 X g for 20 min, solubilized with Buffer A (6 M guanidine HC1, 5 0.1 M Na phosphate, 10 mM Tris HC1, pH 8.0) at l/10th volume of the original culture volume and rocked overnight. Lysate was centrifuged at 10,000 x g for 15 min at 4° C. The supernatant was exposed to 4 ml of Nickel-NTA (nitrilo-tri-acetic acid) resin (Qiagen, 10 Chatsworth, CA) per 1 liter original culture and washed with 10-20 column volumes of Buffer A, 10 column volumes of Buffer B (8 M urea, 0.1 M Na phosphate, 10 mM Tris HC1, pH 8.0), and 5-10 volumes of Buffer C (8 M urea, 0.1 M Na phosphate, 10 mM Tris HC1, pH 6.3) until the A280 15 was <0.01. The recombinant NTN or GDNF protein was eluted with 10-20 ml Buffer E (8 M urea, 0.1 Na phosphate, 10 mM Tris, pH 4.5). Fractions were collected and analyzed by SDS-PAGE. The eluate was immediately diluted to 50 ng/^1 in Buffer B and dialyzed against 4 M 20 Renaturation Buffer (4 M urea, 5 mM cysteine, 0.02% Tween 20, 10% glycerol, 10 mM Tris HC1, 150 mM NaCl, 100 mM Na phosphate, pH 8.3, under argon) at 4° C overnight and then against 2 M Renaturation Buffer (as above except with 2 M urea) 2-3 days with changes every 24 h. 25 Ret phosphorylation assays. PI-PLC treatment TrnRl- and TrnR2-expressing fibroblasts were grown to confluence in DMEM plus 10% calf serum, treated with 50 ng/ml recombinant NTN or GDNF for 10 min, or left untreated, and then lysed. A portion of the lysates was 30 removed and assayed for total Ret expression by western blot analysis using an anti-Ret antibody. The remaining lysates were immunoprecipitated with an anti-phosphotyrosine antibody and analyzed by western blot using the anti-Ret antibody. The results are shown in 35 Figure 3A.

Anti-Ret immunoblot analysis for total Ret shows Printed from Mimosa 47 that expression by the Ret and Ret/TrnR2 expressing clones of the immature (150kD) intracellular Ret protein and the glycosylated mature Ret protein (170 kD) was essentially the same whether treated with NTN or GDNF or 5 left untreated (-) (Total, Fig. 3A).

However, when the lysates were immunoprecipitated with anti-phosphotyrosine antibody before anti-Ret western analysis (IP, Fig. 3A), no Ret protein was observed in lysates from fibroblasts expressing only the 10 Ret tyrosine kinase (Ret, Fig. 3A) whether left untreated or treated with NTN or GDNF at doses ranging from 50 to 3000 ng/ml (Fig. 3A and data not shown). A tyrosine-phosphorylated band of approximately 170 kD was observed only in cells which expressed both Ret and TrnR2 and 15 which were treated with GDNF or NTN, indicating that TRN-induced activation of the mature Ret protein required the presence of both Ret and TrnR2 (IP, Fig. 3A). Similar results were seen for Ret/TrnRl expressing cells (data not shown).

Further evidence that the effects of both GDNF and NTN could be mediated by TrnR2 was obtained by treating fibroblasts expressing Ret/TrnR2 with 1 U/ml PI-PLC for 45-60 min at 37°C and then washing the PI-PLC treated cells prior to NTN or GDNF treatment. As shown in Fig. 25 2B, PI-PLC treatment, which specifically cleaves GPI-linked proteins from the cell surface, significantly depleted the NTN or GDNF induced phosphorylation of Ret (compare +,- lanes with +,+ lanes). These data indicate that TrnR2, a putative GPI-linked protein, can form a 30 functional receptor with Ret for NTN or GDNF. This is analogous to the previously described requirement of TrnRl as a co-receptor with Ret for GDNF signaling (Treanor et al., supra; Jing et al., supra).

To further characterize the activities of these two 35 TRN co-receptors, the effects of 0 to 100 ng/ml of NTN or GDNF on Ret phosphorylation was investigated. As shown Printed from Mimosa 48 in Fig. 3C, the Ret stimulating activity of both co-receptors is dose dependent since more phosphorylation of Ret in either construct was observed at higher levels of GNDF and NTN. However, while the extent of Ret 5 phosphorylation in Ret/TrnRl expressing fibroblasts treated with NTN or GDNF was approximately equivalent at all doses tested, the TrnR2/Ret expressing fibroblasts were more sensitive to NTN treatment than GDNF treatment. In particular. Ret phosphorylation was clearly observed 10 in response to NTN treatment at 0.3 ng/ml; an equivalent response to GDNF was observed at 10 ng/ml. Similar results were obtained with multiple batches of recombinant GDNF and NTN, and with another stable TrnR2/Ret transfectant (data not shown).. • 15 The observed difference in the dose-response curves of the Ret/TrnRl and Ret/TrnR2 fibroblasts to NTN and GDNF suggests that there is a difference in the functional affinity of the ligands for the two receptor complexes, TrnRl/Ret and TrnR2/Ret. The TrnR2/Ret 20 complex may function preferentially as a NTN receptor, whereas the TrnRl/Ret complex responds equivalently to either factor.

Example 2 This example illustrates that the short splice 25 variant of TrnR2 (TrnR2-SV) can mediate signal transduction through Ret like the long splice variant (TrnR2-LV). 3T3 fibroblasts coexpressing Ret and either TrnR2-LV or TrnR2-SV were generated by cotransfecting a clonal 30 Ret-expressing 3T3 cell line with a SV2-His plasmid and a cDNA encoding TrnR2-LV and then selecting the desired transfectants in 2 mM L-histidinol essentially as described in Example 1.

The recombinant fibroblasts were stimulated with 35 either GDNF, neurturin or persephin at 100 ng/ml for 10 minutes and then lysed. To determine the amount of Printed from Mimosa 49 active phopshorylated Ret after stimulation with each growth factor, the lysates were immunoprecipitated using an anti-phosphotyrosine antibody, and then analyzed by western blot using an anti-Ret antibody as described in 5 Example 1. The results are shown in Fig. 3D.

When stimulated with GDNF or neurturin (NTN), the amount of tyrosine-phosphorylated Ret was approximately equal in cells coexpressing TrnR2-SV as that in cells coexpressing TrnR2-LV. In addition, neurturin 10 stimulation produced more Ret phosphorylation than GDNF stimulation in both the TrnR2-SV-expressing and TrnR2-LV-expressing cells, which is consistent with the hypothesis that the, TrnR2-LV/Ret complex has a preferential affinity for -neurturin over GDNF.

These data indicate that the short and long splice variants of TrnR2 are essentially equivalent in mediating GNDF and neurturin signaling through Ret activation.

Thus, it is believed the short splice variant contains all the structural elements necessary for binding to GDNF 20 and neurturin and presenting these ligands to Ret in the appropriate orientation such that Ret is phosphorylated.

Neither cell line responded to persephin (PSP), indicating that this family member acts through a differenct co-receptor complexed with Ret or through a 25 different receptor complex altoghether.

Example 2A This example illustrates that a soluble TmR2 polypeptide specifically binds to the GNDF and NTN members of the TRN family.

A cDNA encoding a soluble TrnR2 receptor- immunoglobulin fusion protein was prepared by fusing a polynucleotide encoding the amino acid sequence from methionine at position -21 through glycine at position 411 of SEQ ID NO:2, i.e., nucleotides 36-1331 of SEQ ID 35 N0:1, to a polynucleotide encoding the Fc region of human IgGi using the plgPlus vector system (Invitrogen, Printed from Mimosa Carlsbad, CA). The resulting construct was transfected into COS cells and stable clones were selected using 1 mg/ml G418. Stable COS clones were grown in conditioned medium from which the secreted TrnR2-Fc fusion protein 5 was purified using protein-A chromotography.

This soluble TrnR2-Fc fusion protein was then used in an ELISA binding assay to determine if soluble TrnR2-LV is capable of binding to GDNF, NTN and PSP. Solutions containing 250 ng/ml of GDNF, NTN or PSP were prepared 10 and 50 pi of each solution was applied to separate Maxisorb ELISA plates (Nunc) (i.e., 12.5 ng growth factor per well) and the growth factor was allowed to bind for 1 hr at room temperature. The wells of each plate were then washed, blocked, and incubated with increasing 15 amounts of the soluble TrnR2-Fc fusion protein. The wells were washed again and bound TrnR2-Fc protein was detected using an anti-human IgG-HRP conjugated secondary antibody (Jackson Immunoresearch), and TMB liquid substrated detection reagent (Sigma). The amount of 20 luminescence was plotted against the amount of TrnR2-Fc protein used in the assay and the results are shown in Fig. 3(E).

In this immunoassay, approximately equal amounts of soluble TrnR2-Fc bound to both GDNF and NTN when the 25 fusion protein was added at less than 1 nM. However, at larger amounts of added fusion protein, much larger amounts of soluble TrnR2 bound to immobilized NTN than to immobilized GDNF, demonstrating that soluble TrnR2 also has greater affinity for NTN than GDNF as was shown in 30 Example 1 for membrane bound TrnR2.

The specificity of TrnR2 for NTN and GDNF is indicated by the lack of its binding to PSP at all amounts of receptor tested.

Example 3 This example illustrates the expression of TrnRl and TrnR2 in various tissues.

Printed from Mimosa 51 TrnR2 expression in adult mouse is more limited than TrnRl expression TrnR2 expression in adult mouse was investigated by Northern blot analysis of total RNA (25 fig) isolated from 5 various adult mouse tissues and electrophoresed in a 1% agarose/formaldehyde gel and blotted onto a nylon membrane (Zetaprobe) using standard procedures (Chomczynski and Sacchi, Rnnal. Biochem 162: 156-159, 1987, incorporated herein by reference). To verify that 10 equal amounts of total RNA were present in each lane, the 28S ribosomal RNA band was visualized by staining with ethidium bromide. RNA homologous to TrnR2 was detected by probing the blot with a 32P-labeled fragment of TrnR2 cDNA. The results are shown in Figure 4. -15 RNA hybridizing to the TrnR2 probe was observed in brain and testis. Two messages were observed in brain, differing only slightly in size. These two bands likely correspond to the two splice forms found in brain while performing RACE PCR to amplify the 5' end of the TrnR2 20 cDNA, the shorter of which is missing 399 nucleotides from the coding region (see Fig. 2). Two different bands were also observed in testis, which were significantly smaller ("1.5-1.8 kb) than either of the transcripts detected in the brain (~4 kb). One of the smaller TrnR2 25 messages in testis may be analogous to a small TrnRl mRNA reported which encodes a truncated protein of 158 amino acids (Treanor et al., 1996). Low-level expression may also be present in the spleen and in the adrenal.

These results indicate that the tissue distribution 30 of TrnR2 is more limited than TrnRl in the adult animal, which has been detected in liver, kidney, and brain of adult rat and mouse (Jing et al., supra).

Analysis of TrnRl. TrnR2. and Ret expression in targets of GDNF and NTN 35 Comparison of TrnR2 and TrnRl expression was also investigated in known sites of GDNF and/or NTN action by Printed from Mimosa 52 in situ hybridization analysis. Mouse tissue samples were obtained and prepared for in situ hybridization as described previously Wanaka et al., Neruron 5: 267-281, 1990, which is incorporated herein by reference). The 5 results of in situ hybridization of these fresh frozen tissue samples with antisense 33P-labeled RNA probes transcribed from fragments of TrnRl, TrnR2 and Ret cDNAs are shown in Figure 5.

In situ hybridization analysis showed only low-level 10 expression of TrnR2 in the substantia nigra in the adult mouse, and in the ventral mesencephalon of an E14 mouse, in contrast to high-level expression of TrnRl and Ret (Fig. 5A and data not shown). Motor neurons in the ventral horn (vh) of the adult spinal cord also express 15 TrnRl and Ret, but not TrnR2 (Fig. 5B). Ret is localized predominately to motor neurons, whereas TrnRl shows additional staining in the intermediate and dorsal horns of the cord. TrnR2 is highly expressed in the developing and adult dorsal root ganglia (drg), along with Ret and 20 TrnRl (Fig. 5C and data not shown). In addition, strong expression of TrnRl and TrnR2, but not Ret, was observed in the exiting nerve root (r) (Fig. 5D). In the developing kidney (k) and gut (g), there is high level expression of TrnRl and Ret, but not TrnR2 (Fig. 5c). 25 Finally, significant expression of both TrnR2 and Ret was observed in the rat superior cervical ganglion (SCG), with only low-level, diffuse staining of TrnRl (Fig. 5d).

