Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

• TrnR2 A Novel Receptor Which Mediates Neurturin And GDNF Signaling Through Ret filed April 17, 1997.
• This invention relates generally to receptors for trophic or growth factors and, more particularly, to a novel receptor for TGF- ⁇ -related neurotrophic factors.
• NGF nerve growth factor
• NGF exists as a non- covalently bound homodimer that promotes the survival and growth of sympathetic, neural crest-derived sensory, and basal forebrain cholinergic neurons.
• 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, the neurotrophin family and the transforming growth factor-beta (TGF- ⁇ ) family.
• TGF- ⁇ 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 the NGF monomer (Barde, Prog Growth Factor Res 2:237-248, 1990 and Liebrock et al. Nature 341:149-152, 1989 which are incorporated by reference).
• BDNF Brain-derived neurotrophic factor
• Trk tyrosine kinase receptors
• NT-3 stimulates phosphorylation of TrkB expressed in fibroblasts with a dose-response 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, although with different specificities than NGF ( Ip et al, supra and Cordon-Cardo et al, Cell 66:173-183, 1991, incorporated herein by reference ) .
• TGF- ⁇ Related Neurotrophic factors are glial cell line-derived neurotrophic factor (GDNF) , neurturin (NTN) , and persephin ( PSP ) .
• GDNF and NTN 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 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).
• 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 ⁇ (GDNFR- ⁇ ) which has no transmembrane domain and is attached to the cell surface via a glycosyl-phosphatidylinositol (GPI) linkage (Durbec et al., Nature 381:789-793, 1996; Jing et al.. Cell 85:1113-1124, 1996; Treanor et al..
• GDNFR- ⁇ glycosyl-phosphatidylinositol
• GDNF also induces activation of the Ret PTK when a soluble form of GDNFR- ⁇ is added to the culture medium along with GDNF, demonstrating that GDNFR- ⁇ does not need to be anchored to the cell membrane to interact with Ret (Jing et al., supra).
• mice deficient in either GDNF or Ret are phenotypically similar and that GDNFR- ⁇ and Ret are expressed together in the developing nephron, 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 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 characterized by structural damage and, possibly, by disease-induced apoptosis, it is likely that neurotrophic 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 the most socially and economically destructive diseases now afflicting our society.
• the present invention is 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 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 co-receptors, referenced herein as TrnR (TGF- ⁇ -related neurotrophic factor Receptors ) .
• TrnR TGF- ⁇ -related neurotrophic factor Receptors
• TrnR co-receptor protein GDNFR- ⁇
• TrnRl co-receptor protein GDNFR- ⁇
• TrnR2 novel protein
• TrnR2 GPI- linked co-receptors for GDNF and neurturin
• GFR ⁇ l previously known as GDNFR ⁇ , TrnRl and RetLl
• GFR ⁇ 2 previously TrnR2, NTNR ⁇ and RetL2
• GFR ⁇ 3 previously TrnR3
• TrnR2 homologs of different mammalian species have at least 85% amino acid sequence identity while amino 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 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 mouse TrnR2 protein have the amino acid sequences shown in SEQ ID N0S:5 and 6 ( Figure 2).
• TrnR2 polypeptides of the invention include variants of human and mouse precursor proteins translated from alternatively spliced TrnR2 mRNA having the amino acid sequences shown in SEQ ID N0S:7 and 8. Soluble TrnR2 polypeptides which lack a GPI anchor are also 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 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 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 N0:4.
• Expression vectors and stably transformed cells are also provided.
• the transformed cells can be used in a method for producing a TrnR2 polypeptide.
• the present invention provides a method for preventing or treating neuronal degeneration comprising administering to a patient in 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 which encodes TrnR2 into a patient ' s tissues which would benefit from increased sensitivity to a TRN such as NTN or GDNF.
• a patient with neuronal degeneration is treated by implanting neuronal cells cultured and expanded by growth in the presence of TrnR2 and NTN or GDNF.
• 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 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.
• the present invention provides isolated and purified TrnR2 antisense polynucleotides.
• the present invention also provides compositions and methods for detecting TrnR2 expression.
• One method detects TmR2 protein using anti-TrnR2 antibodies and other methods are based upon detecting TrnR2 mRNA using recombinant DNA techniques.
• 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 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, 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.
• Figure 1 illustrates the homology of the amino acid sequences for the predicted precursor forms of TrnRl (human, SEQ ID NO: 12; rat, SEQ ID NO: 13) and TrnR2 (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 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 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
• fibroblasts stably transfected with Ret alone (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) before growth factor treatment
• 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 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 TmR2 (TrnR2-LV) or the short splice variant (TrnR2-SV);
• Figure 3E illustrates the binding affinities of soluble TrnR2-LV fused with the Fc region of human IgG, ⁇ (R2-Ig) for GDNF, NTN and PSP as measured in an ELISA binding assay;
• Figure 4 illustrates the tissue distribution of TrnR2 mRNA in adult mouse showing a Northern blot of total RNA probed with a 3z P-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 33 P-labeled RNA probes of tissue samples from (Fig. 5A) E14 mouse (developing) ventral mesencephalon (vm), (Fig. 5B) adult mouse spinal cord, (Fig. 5C) E14 mouse (developing) kidney (k ) , gut (g ) and dorsal root ganglia (drg ) , and (Fig.
• FIG. 5D adult rat superior cervical ganglion (SCG);
• Figure 6 illustrates TrnRl, TrnR2, and Ret 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 primers specific for Ret, TrnR2, neuron-specific enolase NSE, TrnRl, and a Schwann cell marker (S100);
• RT-PCR reverse transcription-polymerase chain reaction
• FIG 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 total RNA isolated from normal sciatic nerve (N) and the distal segment of sciatic nerve seven days post- transection (7D) using 32 P-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 33 P-labeled riboprobes to detect expression of GFR ⁇ -1 (TrnRl) (Fig. 8A), GFR ⁇ -2 (TrnR2) (Fig. 8B), Ret (Fig. 8C) and NTN
• FIG. 8D in which the various regions are abbreviated as Cg—cingulate cortex, Cl—claustrum, DBB—nucleus of the diagonal band of Broca, DEn—dorsal endopiriform nucleus, LS—lateral septal nucleus, MS—medial septal nucleus, Pii?— iriform cortex, Tu—olfactory tubercle, and 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 in situ hybridization using 33 P-labeled riboprobes to detect expression of (Fig. 8) GFR ⁇ -1 (TrnRl), (Fig. 8B) GFR ⁇ -2 (TrnR2), (Fig. 8C) Ret, and (Fig.
• NTN in which the various regions are abbreviated as Th-thalamic nuclei, A—amygdala, H—hypothalamus, LD—laterodorsal nucleus of the thalamus, MD—mediodorsal nucleus of the thalamus, MHb—medial habenula, Rt— eticular 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 darkfield photographs of coronal sections of adult mouse midbrain analyzed by in situ hybridization using 33 P- 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 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.
• Fig. 10A GFRa-1 (TrnRl) in the compacta region of the substantia nigra, the VTA, the oculomotor nucleus and the superficial layers of the superior colliculus
• Fig. 10B GFRa-2 in the compacta region of the substantia nigra, in the VTA,
• IOC IOC Ret mRNA in the SNc and the VTA, in which the various regions are abbreviated as 3—oculomotor nucleus, SN—substantia nigra, SuMM—supramammillary nucleus, MGN—medial geniculate nucleus, and VTA— entral 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 and (Fig. 11B-11C) brightfield photographs at higher magnification which show detection of NTN expression in magnocellular neurons in the supraoptic (Fig. 11B) and paraventricular (Fig. 11C) nuclei with the various regions abbreviated as PV— araventricular nucleus, SO—supraoptic nucleus, and 3V- ⁇ third ventricle;
• Figure 12 illustrates expression of GDNF and GF
• TRN tumor necrosis factor receptor mRNA in adult mouse midbrain and brainstem showing darkfield photographs of coronal sections analyzed by in situ hybridization using 33 P-labeled riboprobes to detect expression of
• Fig. 12A Ret mRNA in 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 motor nucleus and in the dorsal cochlear nucleus (DC),
• Fig. 12A Ret mRNA in cranial nerve nuclei 10 and 12
• Gi gigantocellular reticular nucleus
• Fig. 12B GFRa-2 (TrnR2 ) in cranial nerve nu
• 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 6—abducens nucleus, 7—facial nucleus, 10— agal motor nucleus, 12—hypoglossal nucleus, Gi—gigantocellular reticular nucleus, IG—inferior colliculus, LG—locus coeruleus, Sp5—spinal trigeminal nucleus, Mo5—motor trigeminal nucleus, Tg- ⁇ tegmental nuclei, VG— entral 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 33 P-labeled riboprobes to detect expression of (Fig. 13A) GFR ⁇ -1 (TrnRl), (Fig. 13B) GFR ⁇ - 2 (TmR2), and (Fig. 13C) Ret, with various regions identified as DH— orsal horn, and VH— entral horn;
• Figure 14 illustrates GF (TRN) receptor and NTN mRNA expression in adult cerebellum showing darkfield photographs of sagittal sections analyzed by in situ hybridization using 33 P-labeled riboprobes to detect expression of (Fig. 14A) GFR ⁇ -1 (TrnRl) in cells adjacent to Purkinje neurons in the Purkinje layer, (Fig. 14B) GFR ⁇ -2 (TrnR2) in the granule cell layer and in neurons that appear to be Purkinje cells in the Purkinje layer, (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, Mol—molecular layer.