These data indicate a partially overlapping expression pattern for TrnRl and TrnR2 in embryonic and 30 adult central and peripheral nervous tissue. In several areas of known GDNF action, including nigral and motor neurons, high levels of TrnRl and Ret are expressed, with only low or undetectable levels of TrnR2 expression.

Based on this initial survey, TrnR2 expression is largely 35 limited to neuronal tissue in both embryo and adult, with highest levels of expression in sensory and sympathetic Printed from Mimosa 53 neuronal populations.

Example 4 This example illustrates that both NTN and GDNF promote the survival of newborn rat SCG neurons in 5 culture through their activation of the Ret signaling pathway and this activation is likely mediated by TrnR2, not TrnRl.

Neuronal cultures were prepared from the SCG of postnatal day-1 rats using known procedures (Martin et 10 al., J. Cell Biol. 106:829-844, incorporated herein by reference). The SCG cultures were plated on collagen-coated dishes and maintained in NGF for seven days and then deprived of NGF by switching to medium lacking NGF and containing an anti-NGF antibody (Ruit et al.. Neuron 15 8: 573-587, 1992, incorporated herein by referencey.

After 2-4 h, this medium was replaced with medium containing 50 ng/ml NTN, GDNF, or NGF. After 10 min incubation in these growth factors, lysates were prepared and assayed for total Ret protein and tyrosine-20 phosphorylated Ret as described in Example 1. Tyrosine phosphorylated Ret protein was observed in cells treated with NTN but not in NGF-treated cells (data not shown). Thus, both NTN and GDNF can activate the Ret receptor.

It had been shown that the survival-promoting 25 ability of GDNF on several neuronal populations including SCG neurons was significantly reduced by PI-PLC treatment (Treanor et al., supra). To assess whether NTN-induced Ret activation is similarly affected, SCG cultures were grown in serum-free N2 medium to maximize the activity of 30 PI-PLC and then treated with 1 U/ml PI-PLC prior to the addition of NTN (50 ng/ml). NTN-induced tyrosine phosphorylation of Ret was significantly reduced in cultures treated with PI-PLC (data not shown). Thus, activation of the Ret PTK by NTN and GDNF appears to be 35 mediated by a GPI-linked protein.

As shown in Figure 5D, in situ hybridization Printed from Mimosa 54 analysis indicated that TrnR2 and Ret are expressed at high levels in rat SCG neurons, whereas TrnRl is expressed diffusely and does not appear to be localized to neurons. To further assess the cellular localization 5 of TrnRl, TrnR2 and Ret mRNAs in this ganglion, their expression in primary SCG cultures was analyzed. Because the primary cultures contain a small contaminating population of non-neuronal cells (Schwann cells and fibroblasts), neuronal specific messages can be 10 identified by inducing apoptosis in the neuronal population. Removal of NGF from the culture medium results in near complete death of the neuronal population within 48 hours (Martin et al.," J. Cell Biol. 106:829-844, 1988; Deckwerth et al., J. Cell Biol. 123:1207-1222, 15 1993; Edwards et al., J. Cell Biol. 124:537-546, 1994, which are incorporated herein by reference). During this period, neuronal messages decrease whereas messages from non-neuronal cells remain constant (Freeman et al., Neuron 12:343-355, 1994; Estus et al., J. Cell Biol. 20 127:1717-1727, 1994, each incorporated herein by reference).

The amount of TrnRl, TrnR2, Ret mRNA in neuronal cultures was assessed using semiquantitative reverse transcription-PCR (RT-PCR) as described in Freeman et 25 al., supra and Estus et al., supra. Five day SCG cultures were switched to medium containing anti-NGF antibodies for various times. Polyadenylated RNA was isolated from the cultures at 0, 6, 12, 18, 24 and 36 hours after NGF removal using the QuickPrep Micro Kit 30 (Pharmacia, Piscataway NJ) according to the manufacturer's instructions. Half of the poly-A RNA was converted to cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase with random hexamers (16 fM) as primers. cDNA from approximately 150 35 cells was used in a 50 fi\ PCR reaction using the following primer sets: mouse Ret forward 5'- Printed from Mimosa 55 TGGCACACCTCTGCTCTATG-3' (SEQ ID NO:26) and reverse 5'-TGTTCCCAGGAACTGTGGTC-3' (SEQ ID NO:27); TrnRl forward 5'-GCACAGCTACGGGATGCTCTTCTG-3' (SEQ ID NO:28) and reverse 5'-GTAGTTGGGAGTCATGACTGTGCCAATC-3* (SEQ ID NO:29); TrnR2 5 forward 5*-AGCCGACGGTGTGGCTCTGCTGG-3' (SEQ ID NO:30) and reverse 5'-CCAGTGTCATCACCACCTGCACG-3' (SEQ ID N0:31).

After amplification, the PCR products were separated by electrophoresis on 10% polyacrylamide gels, visualized by autoradiography of the dried gels, and quantified with a 10 Phosphorlmager (Molecular Dynamics, Inc., Sunnyvale CA). The results are shown in Figure 6.

Ret and TrnR2 messages decreased as the neurons died, in a manner similar to neuron-specific enolase (NSE). In contrast, TrnRl levels remained constant, 15 similar to the Schwann cell marker S-100.

These data indicate that Ret and TrnR2 expression is largely limited to neurons in neonatal rat SCG cultures, and likely mediates the functional response of these neurons to NTN and GDNF. This is consistent with in situ 20 hybridization analysis (Fig. 5D) which also indicates that the expressed receptor complex in SCG neurons consists of TrnR2 and Ret. Interestingly, NTN is more potent in promoting the survival of SCG neurons than GDNF (Kotzbauer et al., supra), which is consistent with the 25 higher sensitivity of the TrnR2/Ret receptor complex to NTN treatment (Fig. 3C). Although some low level neuronal expression of TrnRl cannot be excluded by this assay, these data indicate TrnRl is predominantly expressed in the non-neuronal population, consistent with 30 its previously observed expression in Schwann cells (Treanor et al., supra).

Example 5 This example illustrates that TrnRl, but not TrnR2, is up-regulated in distal sciatic nerve after nerve 35 injury.

GDNF is a well characterized trophic factor for both Printed from Mimosa 56 embryonic and adult motor neurons (Henderson et al., Science 266:1062-1064, 1994; Yan et al.. Nature 373:341-344, 1995; Oppenheim et al.. Nature 373:344-346, 1995; Li et al., Proc.Natl.Acad.Scl.USA 92:9771-9775, 1995, all 5 are incorporated herein by reference). In the adult animal, GDNF expression is up-regulated in the distal segment of the sciatic nerve after transection, and in denervated muscle (Trupp et al., J. Cell.Biol. 130:137-148, 1995; Springer et al., Exp.Neurol. 131:47-52, 1995, 10 each is incorporated herein by reference). This is similar to observations regarding NGF and the p75 low affinity neurotrophin receptor (p75NTR), which are both upregulated by Schwann cells in the distal segment of the sciatic nerve after transection (Taniuchi et al., 15 Proc.Natl.Acad.Sci.USA 83:4094-4098, 1986; Heumanr et al., J.Cell.Biol. 104:1623-1631, 1987, each is incorporated herein by reference). Because GDNF is up-regulated after injury, and because TrnRl is expressed by Schwann cells (Treanor et al., supra), the expression of 20 TrnRl and TrnR2 in the distal segment of the rat sciatic nerve before and after transection was examined to determine if one or both of the TRN co-receptors is up-regulated after transection.

Northern blot analysis was performed on total RNA 25 isolated from normal rat sciatic nerve and from the distal segment of the sciatic nerve seven days post-transection using 32P-labeled TrnRl and TrnR2 cDNA fragments as probes. Rat sciatic nerves were transected and recovered as previously described (Araki et al., 30 Neuron 17:353-361, 1996, incorporated herein by reference). Brain total RNA was included on the blot as a positive control for detection of TrnR2 mRNA. The results are shown in Figure 7.

Seven days after nerve transection (7D), the distal 35 portion of the sciatic nerve showed a dramatic increase in the level of TrnRl mRNA from the level observed before Printed from Mimosa 57 transection (N). In contrast, TrnR2 mRNA was not detected in the nerve either before (N) or after transection (7D). The 28S ribosomal RNA band, visualized using ethidium bromide, is shown below to demonstrate 5 equal loading of total RNA samples.

Consistent with the observed differential expression of TrnRl and TrnR2 in Schwann cells, RT-PCR analysis of the JS-1 Schwann cell line also showed expression of TrnRl, but not TrnR2 (data not shown). These results 10 indicate that TrnR2 is unlikely to play a major role in Schwann cell mediated peripheral trophic support of the regenerating nerve. However TrnRl, in conjunction with GDNF produced by the distal sciatic nerve and muscle, could potentially provide a potent trophic substrate for 15 growth of the regenerating nerve. - Example 5 This example illustrates additional analysis by in situ hybridization of the expression of neurturin, GDNF and their receptors in the central nervous system of the 20 adult mouse.

MATERIALS AND METHODS Riboprobes Riboprobes were synthesized from plasmids containing mouse cDNA sequences of neurturin (nucleotides 293-598, 25 441-675 of GenBank accession number U78109), GDNF (nucleotides 256—935 of GenBank accession number D88264), GFRa-1 (TrnRl, nucleotides 574-1069 of GenBank accession number U59486), GFRa-2 (TrnR2, nucleotides 1-569, 1002-1417 of GenBank accession number AF002701) and Ret 30 (nucleotides 207-611 of GenBank accession number X67812). Both GFRa-2 (TrnR2) probes contained sequences that are present in both splice variants of TrnR2 mRNA (Baloh et al.. Neuron 18:793-802, 1997). The Ret probe also included sequence present in all of the known ret splice 35 variants. Plasmids were linearized with appropriate restriction enzymes and transcribed in vitro with 50 pCi Printed from Mimosa 58 of [33P] UTP (NEN Dupont) by using T3, T7, or SP6 RNA polymerase. All experiments were performed within 24 hours of probe synthesis. The probes were clearly specific based on the distinct expression patterns 5 observed for the different mRNA. Two different sense probes were also used to ensure specificity further. Preparation of Tissue Animals were housed and treated in accordance with the guidelines of NIH and Washington University. Young 10 adult (6-8 weeks) female ICR mice that were at mid to late gestation or recently delivered were anesthetized with an overdose of xylazine (20 mg/ml): ketamine (100 mg/ml): acepromazine (10 mg/ml), (3:3:1). Brains (N=5) were removed, frozen on dry ice, and sectioned at 14 fim 15 in the sagittal or coronal plane on a cryostat. Frozen sections were thawed then postfixed in 4% paraformaldehyde in PBS, pH 7.4, for 20 minutes.

Sections were pretreated for hybridization as follows: 3x5 minutes in PBS; 3 minutes in 0.2% glycine in PBS; 5 20 minutes in PBS; 10 minutes in 0.1M triethanolamine, pH 8; 2x10 minutes in 0.025% acetic anhydride/0.1M triethanolamine; and, 5 minutes in PBS. Sections were then dehydrated in a graded series of alcohol and defatted with chloroform. 33P-labeled RNA probes were 25 diluted in hybridization buffer (50% formamide, 50mM Tris-HCL, pH 7.5, 5mM EDTA, lx Denhardt's solution, 10% dextran sulfate) to 1x10s counts per 75 ^1. Slides were incubated overnight at 55°C in a humidified chamber with 75fil of hybridization solution per slide. After 30 hybridization, slides were washed as follows: 4x15 minutes in 4x SSC at room temperature; 20 minutes in 2xSSC at 55°C; 2x15 minutes in RNase buffer (0.5M NaCl, lOmM Tris-HCl, pH 8, ImM EDTA) at 37°C; 30 minutes in 20 pg/ml RNase A in RNase buffer at 37°C; 2x15 minutes in 35 RNase buffer at 37°C; 20 minutes in 2XSSC at 55"C; 20 minutes in lxSSC at 65°C; and, 30 minutes in O.lx SSC at Printed from Mimosa 59 65°C. Slides were then dehydrated and dipped in Kodak NTB2 emulsion. After an exposure time of 14-18 days, slides were developed and counter-stained with hematoxylin and eosin. Neurons were identified based on 5 morphology. Cells with a pale-staining cytoplasm and large-round, pale nuclei were identified as neurons.

Cells with small oval or irregularly shaped, dark-staining nuclei were identified as glial cells. Slides were photographed and figures were prepared from the 10 photographs with Adobe Photoshop 4.0.1. With the exception of dust removal, and dodging/burning to produce uniform tone photographs were not altered.

Labeling was considered specific if it was above the level of background. Background label is defined as 15 labeling obtained with a sense probe or labeling that was not specifically localized to cells. The intensity of labeling for each probe was classified as follows: high—the greatest intensity of labeling observed for a particular probe; moderate-high-to-medium level of 20 labeling; low—easily detected but low level of intensity; barely detected-very low level of labeling in few or scattered cells; or, not detected—no labeling above background. Abbreviations are according to Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates, 25 Academic Press Inc., 1986; and Franklin and Paxinos, The Mouse Brain in Stereotaxic Coordinates, Academic Press Inc., 1997.

RESULTS Forebrain In the forebrain, GFRa-1 (TrnRl), GFRa-2 (TrnR2), and Ret mRNAs were widely expressed in the septum and olfactory system (Figures 8A-C; Table 2). GFRa-1 (TrnRl), GFRa-2 (TrnR2), and Ret mRNAs were expressed in the medial and lateral septal nuclei, the nucleus of the 35 diagonal band of Broca, and the piriform cortex (Figures 8A, 8B). In each of these areas, the receptors were Printed from Mimosa 60 expressed in cells morphologically consistent with neurons. All three receptor components were expressed at higher levels in the lateral septal nucleus than in the medial septal nucleus. Ret was expressed at much lower 5 levels than either GFRa-1 (TrnRl) or GFRa-2 (TrnR2) in the lateral septal nucleus. In the nucleus of the diagonal band, all three receptors were expressed at low levels with GFRa-2 (TrnR2) expressed at the lowest level. GFRa-1 (TrnRl) and GFRa-2 (TrnR2) mRNAs were expressed in 10 the absence of detectable Ret mRNA in the neocortex, claustrum, endopiriform nucleus, the bed nucleus of the stria terminalis, the basal nucleus of Meynert, the ventral pallidum, and the olfactory tubercle (Figure 8, Table 2). GFRa-1 (TrnRl) was expressed at high levels in 15 scattered clusters of neurons in the ventral pallidum. GFRa-2 (TrnR2) was expressed in neurons in the ventral pallidum at much lower levels and in fewer cells than GFRa-1 (TrnRl).

NTN (Figures- 8D, 9D) and GDNF (data not shown) mRNAs 20 were expressed in piriform cortex, in the hippocampus and, at low levels, in neocortex (Figures 8D, 9D; Table 2). In addition, expression of GDNF was seen at moderate levels in the olfactory tubercle and at very low levels in the nucleus accumbens, the globus pallidus, the 25 ventral pallidum, the subiculum, and the striatum (Table 2).

Olfactory Bulb In the olfactory bulb, mRNAs for all three receptors were expressed in the glomerular layer and in the granule 30 layer (Table 2). In the glomerular layer, GFRa-1 (TrnRl) and Ret were expressed at moderate levels and GFRa-2 (TrnR2) was expressed at a lower level. In the granule layer, GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed at higher levels than Ret. In the mitral cell layer, 35 both GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were highly expressed in the absence of ret. GFRa-1 (TrnRl) was Printed from Mimosa wo 98/46622 61 expressed in the absence of Ret and GFRa-2 (TrnR2) in the internal plexiform layer and the external plexiform layer.

NTN and GDNF mRNA expression were not detected in 5 the olfactory bulb (Table 2).

Midbrain Receptors for NTN and GDNF were widely expressed in the midbrain. Most notably, mRNAs for all three receptor components were detected in the pars compacta of the 10 substantia nigra, in which dopaminergic neurons, responsive to both GDNF and NTN in adult mice are located (Figure 10; Table 2) (Lin et al., 1993; Rosenblad et al., Soc. Neurosci. Rbstr. 23:248.12, 1997). Receptor expression in the substantia nigra was predominantly seen 15 in the pars compacta with much sparser labeling in the pars reticulata. In particular, Ret and GFRa-1 (TrnRl) were expressed at highest levels, while GFRa-2 (TrnR2) was expressed at substantially lower levels. All three receptor components were predominantly, but not 20 exclusively, expressed in neurons. Expression of all three receptor components was also found in the ventral tegmental area (VTA), the periaqueductal grey (PAG), the rostral linear raphe (RLi), the interfascicular nucleus (IF), and the Edinger Wesphal nucleus (EW). All three 25 receptor components were also expressed in the large motor neurons of the oculomotor nucleus (3). GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed in the absence of Ret in the superficial layers of the superior colliculus, in the interpeduncular nucleus, and in the 30 cerebral cortex (Figure 10). In addition, GFRa-1 (TrnRl) and Ret, but not GFRa-2 (TrnR2), were expressed in the red nucleus (Table 2).

NTN mRNA was expressed at a barely detectable level in the oculomotor nucleus. NTN expression was not seen 35 in any other areas of the midbrain. GDNF mRNA was expressed at very low levels in the substantia nigra pars Printed from Mimosa compacta, in the superficial layers of the superior colliculus and in the interfascicular nucleus.

Thalamus Receptor components were expressed in several nuclei 5 in the thalamus including both relay and association nuclei (Figures 9A-C; Table 2). The receptors were most prominently expressed in the reticular nucleus (Rt), the zona incerta, and the habenular nuclei (Figures 9A-C). In the reticular nucleus, mRNAs for GFRa-1 (TrnRl) and 10 GFRa-2 (TrnR2) were expressed at high levels while Ret mRNA was expressed at moderate-to-low levels. In the habenular nuclei, GFRa-1 (TrnRl) was expressed at high levels and the other two receptors were expressed at lower levels, particularly in the lateral habenular 15 nucleus. All three receptor components were also expressed at low levels in the medial geniculate nucleus (Figure 10).

Several of the sensory and motor relay nuclei expressed Ret mRNA at very low levels in the absence of 20 GFRa-1 or GFRa-2 (Figure 9; Table 2). These included the two main sensory relay nuclei, the ventroposteromedial (VPM) and the ventroposterolateral (VPL), and the primary motor relays, the ventromedial (VM) and ventrolateral (VL) nuclei. In the other major sensory relay, the 25 posterior nuclear group (Po), both Ret and GFRa-1 (TrnRl) were expressed at low levels but GFRa-2 (TmR2) was not detected. The same pattern was observed in the laterodorsal and lateroposterior nuclei, and the mediodorsal nucleus in which Ret and GFRa-1 (TrnRl) were 30 expressed at low levels and GFRa-2 (TrnR2) was absent.

In addition. Ret and GFRa-2 (TrnR2) were expressed in the absence of GFRa-1 (TrnRl) in the subthalamic nucleus (Figures 9A-C).

NTN was expressed at moderate levels in the 35 anteromedial and anteroventral nuclei of the thalamus (Figure 9D). GDNF mRNA was expressed at low levels in Printed from Mimosa 63 the anteromedial and anteroventral nuclei and, at very low levels in the reticular, ventromedial, ventrolateral, ventroposteromedial, and ventroposterolateral nuclei, and in the posterior nuclear group and the zona incerta 5 (Table 2).

Hvnothalamus In the hypothalamus, GF (TRN) receptor components were prominently expressed in many areas (Figure 9; Table 2). All three receptor components were detected in the 10 periventricular nucleus, the medial preoptic nuclei, both central and median, in the medial and lateral preoptic area, the ventromedial (VM) and dorsomedial (DM) hypothalamic nuclei, the supramammillary nucleus, and the posterior, anterior and lateral hypothalamic areas. 15 GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed, in the absence of Ret, in the paraventricular nucleus and the arcuate nucleus.

NTN was expressed in the supraoptic, and paraventricular nuclei (Figure 11A). NTN expression in 20 the supraoptic and paraventricular nuclei was particularly intense. In the supraoptic and paraventricular nuclei, NTN expression was localized to cells whose morphology is consistent with that of the large secretory neurons that are found in these nuclei 25 (Figures 11B, 11C). GDNF mRNA was expressed at barely detectable levels in the hypothalamus in the dorsomedial, ventromedial, arcuate and medial mammillary nuclei (Table 2).

Brainstem GF (TRN) receptor components were expressed in a number of brainstem nuclei including cranial nerve nuclei (Figure 12; Table 2). All three receptor components were expressed in the facial motor nucleus, in the region of the nucleus ambiguous, the abducens nucleus, the 35 prepositus hypoglossal nucleus, the spinal trigeminal sensory nuclei, the vagal dorsal motor nucleus, the Printed from Mimosa 64 lateral and medial vestibular nuclei, and the dorsal and ventral cochlear nuclei. GFRa-1 (TrnRl) and Ret were also expressed, in the absence of GFRa-2, in the region of the inferior salvitory nucleus and nucleus 5 retroambiguous, and in the hypoglossal nucleus, and in motor neurons in the trigeminal motor nucleus. In the principal sensory trigeminal nucleus. Ret was expressed in the absence of GFRa-1 TrnRl) and GFRa-2 (TrnR2). In the cranial motor nuclei, the receptors were expressed in 10 large motor neurons.

Other areas of the brainstem in which expression of all three receptors was detected included the raphe nuclei, the inferior colliculus, the pontine reticular nucleus, the gigantocellular reticular nucleus, the 15 tegmental nuclei, and the locus coeruleus.

NTN was expressed at very low levels in the region of the nucleus ambiguous. GDNF mRNA was expressed at a low level in the facial motor nucleus and the ventral cochlear nucleus (Figure 12C).

Spinal Cord Transverse sections of cervical, thoracic and lumbar spinal cord were examined. The labeling pattern for each of the receptors was the same at each level studied. GFRa-1 (TrmRl) and Ret were expressed prominently in 25 motor neurons of the ventral horn (Figures 13A, 13C).

Low levels of GFRa-2 (TrnR2) were expressed diffusely in the spinal cord gray matter with highest levels of expression found in the superficial layers of the dorsal horn (Figure 13B). In the ventral horn GFRa-2 (TrnR2) 30 mRNA was expressed in a few scattered motor neurons. Cerebellum In the cerebellum, Ret and GFRa-1 (TrnRl) were expressed at a high level in the Purkinje cell layer (Figures 14A, 14C). Both Ret and GRFa-1 (TrnRl) were 35 expressed in cells adjacent to Purkinje cells, but not in Purkinje cells. GFRa-2 (TrnR2) was strongly expressed in Printed from Mimosa 65 Purkinje cells in a pattern complementary to Ret and GFRa-1 (TrnRl) expression (Figure 14B). GFRa-2 was also expressed at a low level in the granule cell layer and Ret was expressed at a low level in the molecular layer.

In the deep cerebellar nuclei, all three receptor components were detected, with Ret and GFRa-1 (TrnRl) detected at higher levels than GFRa-2 (TrnR2) (Table 2).

Both NTN (Figure 14D) and GDNF (Table 2) were expressed at low levels in the Purkinje cell and granular 10 layers of the cerebellum. NTN and GDNF were not detected in the molecular layer. NTN, but not GDNF, was detected in the deep cerebellar nuclei.

Printed from Mimosa 66 TABLE 11—-Summary of GF Factor and Receptor Expression NTN GDNF GFRa-1 GFRa-2 RET FOREBRAIN Olfactory Bulb OB Glomerular Layer - - ++ + ++ Mitral Cell Layer ~ * - + ++ - Internal Plexiform Layer - - + - - External Plexiform Layer - - + - - Granule Layer - - + + + Olfactory Tubercle - + ++ ++ - Tu Piriform Cortex + ++ ++ ++ + Pir Claustrum - - ++ ++ - CI Dorsal Endopinform Nucleus - - ++ ++ - DEn Medial Septal Nucleus - - + + ++ MS Lateral Septal Nucleus - - ++ ++ + LS Nucleus Diagonal Band - - + ± + DB Bed Nucleus Stria Terminalis - - + + - BST Nucleus Accumbens - + - - - Acb Amygdala - ± + + ± A.