• the present invention is based upon the surprising discovery that NTN, like GDNF, can stimulate Ret PTK through the known co-receptor GDNFR- ⁇ and thereby cause the activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI-3-K) intracellular signaling pathways .
• GDNFR- ⁇ As the first co- receptor known to mediate signaling by at least two members of the TRN family of growth factors, GDNFR- ⁇ is referred to herein as TrnRl .
• TrnR2 NTN and GDNF
• TrnR2 was identified by searching a database of Expressed Sequence Tags (dbEST database) using the Basic local alignment search tool (BLAST, Altschul et al., J.Mol .B ⁇ ol . 215:403-410, 1990 incorporated herein by reference) and the full length rat TrnRl protein sequence (GenBank Accession No. U59486) as a query.
• BLAST Basic local alignment search tool
• BLAST Altschul et al., J.Mol .B ⁇ ol . 215:403-410, 1990 incorporated herein by reference
• BLAST Basic local alignment search tool
• H12981, R02135, W73681 Three human ESTs (H12981, R02135, W73681) which showed only partial, but significant, homology to rat TrnRl were identified by the BLAST search.
• the 5 ' end of the cDNA was obtained by the rapid 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- 2220, 1994, incorporated herein by reference.
• RACE rapid amplification of cDNA ends
• PCR Klentaq LA polymerase chain reaction
• TrnR2 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 (TrnR2-LV) (residues 75 to 473 of SEQ ID N0:1).
• 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 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 N0:1.
• the corresponding mouse cDNAs for both the long and short splice variants were also obtained by PCR, 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 ORF encoding a predicted 330 amino acid polypeptide (SEQ ID NO: 8) which would be encoded by nucleotides 1-39 and 438-1389 of SEQ ID NO: 4.
• ORF long open reading frame
• TrnR2 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 significant homology with TrnRl. (The human and rat TrnRl sequences (SEQ ID Nos.
• 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 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 hydrophobic regions indicates that mature TrnR2 is potentially a GPI-linked protein (Udenfriend and Kodukula, Ann. Rev. Biochem.
• 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 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.
• TrnR2 and TrnRl are expressed in a partially overlapping manner.
• TrnR2 While both co- receptors are expressed in the dorsal root ganglia (DRG) 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. In contrast, TrnR2 and Ret appear to comprise the expressed receptor complex in SCG neurons .
• TrnR2 TrnR2
• TrnRl TrnRl or other as yet unidentified members of 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.
• the invention provides a 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 TmR2 polypeptides characterized and 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.
• compositions of the present invention are capable of demonstrating some or all of the same signal mediating properties in a similar fashion, not necessarily to the same degree, as the recombinantly produced human or mouse TrnR2.
• substantially homologous it is meant that the 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- ⁇ ).
• Sequence identity or percent identity is intended to mean the percentage of identical residues between two 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 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 ) .
• multiple alignments are carried out 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 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 alignment contribute to the score.
• the residue weight table used 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 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 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 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 species such as avian species, it is believed that the degree of homology with TrnR2 is at least about 65% identity.
• the variations between members of the TrnR family of receptors can be seen by comparing TrnRl and TmR2 ( Fig. 1 ) .
• Human and mouse precursor TrnR2 share about 94% identical amino acids and have about 53% and 52% sequence conservation with human and rat precursor TrnRl, respectively.
• TrnR family members similarly have a sequence identity of about 40% to that of TmR2 and about 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.
• a given non-TrnR2 and non-TrnRl family member from one species would be expected to show lesser sequence 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 .
• 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 unusual amino acid and modifications such as glycosylations so long as the hybrid or modified form retains TrnR2 biological activity.