Basal Nucleus of Meynert - - ± + - B Ventral Pallidum - + ++ ± - VP Globus Pallidus - ± - - - GP Striatum ± ± - - - Cpu MIDBRAIN Substantia Nigra SN Compacta - ± +++ ++ +++ SNc Reticulata - - + + + SNr Ventral Tegmental Area - - +++ ++ +++ VTA Periaqueductal Gray - - +++ ++ +++ PAG Rostral Linear Raphe - - + + + RLi Dorsal Raphe - - ++ + + DR Oculomotor Nucleus ± + ++ ++ ++ 3 Edinger Westphal - - ++ + + EW Superior Colliculus - ± ++ ++ - SC Red Nucleus - - ± - + R Interfascicular Nucleus - ± ++ ++ ++ IF Interpeduncular Nucleus - - ++ ++ - IP CORTEX Neocortex + ± ++ +++ - Cingulate Cortex + + + ++ - Cg Hippocampus Subiculum - + ++ + - S CA1 + ++ ++ ++ CA2 ++ ++ ++ ++ CA3 ++++++ ++ Dentate + ++ ++ ++ - DG Printed from Mimosa 67 TABLE 1—Summary ot GF Factor and Receptor Expression NTN GDNF GFRa-1 GFRa-2 RET CEREBELLUM Granule Cell Layer + + - + - GR Purkinje Cell Layer + + ++ -H- ++ P Molecular Layer - - - - + Mol Deep Nuclei ± ' - -H- + ++ THALAMUS Medial Habenula - - +++ + + MHb Lateral Habenula - - -H- + + LHb Reticular Nucleus - - +++ +++ ++ Rt Mediodorsal Nucleus - - + - ++ MD Anteromedial Nucleus ++ + - - - AM Anteroventral Nucleus ++ + - - - AV Laterodorsal Nucleus - + ± - ± LD Lateral Posterior Nucleus - + ± - ± LP Zona Incerta - ± ++ ++ ++ ZI Subthalamic Nucleus - - - ++ ++ STh Ventromedial - ± - - ± VM Ventrolateral - ± - - ± VL Ventral Anterior ± - - - VA Posterior Nuclear Group - ± ± - ± Po Ventroposterolateral - + - - ± VPL Ventroposteromedial - + - - ± VPM Lateral Geniculate - - + - + LGN Medial Geniculate - - + + + MGN HYPOTHALAMUS Medial Preoptic Nucleus MP Central - - ++ ++ ++ Median - - -H- . ++ Preoptic Area PA Medial - - ++ ++ ++ Lateral - - ++ -H- ++ Periventricular Nucleus - - + + + Pe Tubercinerium - - ++ + ± T Lateral Hypothalamic Area - - + +++ ± LHA Anterior Hypothalamic Area - - ++ + + AHA Posterior Hypothalamic Area - - + + + PHA Ventromedial Hypothalamic Nucleus - ± + ++ ± VM Dorsomedial Nucleus - ± + ++ + DM Supraoptic Nucleus ++ - - - - SO Arcuate Nucleus - ± + ++ - Arc Medial Mammillary Nucleus - ± - - - MM Supramammillary Nucleus - - ± -H- + SuM Paraventricular Nucleus ++ - ++ + - PV Printed from Mimosa 68 TABLE 11—Summary of GF Factor and Receptor Expression NTN GDNF GFRa-1 GFRa-2 RET BRAINSTEM Median Raphe Raphe Pallidus Raphe Magnus Inferior Colliculus Preolivary Region Trigeminal Principalis Spinal Trigeminal Motor Trigeminal Nucleus Ambiguous Nucleus Retroambiguous Vagal Dorsal Motor Nucleus Hypoglossal Prepositus Hypoglossal Abducens Facial Nucleus Dorsal Cochlear Nucleus Ventral Cochlear Lateral Vestibular Medial Vestibular Inferior Salvitory Pontine Reticular Nucleus Gigantocellular reticular Tegmental Nuclei Locus Coeruleus + + + + ++ + ± + + + ++ + + + + + ++ + + + + + + + + + ± + + + MnR Rpa RMg IC Pr5 Mo5 12 6 7 DC VC Gi Tg LC SPINAL CORD Ventral Horn Dorsal Horn VH DH 1 Level of expression- +++, high; ++, moderate; +, low; ±, barely detected; not detected.

Printed from Mimosa 69 DISCUSSION In the experiments described in this example, we studied the expression of NTN and GDNF and their receptors in the adult mouse brain. GF (TRN) receptors 5 are widely expressed throughout the adult brain. In many areas of the brain, including all the areas in which GF-responsive neurons are present, Ret and either GFRa-1 (TrnRl) or GFRa-2 (TrnR2) mRNA are present. In some areas of the brain, most notably the cerebral cortex and 10 the hippocampus, co-receptors GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed without Ret.

NTN and GDNF mRNA were expressed at lower levels and in fewer areas of the adult mouse brain than GF receptor (TrnR) mRNA. NTN and GDNF were expressed in several 15 areas of the brain that receive projections from neurons expressing receptor mRNA. This expression pattern suggests that NTN and GDNF function through a classical target-derived mechanism of trophic factor action to maintain neuronal circuits in the mature CNS.

Neurons That Respond to NTN and/or GDNF in Adult Rodents Express GF (TRN> Receptors.

Several central neuronal populations respond to GDNF in the adult rodent including spinal motor neurons (Li et al., Proc. Natl. Read. Set. 92:9771-9775, 1995), facial 25 motor neurons (Yan et al., Nature 373 (6512):341-344, 1995), dopaminergic neurons of the ventral midbrain (Bowenkamp et al., J. Comp. Neurol. 355:479-489, 1995; Keams and Gash, Brain Res. 672:104-111, 1995; Tomac et al.. Nature 373:335-339, 1995; Cass, J. Neurosci. 30 16(24):8132-8139, 1996; Bowenkamp et al., Exp. Neurol. 145:104-117, 1997; Choi-Lunberg et al.. Science 275:838-841, 1997; Lapchak et al., Neurosci. 78(1):61-72, 1997), noradrenergic neurons of the locus coeruleus (Arenas et al.. Neuron 15:1465-1473, 1995), and cholinergic neurons 35 of the basal forebrain (Williams et al., J. Pharm. Exper. Ther. 277:1140-1151, 1996). In addition, dopaminergic Printed from Mimosa 70 midbrain neurons are responsive to NTN in adult rats (Rosenblad et al., supra).

In the experiments described in this example, we observed expression of GF (TRN) receptors in areas of the 5 brain in which responsive neurons are located. For example. In vivo, GDNF is a potent neurotrophic factor for spinal (Li et al., supra) and facial (Yan et al., supra) motoneurons in adult rodents. Both facial motor neurons and spinal motor neurons expressed Ret and GFRa-1 10 (TrnRl). In adult rodents, dopaminergic neurons in the pars compacta of the substantia nigra and in the VTA are protected from chemical and mechanical injury by NTN (Rosenblad et al., supra) or GDNF (Kearns and Gash, supra; Bowenkamp et al., 1997, supra). Ret, GFRa-1 15 (TrnRl) and GFRa-2 (TrnR2) were all expressed in neurons in these areas of the ventral midbrain. All three receptor components were also expressed in the interfascicular nucleus and the rostral linear raphe. In adult mice, dopaminergic neurons in these nuclei are 20 protected from MPTP (l-methyl-4-phenyl-l,2,3, 6- tetrahydropyridine) treatment by GDNF (Tomac et al., supra). GDNF treatment also protects cholinergic neurons of the septal/diagonal band nuclei from axotomy by fimbria/fornix section in adult rats (Williams et al., 25 supra). In our experiments, we found GF (TRN) receptors in both the medial septal nucleus and in the nucleus of the diagonal band of Broca in neurons morphologically consistent with large cholinergic neurons characteristic of this area. In the locus coeruleus, GDNF treatment 30 protects noradrenergic neurons from death (Arenas et al., supra). Neurons in the locus coeruleus expressed all three GF receptors. In summary, there is a strong positive coorelation between GF (TRN) receptor expression in mature neurons and responsiveness of these neurons to 35 NTN and GDNF.

GF (TRN) receptors were also expressed in areas of Printed from Mimosa 71 ■the adult brain In which responsiveness to NTN and GDNF has not yet been directly evaluated. Areas of the brain in which Ret and one or both of the co-receptors were expressed include the oculomotor nucleus, the subthalamic 5 nucleus, and several nuclei in the thalamus and hypothalamus. The strong correlation between receptor expression and responsiveness to NTN and GDNF suggests that neurons in these areas may respond to one or both factors.

Our finding that GF (TRN) receptors were expressed in areas of the hypothalamus involved in the control of feeding behavior and regulation of body weight, including the lateral hypothalamic area and the ventromedial and dorsomedial nuclei of the hypothalamus, suggests that NTN 15 and/or GDNF may be involved in these processes. Previous studies that show weight loss in rats (Hudson et al., Brain Res. Bull. 35;425-432, 1995) and monkeys (Gash et al., J. Comp. Neurol. 363:345-358, 1995) treated with GDNF support such a role for GDNF. Other neurons that 20 appear especially likely to benefit from the GF (TRN) factors are the motoneurons in the oculomotor nucleus; these neurons expressed all three GF (TRN) receptor components and their loss would be consistent with the ptosis observed in the NTN-deficient mice (Heuckeroth et 25 al., Soc. Neurosci. Rbstr. 23:668.1, 1997).

NTN and GDNF Expression Both NTN and GDNF were expressed in the targets of neurons that respond to these factors. As shown previously (Trupp et al., J. Neurosci. 17(10):3554-3567, 30 1997) and confirmed by the results reported here, GDNF mRNA is expressed in targets of dopaminergic neurons of the substantia nigra compacta (SNc); these targets include the olfactory tubercle, the nucleus accumbens, the striatum, and the globus pallidus. In addition, we 35 found low-level expression of GDNF mRNA in other SN targets including the piriform cortex, and the arcuate.

Printed from Mimosa 72 dorsomedial, and ventromedial nuclei of the hypothalamus. We found GDNF mRNA in the compacta region of the substantia nigra, where GDNF protein may support dopaminergic neurons through an autocrine or paracrine 5 mechanism. The present study also showed NTN mRNA expression in nigral targets including the arcuate and paraventricular nuclei of the hypothalamus. This distribution of NTN and GDNF mRNA is consistent with both of these growth factors functioning as target-derived 10 trophic factors for nigral dopaminergic neurons in the mature brain.

NTN and GDNF were expressed diffusely in all layers of neocortex. The cerebral cortex may serve as a source of NTN and GDNF for a number of cortical-projecting 15 neurons that express NTN and GDNF receptors; these include neurons in the nuclei of the medial septum and diagonal band of Broca that expressed Ret and GFRa-1 (TrnRl) and neurons in the locus coeruleus that express Ret and GFRa-1 (TrnRl) and GFRa-2 (TrnR2). NTN and GDNF 20 mRNA were also expressed in a number of areas of the hippocampal formation including all three fields of Amnion's horn (CA1-3) within the hippocampus proper and in the dentate gyrus. In addition, GDNF mRNA was expressed in the subiculum. Like the neocortex, the hippocampus is 25 a potential source of NTN and GDNF for neurons that project to this region of the brain and express GF receptors. Similarly, the medial septal nucleus and the nucleus of diagonal band of Broca, which also expressed GF (TRN) receptors, have large efferent projections to 30 all fields of the hippocampal formation. The supramammillary nucleus of the hypothalamus in which all three GF (TRN) receptors are expressed could obtain NTN or GDNF by way of its efferents to the dentate gyrus and the hippocampus proper. The dentate gyrus and the 35 hippocampus proper also receive a prominent projection from the locus coeruleus, which also expressed GF (TRN) Printed from Mimosa receptors.

The strongest and most circumscribed NTN expression in the adult brain was seen in the lateral and medial magnocellular paraventricular nucleus and in the 5 supraoptic nucleus of the hypothalamus. Within these nuclei are large magnocellular secretory neurons; these cells secrete oxytocin and vasopressin directly into the general circulation via the posterior pituitary. What function NTN may serve in these neurons is unclear. 10 Other growth factors, including fibroblast growth factor-2 (Gonzales et al., Endocrinology 134(5):2289-2297, 1994) and insulin-like growth factor I (Aguado et al., Neuroendocrinal. 56:856-863, 1992) are found in hypothalamic magnocellular neurons and are believed to be 15 involved in neuroendocrine function; NTN may serve a similar role. Alternatively, NTN in the supraoptic and paraventricular nuclei may be a source of target-derived trophic support for a number of neuronal populations that express GF (TRN) receptors and have efferent projections 20 -to these hypothalamic nuclei. Fox example, neurons in the medial preoptic nucleus and the medial and lateral septal nuclei project heavily to the supraoptic nucleus. Cells in these nuclei expressed Ret mRNA as well as mRNA for GFRa-1 (TrnRl) and GFRa-2 (TrnR2). Afferent 25 projections to the magnocellular neurons of the paraventricular nucleus are less well defined but are believed to include projections from the medial preoptic nucleus and the median and dorsal raphe nuclei. We found GF (TRN) receptor expression in each of these nuclei. 30 Another possibility is that NTN acts at some site distant to the hypothalamus and is secreted directly into the peripheral circulation like oxytocin and vasopressin. Brain Regions That Do Not Contain Complete Receptor Complexes In our experiments as well as in previous studies (Nosrat et al., Exp. Brain Res. 225;410-422, 1997; Tomac et al., supra) of GF (TRN) receptor expression, Printed from Mimosa 74 incomplete receptor complexes have been found in some areas of the brain. For instance, in the thalamus and the trigeminal principal sensory nucleus, ret was expressed in the absence of GFRa-1 (TrnRl) and GFRa-2 (TrnR2). In other regions, most strikingly neocortex and hippocampus, GFRa-1 (TrnRl) or GFRa-2 5 (TrnR2) were expressed in the absence of Ret. These expression patterns raise the possibility that additional, as yet unidentified, receptor components for GDNF and NTN exist. Additional ligand-binding receptor components or other signaling components may exist in addition to Ret. This latter possibility seems less probable since all neurons that respond to NTN and GDNF express Ret. 10 An alternative possibility, which has been suggested previously (Trupp et al., supra), is that, in some cases, responsive neurons express Ret only and the ligand-binding component is supplied in trans perhaps by the target of the responsive neuron. Several patterns of NTN and GDNF receptor expression are consistent with this possibility. For example, in the principle thalamic motor (VL, 15 VM) and sensory (VPM, VPL) relay nuclei, Ret was expressed in the absence of GFRa-1 (TmRl) or GFRa-2 (TmR2). Cells in these nuclei send projections to cerebral cortex where both GFRa-1 (TrnRl) and GFRa-2 (TrnR2) were expressed at high levels. NTN and GDNF were also expressed in cerebral cortex. Therefore, thalamic neurons that express Ret may obtain NTN or GDNF as well as the 20 ligand-binding receptor component from their target cortical neurons Another example of incomplete receptor expression was found in the Purkinje neurons of the cerebellum. In these neurons, only GFRa-2 (TrnR2) mRNA was expressed. Ret mRNA was expressed in other cells in the Purkinje layer, possibly basket cells or glial cells. The reason for this segregated expression 25 in Purkinje neurons is unclear. Whether mature Purkinje neurons respond to NTN or GDNF is unknown, although embryonic Purkinje neurons respond to GDNF in vitro (Mount et al., Proc. Natl. Acad. Sci. 92:9092-9096, 1995).

Our findings show that GF (TRN) receptors are expressed in areas of the adult brain in which neurons that respond to these factors are located. In addition, 30 GF (TRN) receptor expression in other areas suggests that additional NTN- or GDNF-responsive populations exist. In summary, the mRNA expression pattern Printed from Mimosa 75 for NTN, GDNF, and GF (TRN) receptors in the adult brain strongly suggests a role for these proteins as target-derived trophic factors for mature neurons.

In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained. 5 As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Printed from Mimosa 76 SEQUENCE LISTING (1) GENERAL INFORMATION. (i) APPLICANT: MILBRANDT, JEFFREY D JOHNSON JR, EUGENE M BALOH, ROBERT H (li) TITLE OF INVENTION TrnR2, A Novel Receptor Which Mediates Neurturin and GDNF Signaling Through Ret (ill) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE ADDRESS.

(A) ADDRESSEE: HOWELL & HAFERKAMP, LC (B) STREET: 7733 FORSYTH BLVD (C) CITY. ST LOUIS (D) STATE. MO (E) COUNTRY: USA (F) ZIP: 63105 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER. IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: -(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HOLLAND, DONALD R (B) REGISTRATION NUMBER: 3 5197 (C) REFERENCE/DOCKET NUMBER: 976328 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 314-727-5188 (B) TELEFAX: 314-727-6092 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1543 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION- 36..1427 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 36. 98 Printed from Mimosa WO 98/46622 PCT/US98/07996 77 (XI) SEQUENCE DESCRIPTION. SEQ ID NO:1.

GAGAAAGACA AAAAAAACGG TGGGATTTAT TTAAC ATG ATC TTG GCA AAC GTC 53 Met He Leu Ala Asn Val -21 -20 TTC TGC CTC TTC TTC TTT CTA GAC GAG ACC CTC CGC TCT TTG GCC AGC 101 Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr Leu Arg Ser Leu Ala Ser -15 -10 -5 1 CCT TCC TCC CTG CAG GGC CCC GAG CTC CAC GGC TGG CGC CCC CCA GTG 14 9 Pro Ser Ser Leu Gin Gly Pro Glu Leu His Gly Trp Arg Pro Pro Val 5 10 15 GAC TGT GTC CGG GCC AAT GAG CTG TGT GCC GCC GAA TCC AAC TGC AGC 197 Asp Cys Val Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn Cys Ser 20 25 30 TCT CGC TAC CGC ACT CTG CGG CAG TGC CTG GCA GGC CGC GAC CGC AAC 245 Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu Ala Gly Arg Asp Arg Asn 35 40 45 ACC ATG CTG GCC AAC AAG GAG TGC CAG GCG GCC TTG GAG GTC TTG CAG 293 Thr Met Leu Ala Asn Lys Glu Cys Gin Ala Ala Leu Glu Val Leu Gin 50 55 60 65 GAG AGC CCG CTG TAC GAC TGC CGC TGC AAG CGG GGC ATG AAG AAG GAG 341 Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 70 75 80 CTG CAG TGT CTG CAG ATC TAC TGG AGC ATC CAC CTG GGG CTG ACC GAG 389 Leu Gin Cys Leu Gin lie Tyr Trp Ser lie His Leu Gly Leu Thr Glu 85 90 95 GGT GAG GAG TTC TAC GAA GCC TCC CCC TAT GAG CCG GTG ACC TCC CGC 4 37 Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser Arg 100 105 110 V CTC TCG GAC ATC TTC AGG CTT GCT TCA ATC TTC TCA GGG ACA GGG GCA 485 Leu Ser Asp lie Phe Arg Leu Ala Ser lie Phe Ser Gly Thr Gly Ala 115 120 125 GAC CCG GTG GTC AGC GCC AAG AGC AAC CAT TGC CTG GAT GCT GCC AAG 533 Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala Lys 130 135 140 145 GCC TGC AAC CTG AAT GAC AAC TGC AAG AAG CTG CGC TCC TCC TAC ATC 581 Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr lie 150 155 160 TCC ATC TGC AAC CGC GAG ATC TCG CCC ACC GAG CGC TGC AAC CGC CGC 629 Ser lie Cys Asn Arg Glu lie Ser Pro Thr Glu Arg Cys Asn Arg Arg 165 170 175 AAG TGC CAC AAG GCC CTG CGC CAG TTC TTC GAC CGG GTG CCC AGC GAG 677 Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser Glu 180 185 190 Printed from Mimosa WO 98/46622 PCT/US98/07996 78 TAC ACC TAC CGC ATG CTC TTC TGC TCC TGC CAA GAC CAG GCG TGC GCT Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys Ala 195 200 205 725 GAG CGC CGC CGG CAA ACC ATC CTG CCC AGC TGC TCC TAT GAG GAC AAG Glu Arg Arg Arg Gin Thr lie Leu Pro Ser Cys Ser Tyr Glu Asp Lys 210 215 220 225 773 GAG AAG CCC AAC TGC CTG GAC CTG CGT GGC GTG TGC CGG ACT GAC CAC Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr Asp Hxs 230 235 240 821 CTG TGT CGG TCC CGG CTG GCC GAC TTC CAT GCC AAT TGT CGA GCC TCC Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala Ser 245 250 255 869 TAC CAG ACG GTC ACC AGC TGC CCT GCG GAC AAT TAC CAG GCG TGT CTG Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys Leu 260 265 270 917 GGC TCT TAT GCT GGC ATG ATT GGG TTT GAC ATG ACA CCT AAC TAT GTG Gly Ser Tyr Ala Gly Met lie Gly Phe Asp Met Thr Pro Asn Tyr Val 275 280 285 965 GAC TCC AGC CCC ACT GGC ATC GTG GTG TCC CCC TGG TGC AGC TGT CGT Asp Ser Ser Pro Thr Gly lie Val Val Ser Pro Trp Cys Ser Cys Arg 290 295 300 305 1013 GGC AGC GGG AAC ATG GAG GAG GAG TGT GAG AAG TTC CTC AGG GAC TTC Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp Phe 310 315 320 1061 ACC GAG AAC CCA TGC CTC CGG AAC GCC ATC CAG GCC TTT GGC AAC GGC Thr Glu Asn Pro Cys Leu Arg Asn Ala lie Gin Ala Phe Gly Asn Gly 325 330 335 1109 ACG GAC GTG AAC GTG TCC CCA AAA GGC CCC TCG TTC CAG GCC ACC CAG Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gin Ala Thr Gin 340 345 350 1157 GCC CCT CGG GTG GAG AAG ACG CCT TCT TTG CCA GAT GAC CTC AGT GAC Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser Asp 355 360 365 1205 AGT ACC AGC TTG GGG ACC AGT GTC ATC ACC ACC TGC ACG TCT GTC CAG Ser Thr Ser Leu Gly Thr Ser Val lie Thr Thr Cys Thr Ser Val Gin 370 375 380 385 1253 GAG CAG GGG CTG AAG GCC AAC AAC TCC AAA GAG TTA AGC ATG TGC TTC Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys Phe 390 395 400 1301 ACA GAG CTC ACG ACA AAT ATC ATC CCA GGG AGT AAC AAG GTG ATC AAA Thr Glu Leu Thr Thr Asn lie lie Pro Gly Ser Asn Lys Val lie Lys 405 410 415 1349 CCT AAC TCA GGC CCC AGC AGA GCC AGA CCG TCG GCT GCC TTG ACC GTG 1397 Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu Thr Val 420 425 430 V 'N CTG TCT GTC CTG ATG CTG AAA CAG GCC TTG TAGGCTGTGG GAACCGAGTC 1447 Leu Ser Val Leu Met Leu Lys Gin Ala Leu 435 440 AGAAGATTTT TGAAAGCTAC GCAGACAAGA ACAGCCGCCT GACGAAATGG AAACACACAC 1507 Printed from Mimosa WO 98/46622 PCT/US98/07996 79 AGACACACAC ACACCTTGCA AAAAAAAAAA AAAAAA 1543 (2) INFORMATION FOR SEQ ID NO:2. (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 464 amino acids (B) TYPE, amino acid (D) TOPOLOGY: linear (li) MOLECULE TYPE- protein (Xl) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met lie Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr -21 -20 -15 -10 Leu Arg Ser Leu Ala Ser Pro Ser Ser Leu Gin Gly Pro Glu Leu His -5 15 10 Gly Trp Arg Pro Pro Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala 15 20 25 Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu 30 35 40 Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala 45 50 55 Ala Leu Glu Val Leu Gin Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 60 65 70 75 Arg Gly Met Lys Lys Glu Leu Gin Cys Leu Gin lie Tyr Trp Ser lie 80 85 90 His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr 95 100 105 Glu Pro Val Thr Ser Arg Leu Ser Asp lie Phe Arg Leu Ala Ser lie 110 115 120 Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His 125 130 135 Cys Leu Asp Ala Ala Lye Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys 140 145 150 155 Leu Arg Ser Ser Tyr lie Ser lie Cys Asn Arg Glu lie Ser Pro Thr 160 165 170 Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe 175 180 185 Abp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys 190 195 200 Gin Asp Gin Ala Cys Ala Glu Arg Arg Arg Gin Thr lie Leu Pro Ser 205 210 215 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly 220 225 230 235 Val Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His 240 245 250 Printed from Mimosa WO 98/46622 PCT/US98/07996 80 Ala Asn Cys Arg Ala Ser Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp 255 260 265 Asn Tyr Gin Ala Cys Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp 270 275 280 Met Thr Pro Asn Tyr Val Asp Ser Ser Pro Thr Gly lie Val Val Ser 285 290 295 Pro Trp Cys Ser Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu 300 305 310 315 Lys Phe Leu Arg Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie 320 325 330 Gin Ala Phe Gly Asn Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro 335 340 345 Ser Phe Gin Ala Thr Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu 350 355 360 Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val lie Thr 365 370 375 Thr Cys Thr Ser Val Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys 380 385 390 395 Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn lie lie Pro Gly 400 405 410 Ser Asn Lys Val lie Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro 415 420 425 Ser Ala Ala Leu Thr Val Leu Ser Val Leu Met Leu Lys Gin Ala Leu 430 435 440 (2) INFORMATION FOR SEQ ID NO:3: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Ser Pro Ser Ser Leu Gin Gly Pro Glu Leu His Gly Trp Arg Pro Pro 15 10 15 Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn Cys 20 25 30 Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu Ala Gly Arg Asp Arg 35 40 45 Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala Ala Leu Glu Val Leu 50 55 60 Printed from Mimosa WO 98/46622 PCT/US98/07996 81 Gin Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys 65 70 75 80 Glu Leu Gin Cys Leu Gin lie Tyr Trp Ser lie His Leu Gly Leu Thr 85 90 95 Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser 100 105 110 Arg Leu Ser Asp lie Phe Arg Leu Ala Ser lie Phe Ser Gly Thr Gly 115 120 125 Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 130 135 140 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr 145 150 155 160 lie Ser lie Cys Asn Arg Glu lie Ser Pro Thr Glu Arg Cys Asn Arg 165 170 175 Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser 180 185 190 Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys 195 200 205 Ala Glu Arg Arg Arg Gin Thr lie Leu Pro Ser Cys Ser Tyr Glu Asp 210 215 220 Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Gly Val Cys Arg Thr Asp 225 230 235 240 His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala 245 250 255 Ser Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys 260 265 270 Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp Met Thr Pro Asn Tyr 275 280 285 Val Asp Ser Ser Pro Thr Gly lie Val Val Ser Pro Trp Cys Ser Cys 290 295 300 Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp 305 310 315 320 Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie Gin Ala Phe Gly Asn 325 330 335 ( Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gin Ala Thr 340 345 350 Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser 355 360 365 Asp Ser Thr Ser Leu Gly Thr Ser Val lie Thr Thr Cys Thr Ser Val 370 375 380 Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys 385 390 395 400 Phe Thr Glu Leu Thr Thr Asn lie lie Pro Gly 405 410 Printed from Mimosa WO 98/46622 PCT/US98/07996 82 (2) INFORMATION FOR SEQ ID NO-4- (l) SEQUENCE CHARACTERISTICS (A) LENGTH 13 92 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ll) MOLECULE TYPE. cDNA (ill) HYPOTHETICAL NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY. CDS (B) LOCATION' 1. .1389 (ix) FEATURE: (A) NAME/KEY. sig_peptide (B) LOCATION. 1 .63 (ix) FEATURE: (A) NAME/KEY: matjpeptide (B) LOCATION: 64..1389 (xi) SEQUENCE DESCRIPTION: SEQ ID NO-4.