• TrnR2 biological activity it is meant that Ret PTK is activated in the presence of the hybrid or modified TrnR2 and NTN or GDNF 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.
• 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 isolated through degenerate PCR or by hybridization with the complementary sequence of genomic or subgenomic 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 invention.
• TrnR2 polypeptide As used herein.
• TrnR2 The predicted amino acid sequence and biological function of TrnR2 indicate that it is an externally disposed plasma membrane protein anchored to the extracellular surface of the cell membrane by a gly ⁇ osyl- phosphatidyl (GPI) linkage.
• GPI gly ⁇ osyl- phosphatidyl
• TrnR2 polypeptide also includes soluble TrnR2 polypeptides 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 NO: 3, SEQ ID NO: 5, and SEQ ID NO: 6.
• a more preferred TrnR2 polypeptide is human mature TrnR2 protein which has the amino acid sequence set forth in SEQ ID NO: 3.
• TrnR2 polypeptide is intended to include fragments which have one or more of the biological activities of precursor or mature TrnR2 protein. Such 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 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 hydrophobic domain for the GPI-attachment site which are referred to herein as soluble TrnR2 fragments.
• 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 (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 thereorf .
• 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 polypeptide may be prepared by recombinant DNA technology. By “pure form” or “purified form” or “substantially purified form” it is meant that a TmR2 composition is substantially free of other proteins which are not TrnR2.
• TrnR2 polypeptide substantially free of other proteins including immunochromatography, size-exclusion chromatography, HPLC, ion-exchange chromatography, and ligand affinity chromatography.
• an example of one way to obtain TrnR2 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 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 expressing the DNA sequences encoding TrnR2 in a suitable transformed host cell. Using methods well known in the art, the DNA encoding the TrnR2 polypeptide may be linked to an expression vector, transformed into a host cell and conditions established that are suitable for expression of the TrnR2 polypeptide by the transformed cell.
• TrnR2 Any suitable expression vector may be employed to produce recombinant TrnR2 such as, for example, the mammalian expression vector pCMV-neo (Brewer, eth.Cell Biol. 43:233-245, 1994, incorporated herein by reference) which was used herein or the E. coli pET expression vectors, in particular, pET-30a (Studier et al.. Methods Enz mol. 185:60-89, 1990 which is incorporated by reference).
• Other suitable expression vectors for expression in mammalian and bacterial cells are known in the art as are expression vectors for use in yeast or insect cells. Baculovirus expression systems can also be employed.
• the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence that encodes a TrnR2 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 nucleotides 99-1331 encode a human mature TrnR2.
• 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 fragments are also contemplated, particularly soluble
• TrnR2 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), residues 64-1296 of SEQ ID NO: 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 also intended to be included within the present invention.
• TrnR2 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 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.
• nucleotide sequences that are substantially the same as a nucleic acid sequence encoding TrnR2. Substantially the same sequences may, for example, be substituted with codons more readily expressed in a given host cell such as E. coli according to well known and standard procedures. Such modified nucleic acid sequences would be included within the scope of this invention.
• 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 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 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.
• the cDNA sequences provided herein allow genomic clones for the TrnR2 gene to be readily isolated.
• genomic clones One use for genomic clones is for chromosome localization studies.
• human and mouse genomic clones for the TrnR2 gene were obtained by screening PI (mouse) and 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 chromosome 8 in region pl2-21 by fluorescence in situ hybridization analysis (FISH).
• FISH fluorescence in situ hybridization analysis
• a search of the database for neurological diseases genetically mapped to the human locus revealed only one such disease, SPG5A, an autosomal recessive form of 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 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 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 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.
• recombinant cells expressing both 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 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 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 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.
• TRN growth factor such as NTN, GDNF, or persephin
• Compounds which increase Ret PTK 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.
• 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 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.
• MAPK mitogen-activated protein kinase
• a reporter gene operably linked to the 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 those skilled in the art and can be readily isolated.
• reporter genes which encode for readily detectable products include, but are not limited to, ⁇ - galactosidase, chloramphenicol acetyl transferase, luciferase and ⁇ -glucuronidase. Detection of the 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.
• TrnR2 polypeptides Preparation can be by isolation from a variety of cell types so long as the cell type expresses TrnR2 protein.
• 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 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.
• 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 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 has aberrant TrnR2 expression or expresses a biologically inactive TrnR2 mutant. TrnR2 expression may be detected with probes which react with TmR2 mRNA or TrnR2 protein.