ATG ATC TTG GCA AAC GCC TTC TGC CTC TTC TTC TTT TTA GAC GAA ACC 48 Met He Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr -21 -20 -15 -10 CTC CGC TCT TTG GCC AGC CCT TCC TCT CCG CAG GGC TCT GAG CTC CAC 96 Leu Arg Ser Leu Ala Ser Pro Ser Ser Pro Gin Gly Ser Glu Leu His -5 15 10 GGC TGG CGC CCC CAA GTG GAC TGT GTC CGG GCC AAT GAG CTG TGT GCG 144 Gly Trp Arg Pro Gin Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala 15 20 25 GCT GAA TCC AAC TGC AGC TCC AGG TAC CGC ACC CTT CGG CAG TGC CTG 192 Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu 30 35 40 GCC GGC CGG GAT CGC AAT ACC ATG CTG GCC AAT AAG GAG TGC CAG GCG 240 Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala 45 50 55 GCC CTG GAG GTC TTG CAG GAA AGC CCA TTG TAT GAC TGC CGC TGC AAG 288 Ala Leu Glu Val Leu Gin Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 60 65 70 75 CGG GGC ATG AAG AAG GAG CTG CAG TGT CTG CAG ATC TAT TGG AGC ATC 336 Arg Gly Met Lys Lys Glu Leu Gin Cys Leu Gin lie Tyr Trp Ser lie 80 85 90 CAT CTG GGG CTG ACG GAG GGT GAG GAG TTC TAC GAA GCT TCG CCC TAT 384 His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr 95 100 105 GAG CCT GTG ACC TCC CGC CTC TCG GAC ATC TTC AGG CTC GCT TCA ATC 432 Glu Pro Val Thr Ser Arg Leu Ser Asp lie Phe Arg Leu Ala Ser lie 110 115 120 Printed from Mimosa WO 98/46622 PCT/US98/07996 83 TTC TCA GGG ACA GGG GCA GAC CCG GTG GTC AGT GCC AAG AGC AAC CAC 480 Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala Lys Ser Asn His 125 130 135 TGC CTG GAT GCC GCC AAG GCC TGC AAC CTG AAC GAC AAC TGC AAG AAG 528 Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys 140 145 150 155 CTC CGC TCC TCC TAC ATC TCC ATC TGC AAC CGC GAG ATC TCT CCC ACT 576 Leu Arg Ser Ser Tyr lie Ser lie Cys Asn Arg Glu lie Ser Pro Thr 160 165 170 GAA CGC TGC AAC CGC CGC AAG TGC CAC AAG GCC CTG CGC CAG TTC TTC 624 Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe 175 180 185 GAC CGT GTG CCC AGC GAG TAT ACC TAC CGC ATG CTC TTC TGC TCC TGT 672 Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys 190 195 200 CAG GAC CAG GCA TGC GCC GAG CGT CGC CGG CAA ACC ATC CTG CCC AGC 720 Gin Asp Gin Ala Cys Ala Glu Arg Arg Arg Gin Thr lie Leu Pro Ser 205 210 215 TGT TCC TAT GAG GAC AAG GAG AAG CCC AAC TGC TTG GAC CTG CGC AGC 768 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser 220 225 230 235 CTG TGT CGT ACA GAC CAC TTG TGC CGG TCC CGC CTG GCA GAC TTC CAC 816 Leu Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His 240 245 250 GCC AAC TGT CGA GCC TCC TAC CGG ACA ATC ACC AGC TGC CCT GCG GAC 864 Ala Asn Cys Arg Ala Ser Tyr Arg Thr lie Thr Ser Cys Pro Ala Asp 255 260 265 AAC TAC CAG GCA TGT CTG GGC TCC TAT GCT GGC ATG ATT GGG TTT GAT 912 Asn Tyr Gin Ala Cys Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp 270 275 280 ATG ACA CCG AAC TAT GTG GAC TCC AAC CCC ACG GGC ATC GTG GTG TCT 960 Met Thr Pro Asn Tyr Val Asp Ser Asn Pro Thr Gly lie Val Val Ser 285 290 295 CCC TGG TGC AAT TGT CGT GGC AGT GGG AAC ATG GAA GAA GAG TGT GAG 1008 Pro Trp Cys Asn Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu 300 305 310 315 AAG TTC CTC AAG GAC TTC ACA GAA AAC CCA TGC CTC CGG AAT GCC ATT 1056 Lys Phe Leu Lys Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie 320 325 330 CAA GCC TTT GGC AAT GGC ACA GAT GTG AAC ATG TCT CCC AAA GGC CCC 1104 Gin Ala Phe Gly Asn Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro 335 340 345 ACA TTT TCA GCT ACC CAG GCC CCT CGG GTA GAG AAA ACT CCT TCA CTG 1152 Thr Phe Ser Ala Thr Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu 350 355 360 CCA GAT GAC CTC AGT GAT AGC ACC AGC CTG GGG ACC AGT GTC ATC ACC 1200 Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val lie Thr 365 370 375 Printed from Mimosa WO 98/46622 PCT/US98/07996 84 ACC TGC ACA TCT ATC CAG GAG CAA GGG CTG AAG GCC AAC AAC TCC AAA 1248 Thr Cys Thr Ser lie Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys 380 385 390 395 GAG TTA AGC ATG TGT TTC ACA GAG CTC ACG ACA AAT ATC AGC CCA GGG 12 96 Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn lie Ser Pro Gly 400 405 410 AGT AAA AAG GTG ATC AAA CTT TAC TCA GGC TCC TGC AGA GCC AGA CTG 1344 Ser Lys Lys Val lie Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu 415 420 425 TCG ACT GCC TTG ACT GCC CTC CCA CTC CTG ATG GTG ACC TTG GCC 1389 Ser Thr Ala Leu Thr Ala Leu Pro Leu Leu Met Val Thr Leu Ala 430 435 440 TAG 1392 (2) INFORMATION FOR SEQ ID NO-5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 463 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (li) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Met lie Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Asp Glu Thr -21 -20 -15 -10 Leu Arg Ser Leu Ala Ser Pro Ser Ser Pro Gin Gly Ser Glu Leu His -5 15 10 Gly Trp Arg Pro Gin Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala 15 20 25 Ala Glu Ser Asn Cys Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu 30 35 40 Ala Gly Arg Asp Arg Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala 45 50 55 Ala Leu Glu Val Leu Gin Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys 60 65 70 75 Arg Gly Met Lys Lys Glu Leu Gin Cys Leu Gin lie Tyr Trp Ser lie 80 85 90 His Leu Gly Leu Thr Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr 95 100 105 Glu Pro Val Thr Ser Arg Leu Ser Asp lie Phe Arg Leu Ala Ser lie 110 115 120 Phe Ser Gly Thr Gly Ala Asp Pro Val Val Ser Ala LyB Ser ABn His 125 130 135 Cys Leu Asp Ala Ala Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys 140 145 150 155 Leu Arg Ser Ser Tyr lie Ser lie Cys Asn Arg Glu lie Ser Pro Thr 160 165 170 Printed from Mimosa 85 Glu Arg Cys Asn Arg Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe 175 180 185 Asp Arg Val Pro Ser Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys 190 195 200 Gin Asp Gin Ala Cys Ala Glu Arg Arg Arg Gin Thr lie Leu Pro Ser 205 210 215 Cys Ser Tyr Glu Asp Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser 220 225 230 235 Leu Cys Arg Thr Asp His Leu Cys Arg Ser Arg Leu Ala Asp Phe His 240 245 250 Ala Asn Cys Arg Ala Ser Tyr Arg Thr lie Thr Ser Cys Pro Ala Asp 255 260 265 Asn Tyr Gin Ala Cys Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp 270 275 280 Met Thr Pro Asn Tyr Val Asp Ser Asn Pro Thr Gly lie Val Val Ser 285 290 295 Pro Trp Cys Asn Cys Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu 300 305 310 315 Lys Phe Leu Lys Asp Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie 320 325 330 Gin Ala Phe Gly Asn Gly Thr Asp Val Asn Met Ser Pro Lys Gly Pro 335 340 345 Thr Phe Ser Ala Thr Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu 350 355 360 Pro Asp Asp Leu Ser Asp Ser Thr Ser Leu Gly Thr Ser Val lie Thr 365 370 375 Thr Cys Thr Ser lie Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys 380 385 390 395 Glu Leu Ser Met Cys Phe Thr Glu Leu Thr Thr Asn lie Ser Pro Gly 400 405 410 Ser Lys Lys Val lie Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu 415 420 425 Ser Thr Ala Leu Thr Ala Leu Pro Leu Leu Met Val Thr Leu Ala 430 435 440 (2) INFORMATION FOR SEQ ID N0:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 411 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: YES Printed from Mimosa WO 98/46622 PCT/US98/07996 86 (xi) SEQUENCE DESCRIPTION. SEQ ID NO 6: Ser Pro Ser Ser Pro Gin Gly Ser Glu Leu His Gly Trp Arg Pro Gin 15 10 15 Val Asp Cys Val Arg Ala Asn Glu Leu Cys Ala Ala Glu Ser Asn Cys 20 25 30 Ser Ser Arg Tyr Arg Thr Leu Arg Gin Cys Leu Ala Gly Arg Asp Arg 35 40 45 Asn Thr Met Leu Ala Asn Lys Glu Cys Gin Ala Ala Leu Glu Val Leu 50 55 60 Gin Glu Ser Pro Leu Tyr Asp Cys Arg Cys Lys Arg Gly Met Lys Lys 65 70 75 80 Glu Leu Gin Cys Leu Gin lie Tyr Trp Ser lie His Leu Gly Leu Thr 85 90 95 Glu Gly Glu Glu Phe Tyr Glu Ala Ser Pro Tyr Glu Pro Val Thr Ser 100 105 110 Arg Leu Ser Asp lie Phe Arg Leu Ala Ser lie Phe Ser Gly Thr Gly 115 120 125 Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 130 135 140 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr 145 150 155 160 lie Ser lie Cys Asn Arg Glu He Ser Pro Thr Glu Arg Cys Asn Arg 165 170 175 Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser 180 185 190 Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys 195 200 205 Ala Glu Arg Arg Arg Gltl Thr lie Leu Pro Ser Cys Ser Tyr Glu Asp 210 215 220 Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser Leu Cys Arg Thr Asp 225 230 235 240 His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala 245 250 255 Ser Tyr Arg Thr lie Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys 260 265 270 Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp Met Thr Pro Asn Tyr 275 280 285 Val Asp Ser Asn Pro Thr Gly lie Val Val Ser Pro Trp Cys Asn Cys 290 295 300 Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Lys Asp 305 310 315 320 Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie Gin Ala Phe Gly Asn 325 330 335 Printed from Mimosa 87 Gly Thr Asp Val Asn Met Ser Pro 340 Gin Ala Pro Arg Val Glu Lys Thr 355 360 Asp Ser Thr Ser Leu Gly Thr Ser 370 375 Gin Glu Gin Gly Leu Lys Ala Asn 385 390 Phe Thr Glu Leu Thr Thr Asn lie 405 Lys Gly Pro Thr Phe Ser Ala Thr 345 350 Pro Ser Leu Pro Asp Asp Leu Ser 365 Val lie Thr Thr Cys Thr Ser lie 380 Asn Ser Lys Glu Leu Ser Met Cys 395 400 Ser Pro Gly 410 (2) INFORMATION FOR SEQ ID NO:7- (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 331 amino acids (B) TYPE, amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (lii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met lie Leu Ala Asn Val Phe Cys Leu Phe Phe Phe Leu Gly Thr Gly 15 10 15 Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 20 25 30 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr 35 40 45 lie Ser lie Cys Asn Arg Glu lie Ser Pro Thr Glu Arg Cys Asn Arg 50 55 60 Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser 65 70 75 80 Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys 85 90 95 Ala Glu Arg Arg Arg Gin Thr lie Leu Pro Ser Cys Ser Tyr Glu Asp 100 105 110 Lys Glu Lys Pro Asn CyB Leu Asp Leu Arg Gly Val Cys Arg Thr Asp 115 120 125 His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala 130 135 140 Ser Tyr Gin Thr Val Thr Ser Cys Pro Ala Asp Aen Tyr Gin Ala Cys 145 150 155 160 Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp Met Thr Pro Asn Tyr 165 170 175 Printed from Mimosa WO 98/46622 PCT/US98/07996 88 Val Asp Ser Ser Pro Thr Gly lie Val Val Ser Pro Trp Cys Ser Cys 180 185 190 Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Arg Asp 195 200 205 Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie Gin Ala Phe Gly Asn 210 215 220 Gly Thr Asp Val Asn Val Ser Pro Lys Gly Pro Ser Phe Gin Ala Thr 225 230 235 240 Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser 245 250 255 Asp Ser Thr Ser Leu Gly Thr Ser Val lie Thr Thr Cys Thr Ser Val 260 265 270 Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys 275 280 285 Phe Thr Glu Leu Thr Thr Asn lie lie Pro Gly Ser Asn Lys Val lie 290 295 300 Lys Pro Asn Ser Gly Pro Ser Arg Ala Arg Pro Ser Ala Ala Leu Thr 305 310 315 320 Val Leu Ser Val Leu Met Leu Lys Gin Ala Leu 325 330 (2) INFORMATION FOR SEQ ID NO:8. (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 330 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met lie Leu Ala Asn Ala Phe Cys Leu Phe Phe Phe Leu Gly Thr Gly 15 10 15 Ala Asp Pro Val Val Ser Ala Lys Ser Asn His Cys Leu Asp Ala Ala 20 25 30 Lys Ala Cys Asn Leu Asn Asp Asn Cys Lys Lys Leu Arg Ser Ser Tyr 35 40 45 lie Ser lie Cys Asn Arg Glu lie Ser Pro Thr Glu Arg Cys Asn Arg 50 55 60 Arg Lys Cys His Lys Ala Leu Arg Gin Phe Phe Asp Arg Val Pro Ser 65 70 75 80 Glu Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys 85 90 95 Ala Glu Arg Arg Arg Gin Thr lie Leu Pro Ser Cys Ser Tyr Glu Asp 100 105 110 Printed from Mimosa 89 Lys Glu Lys Pro Asn Cys Leu Asp Leu Arg Ser Leu Cys Arg Thr Asp 115 120 125 His Leu Cys Arg Ser Arg Leu Ala Asp Phe His Ala Asn Cys Arg Ala 130 135 140 Ser Tyr Arg Thr lie Thr Ser Cys Pro Ala Asp Asn Tyr Gin Ala Cys 145 150 155 160 Leu Gly Ser Tyr Ala Gly Met lie Gly Phe Asp Met Thr Pro Asn Tyr 165 170 175 Val Asp Ser Asn Pro Thr Gly lie Val Val Ser Pro Trp Cys Asn Cys 180 185 190 Arg Gly Ser Gly Asn Met Glu Glu Glu Cys Glu Lys Phe Leu Lys Asp 195 200 205 Phe Thr Glu Asn Pro Cys Leu Arg Asn Ala lie Gin Ala Phe Gly Asn 210 215 220 Glv Thr Asp Val Asn Met Ser Pro Lys Gly Pro Thr Phe Ser Ala Thr 225 230 235 240 Gin Ala Pro Arg Val Glu Lys Thr Pro Ser Leu Pro Asp Asp Leu Ser 245 250 255 Asp Ser Thr Ser Leu Gly Thr Ser Val lie Thr Thr Cys Thr Ser lie 260 265 270 Gin Glu Gin Gly Leu Lys Ala Asn Asn Ser Lys Glu Leu Ser Met Cys 275 280 285 Phe Thr Glu Leu Thr Thr Asn lie Ser Pro Gly Ser Lys Lys Val lie 290 295 300 Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu Ser Thr Ala Leu Thr 305 310 315 320 Ala Leu Pro Leu Leu Met Val Thr Leu Ala 325 330 (2) INFORMATION FOR SEQ ID N0:9: <i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 1 1 5 10 Printed from Mimosa 90 (2) INFORMATION FOR SEQ ID NO.10: (l) SEQUENCE CHARACTERISTICS- (A) LENGTH. 11 amino acids (B) TYPE, amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION. SEQ ID NO.10 Cys Asn Arg Arg Lys Cys His Lys Ala Lys Arg 15 10 (2) INFORMATION FOR SEQ ID NO-11: (l) SEQUENCE CHARACTERISTICS- (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE- internal (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /product= "OTHER" /note= "Xaa is Lys or Arg" (xi) SEQUENCE DESCRIPTION: SEQ ID NO.-ll- Cys Leu Xaa Asn Ala lie Glu Ala Phe Gly Asn Gly 15 10 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 465 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION SEQ ID NO-.12.

Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 15 10 15 Printed from Mimosa WO 98/46622 PCT/US98/07996 91 Leu Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 Leu Arg Gin Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Ala Ser 50 55 60 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu 85 90 95 Lys Asn Cys Leu Arg lie Tyr Trp Ser Met Tyr Gin Ser Leu Gin Gly 100 105 110 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125 Ser Asp lie Phe Arg Val Val Pro Phe lie Ser Asp Val Phe Gin Gin 130 135 140 Val Glu His lie Pro Lys Gly Asn Asn Cys Leu Asp Ala Ala -Lys Ala 145 150 155 160 Cys Asn Leu Asp Asp lie Cys Lys Lys Tyr Arg Ser Ala Tyr lie Thr 165 170 175 Pro Cys Thr Thr Ser Val Ser Asn Asp Val Cys Asn Arg Arg Lys Cys 180 185 190 His Lys Ala Leu Arg Gin Phe Phe Asp Lys Val Pro Ala Lys His Ser 195 200 205 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp lie Ala Cys Thr Glu Arg 210 215 220 Arg Arg Gin Thr lie Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Lys 225 230 235 240 Pro Asn Cys Leu Ser Leu Gin Asp Ser Cys Lys Thr Asn Tyr lie Cys 245 250 255 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gin Pro Glu Ser Arg 260 265 270 Ser Val Ser Ser Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala 275 280 285 Tyr Ser Gly Leu lie Gly Thr Val Met Thr Pro Asn Tyr lie Asp Ser 290 295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 Asp Leu Glu Glu Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 Cys Leu Lys Asn Ala lie Gin Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 Val Trp Gin Pro Ala Pro Pro Val Gin Thr Thr Thr Ala Thr Thr Thr 355 360 365 Printed from Mimosa 92 Thr Ala Leu Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 Asn Glu lie Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gin Ala 385 390 395 400 Gin Lys Leu Lys Ser Asn Val Ser Gly Asn Thr His Leu Cys lie Ser 405 410 415 Asn Gly Asn Tyr Glu Lys Glu Gly Leu Gly Ala Ser Ser His lie Thr 420 425 430 Thr Lys Ser Met Ala Ala Pro Pro Ser Cys Gly Leu Ser Pro Leu Leu 435 440 445 Val Leu Val Val Thr Ala Leu Ser Thr Leu Leu Ser Leu Thr Glu Thr 450 455 460 Ser 465 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 468 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO.13: Met Phe Leu Ala Thr Leu Tyr Phe Ala Leu Pro Leu Leu Asp Leu Leu 15 10 15 Met Ser Ala Glu Val Ser Gly Gly Asp Arg Leu Asp Cys Val Lys Ala 20 25 30 Ser Asp Gin Cys Leu Lys Glu Gin Ser Cys Ser Thr Lys Tyr Arg Thr 35 40 45 Leu Arg Gin Cys Val Ala Gly Lys Glu Thr Asn Phe Ser Leu Thr Ser 50 55 60 Gly Leu Glu Ala Lys Asp Glu Cys Arg Ser Ala Met Glu Ala Leu Lys 65 70 75 80 Gin Lys Ser Leu Tyr Asn Cys Arg Cys Lys Arg Gly Met Lys Lys Glu BS 90 95 Lys Asn Cys Leu Arg lie Tyr Trp Ser Met Tyr Gin Ser Leu Gin Gly 100 105 110 Asn Asp Leu Leu Glu Asp Ser Pro Tyr Glu Pro Val Asn Ser Arg Leu 115 120 125 Ser Asp lie Phe Arg Ala Val Pro Phe lie Ser Asp Val Phe Gin Gin 130 135 140 Printed from Mimosa WO 98/46622 PCT/US98/07996 93 Val Glu His lie Ser Lys Gly Asn Asn Cys Leu Asp Ala Ala Lys Ala 145 150 155 160 Cys Asn Leu Asp Asp Thr Cys Lys Lys Tyr Arg Ser Ala Tyr lie Thr 165 170 175 Pro Cys Thr Thr Ser Met Ser Asn Glu Val Cys Asn Arg Arg Lys Cys 180 185 190 His Lys Ala Leu Arg Gin Phe Phe Asp Lys Val Pro Ala Lys His Ser 195 200 205 Tyr Gly Met Leu Phe Cys Ser Cys Arg Asp lie Ala Cys Thr Glu Arg 210 215 220 Arg Arg Gin Thr lie Val Pro Val Cys Ser Tyr Glu Glu Arg Glu Arg 225 230 235 240 Pro Asn Cys Leu Ser Leu Gin Asp Ser Cys Lys Thr Asn Tyr lie Cys 245 250 255 Arg Ser Arg Leu Ala Asp Phe Phe Thr Asn Cys Gin Pro Glu Ser Arg 260 265 270 Ser Val Ser Asn Cys Leu Lys Glu Asn Tyr Ala Asp Cys Leu Leu Ala. 275 280 285 Tyr Ser Gly Leu lie Gly Thr Val Met Thr Pro Asn Tyr Val Asp Ser 290 295 300 Ser Ser Leu Ser Val Ala Pro Trp Cys Asp Cys Ser Asn Ser Gly Asn 305 310 315 320 Asp Leu Glu Asp Cys Leu Lys Phe Leu Asn Phe Phe Lys Asp Asn Thr 325 330 335 Cys Leu Lys Asn Ala lie Gin Ala Phe Gly Asn Gly Ser Asp Val Thr 340 345 350 Met Trp Gin Pro Ala Pro Pro Val Gin Thr Thr Thr Ala Thr Thr Thr 355 360 365 Thr Ala Phe Arg Val Lys Asn Lys Pro Leu Gly Pro Ala Gly Ser Glu 370 375 380 Asn Glu lie Pro Thr His Val Leu Pro Pro Cys Ala Asn Leu Gin Ala 385 390 395 400 Gin Lys Leu Lys Ser Asn Val Ser Gly Ser Thr His Leu Cys Leu Ser 405 410 415 Asp Ser Asp Phe Gly Lys Asp Gly Leu Ala Gly Ala Ser Ser His lie 420 425 430 Thr Thr Lys Ser Met Ala Ala Pro Pro Ser CyB Ser Leu Ser Ser Leu 435 440 445 Pro Val Leu Met Leu Thr Ala Leu Ala Ala Leu Leu Ser Val Ser Leu 450 455 460 Ala Glu Thr Ser 465 Printed from Mimosa 94 (2) INFORMATION FOR SEQ ID NO 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY, linear Cii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "TrnRl primer 1" (ill) HYPOTHETICAL: NO (iv) ANTI-SENSE. NO (xi) SEQUENCE DESCRIPTION. SEQ ID NO:14: GCGGTACCAT GTTCCTAGCC ACTCTGTACT TCGC (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairp (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION, /desc = "TrnRl primer 2" (iii) HYPOTHETICAL:" NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GCTCTAGACT ACGACGTTTC TGCCAACGAT ACAG (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "Tm2 primer 1" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCGGTACCAT GATCTTGGCA AACGTCTGC Printed from Mimosa 95 (2) INFORMATION FOR SEQ ID NO:17. (i) SEQUENCE CHARACTERISTICS: (A) LENGTH- 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS. single (D) TOPOLOGY, linear (ii) MOLECULE TYPE- other nucleic acid (A) DESCRIPTION: /desc = "TrnR2 primer 2" (lil) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO.17: GCTCTAGAGT CAGGCGGCTG TTCTTGTCTG CG (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS. single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "NTN Primer 1" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ^D NO-.18.

GCATATGCCG GGTGCTCGTC CGTGCGGCCT GCGTGCAACT GGAAGTTCGT GTTTCTGAAC TGGGTCTGGG TTACACTTCT GACGAAACTG T (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "NTN Primer 2" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION. SEQ ID NO:19-GCTGACGCAG ACGACGCAGA CCCAGGTCGT AGATACGGAT AGCAGCTTCG CATGCACCAG CGCAGTAACG GAACAGAACA GTTTCGT Printed from Mimosa 96 (2) INFORMATION FOR SEQ ID NO:20 (l) SEQUENCE CHARACTERISTICS (A) LENGTH- 87 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION, /desc = "NTN Primer 3" (iii) HYPOTHETICAL. NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20: CTGCGTCAGC GTCGTCGTGT TCGTCGTGAA CGTGCTCGTG CTCACCCGTG CTGCCGTCCG ACTGCTTACG AAGACGAAGT TTCTTTC (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 86 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "NTN Primer 4" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CGGATCCTTA AACGCAAGCG CATTCACGAG CAGACAGTTC CTGCAGAGTG TGGTAACGAG AGTGAACGTC CAGGAAAGAA ACTTCG (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OLIGOLINKER A" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION. SEQ ID NO.22: TATGCACCAT CATCATCATC ATGACGACGA CGACAAGGC Printed from Mimosa 97 (2) INFORMATION FOR SEQ ID NO.23: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "OLIGOLINKER B" (iii) HYPOTHETICAL. NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23-TAGCCTTGTC GTCGTCGTCA TGATGATGAT GATGGTGCA 39 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs ( (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "GDNF PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CAGCATATGT CACCAGATAA ACAAGCGGCG GCACT 35 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 baBe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "GDNF PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: CAGGGATCCG GGTCAGATAC ATCCACACCG TTTAGC 36 Printed from Mimosa 98 (2) INFORMATION FOR SEQ ID NO:26: (l) SEQUENCE CHARACTERISTICS (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "MOUSE RET FORWARD PCR PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO-26: TGGCACACCT CTGCTCTATG (2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "MOUSE RET REVERSE PCR PRIMER" (lli) HYPOTHETICAL: NO (IV) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: TGTTCCCAGG AACTGTGGTC (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "TrnRl FORWARD PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION SEQ ID NO 28. GCACAGCTAC GGGATGCTCT TCTG Printed from Mimosa 99 (2) INFORMATION FOR SEQ ID NO:29: (x) SEQUENCE CHARACTERISTICS- (A) LENGTH: 28 base paxrs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "TrnRl REVERSE PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO-29: GTAGTTGGGA GTCATGACTG TGCCAATC (2) INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "TrnR2 FORWARD PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE. NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: AGCCGACGGT GTGGCTCTGC TGG (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "TmR2 REVERSE PRIMER" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: CCAGTGTCAT CACCACCTGC ACG Printed from Mimosa

Claims (6)

WO 98/46622 PCI E/07996 1? 100 What is Claimed is: INTELLECTUAL PROPERTY OFFICE OF NZ. 2 8 NOV 2001

1. An isolated and purified polypeptide comprising a TFG-(i> related neurotrophic receptor 2 (TrnR2) polypeptide wherein the TrnR2 polypeptide has a human amino acid sequence selected from the group consisting of SEQ b ID NO:2, SEQ ID NO:3, and SEQ ID NO:7 or wherein the TrnR2 polypeptide has a mouse amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8

2. The isolated and purified polypeptide of claim 1 wherein the TrnR2 polypeptide is a soluble TrnR2 polypeptide or soluble TrnR2 fragment.

3. A composition comprising the isolated and purified polypeptide of claim 1 in a pharmaceutically acceptable carrier.

The composition of claim 3 further comprising a TGF-p Related Neurotrophic (TRN) growth factor.

5 • An isolated and purified polypeptide that is a member of the TGF-p related neurotrophic receptor family (TrnR) comprising an amino acid sequence having at least 47% sequence identity with TGF-p 5 related neurotrophic receptor 1 (TrnRl) and at least 47% sequence identity with the TrnR2 polypeptide of claim 1.

6. The isolated and purified polypeptide of claim 6 wherein said TrnR family member is comprised of a conserved region amino acid sequence having at least 90% sequence identity with SEQ ID NO:9 or at least 90% 5 sequence identity with SEQ ID NO:10 or at least 90% sequence identity with SEQ ID NO:11.

7. An isolated and purified polynucleotide comprising a nucleotide sequence encoding the TrnR2 polypeptide of claim 1.

8. The isolated and purified polynucleotide of claim 7 wherein the TrnR2 polypeptide has a human amino acid sequence selected from the group consisting of SEQ ID WO 98/46622 PC^/US98/07996 101 NO:2, SEQ ID NO:3, and SEQ ID NO:7 or a mouse amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:8. 9 • The isolated and purified polynucleotide of claim 8 wherein the TrnR2 polypeptide is a human or mouse soluble TrnR2 polypeptide or soluble TrnR2 fragment.

10. An isolated and purified polynucleotide comprising a nucleotide sequence which hybridizes to a nucleotide sequence selected from the group consisting of: 5 (a) a coding sequence for an amino acid sequence of a human precursor TrnR2 as set forth in SEQ ID NO:2; (b) a coding sequence for an amino acid sequence for a mature human TrnR2 as set forth in SEQ ID NO:3; (c) a sequence complementary to the coding sequence 10 of (a); and (d) a sequence complementary to the coding sequence of (b).

11. A vector comprising a recombinant DNA molecule comprising expression regulatory elements operably linked to a nucleotide sequence encoding the TrnR2 polypeptide of claim 1. 12- a host cell transformed with the vector of claim -11.

13. A recombinant cell transformed with nucleotide sequences encoding for the expression of the TrnR2 polypeptide of claim 1 and a Ret protein tyrosine kinase receptor. WO 98/46622 102 R EfSEIVED 2 8 NOV 2001 m 42

14. A method of producing a TrnR2 polypeptide comprising- (a) subcloning a DNA sequence encoding the TrnR2 polypeptide of claim 1 into an expression vector which comprises regulatory elements needed to express the DNA 5 sequence; (b) transforming a host cell with said expression vector; (c) growing the host cell to produce a host cell culture; and 10 (d) harvesting the TrnR2 polypeptide and/or the DNA sequence from the host cell culture.

15. An isolated and purified antibody which is capable of reacting with the TrnR2 polypeptide of claim 1 or an epitope thereof. 1 6. The use, in the manufacture of a medicament of the TrnR2 polypeptide of claim 1, in a composition for preventing or treating cellular degeneration or insufficiency together with a pharmaceutically acceptable diluent, carrier or excipient.

17. a method for detecting TrnR2 expression in a sample from a patient comprising detecting in the sample the presence of the TrnR2 polypeptide of claim 1 or detecting the presence of a mRNA encoding the TrnR2 5 polypeptide of claim 1 or a biologically inactive mutant thereof. 18 • A method for promoting the growth and/or differentiation of a cell in a culture medium comprising administering to the cell the TrnR2 polypeptide of claim 1 and a TRN growth factor. 1'9. An isolated and purified TrnR2 antisense polynucleotide comprising a nucleotide sequence which is complementary to and hybridizes to a naturally-occurring DNA or mRNA polynucleotide sequence encoding a TrnR2 5 polypeptide to prevent transcription and/or translation of the encoded TmR2 polypeptide. 20 The use in the manufacture of a medicament of an inhibitory effective amount of the isolated and purified antisense TrnR2 polynucleotide of claim 19 in a composition for treating a disease condition mediated by the expression of a TrnR2 polypeptide in a cell together with a pharmaceutically acceptable diluent, carrier or excipient. WO 98/46622 103

21. The use in the manufacture of a medicament of a therapeutically effective amount of an isolated and purified anti-TrnR2 antibody which blocks Ret activation in the presence of endogenous neurturin (NTN) and/or glial-derive neurotrophic factor (GDNF) in a composition for treating a disease condition in a 5 patient mediated by the expression of the TrnR2 polypeptide of claim 1 together with a pharmaceutically acceptable diluent, carrier or excipient.

22. The use in the manufacture of a medicament of a therapeutically effective amount of the isolated and purified soluble TrnR2 polypeptide or soluble TrnR2 fragment of claim 2 in a composition for treating a disease condition in a patient mediated by the expression of a TRN growth factor together with a 5 pharmaceutically acceptable diluent, carrier or excipient.

23. A method for screening compounds for TRN agonistic or antagonistic activity comprising incubating a test compound with the recombinant cell of claim 13 in the presence or absence of a TRN and assaying for Ret 5 protein tyrosine kinase activity. 24 •, An isolated and purified polypeptide comprising a TGF-p related neurotrophic receptor 2 (TrnR2) polypeptide which comprises a human amino acid sequence as set forth in SEQ ID NO:7 or a mouse amino acid 10 sequence as set forth in SEQ ID NO:8.

25. An isolated and purified polypeptide comprising a soluble TGF-p related neurotrophic receptor 2 (TrnR2) polypeptide which comprises a human amino acid sequence as set forth in amino acids 1-299 of SEQ ID NO:7 or a 15 mouse amino acid sequence as set forth in amino acids 1-299 of SEQ ID NO:8. R E G E J y £ u 2 8 NOV 2001