• 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 nucleotides which are complementary to the TrnR2 target sequence.
• Oligonucleotide probes may be prepared by any method known in the art such as, for example, excision, transcription or chemical synthesis.
• the polynucleotide probe may comprise a cDNA encoding TrnR2 or a fragment thereof as a probe.
• 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 be accomplished by any method known in the art such as by PCR, random priming, end labelling, nick translation or the like.
• methods not employing a labelled probe can be used to determine the hybridization. Examples of methods 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 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 in the hybridization and washing steps. Such factors are well known to those 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 amplify cDNA transcribed from TrnR2 mRNA using primers specific for a TrnR2-encoding nucleotide sequence (see example 4 and Fig. 6 below).
• RT/PCR reverse transcription/polymerization chain reaction
• 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 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, immunoelectrophoresis, immunochemical methods, binder- ligand assays, immunohistochemical techniques, agglutination and complement assays .
• 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.
• a derivative As used herein, a derivative.
• 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 polypeptide derivative cross-reacts with antibodies raised against the TrnR2 protein.
• cross-reaction it is meant that an antibody reacts with an antigen other than the one that induced its formation.
• 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 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 (RIA), enzyme immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays and 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 art.
• 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.
• an epitope consists of at least 5 such amino acids.
• Methods of determining the 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 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 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 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 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 Chem Soc 85:2149, 1963 which is incorporated by reference) or the FMOC strategy on a Rapid Automated 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 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. 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 such as myeloma or lymphoma cells.
• TrnR2 may be a biologically inactive mutant.
• 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 need thereof a therapeutically effective amount of a TrnR2 polypeptide.
• Certain degeneration disorders may be related to a lack of or reduced expression of biologically active TrnR2, while NTN or GDNF expression is normal.
• TrnR2 levels it may be desirable under certain circumstances 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 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 be achieved by administering NTN and/or GDNF along with the TrnR2 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 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.
• TrnR2 expression of TrnR2 could be increased in tissues defective in such expression by gene therapy.
• Patients may be implanted with vectors or cells capable of producing a biologically-active TrnR2 polypeptide.
• cells that secrete soluble TrnR2 may be encapsulated into semipermeable membranes for implantation into a patient.
• the cells can be those that normally express a TrnR2 protein or the cells can be transformed to express a TrnR2 polypeptide.
• the TrnR2 be human TrnR2.
• the formulations and methods herein can be used for veterinary as well as human applications and the term "patient" as used herein is intended to include human and veterinary patients.
• TrnR2 in combination with NTN or GDNF can be 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 nervous system as well, including gut and kidney.
• a composition comprising TrnR2 and NTN or GDNF is used to promote the ex vivo expansion of Ret-expressing cells for transplantation or engraftment.
• compositions and methods are useful for treating a number of degenerative diseases.
• the diseases include, but are not limited to peripheral neuropathy, amyotrophic lateral sclerosis, Alzheimer's 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 dysfunctions and damage caused by infectious agents.
• the diseases include, but are not limited to disorders of insufficient blood cells such as, for example, leukopenias including eosinopenia and/or basopenia, lymphopenia, monocytopenia, neutropenia, anemias, thrombocytopenia as well as an insufficiency of stem cells for any of the above.
• leukopenias including eosinopenia and/or basopenia
• lymphopenia including eosinopenia and/or basopenia
• lymphopenia including eosinopenia and/or basopenia
• lymphopenia monocytopenia
• neutropenia neutropenia
• anemias 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.
• compositions and methods herein can also be useful to prevent degeneration and/or promote survival in other non-neuronal tissues as well.
• One skilled in the 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.
• TrnR2 and a TRN such as NTN or GDNF.
• TrnR2 antibodies or other compounds having TRN antagonist properties may involve administration of TrnR2 antibodies or other compounds having TRN antagonist properties, or the use of antisense polynucleotides to modulate TrnR2 expression.
• 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 known in the art.
• 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 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 art. See for example, Hayden et al., Curr. Opin.
• TrnR2 protein or an immunologically active fragment thereof, or an anti- idiotypic antibody, or fragment thereof can be administered to an animal to elicit the production of antibodies capable of recognizing and binding to the TrnR2 protein.
• 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.
• a soluble TrnR2 administered in excess would GDNF, NTN, and possibly other TRN growth factors, thereby sequestering the TRN growth factor from the anchored TrnR2 receptors on target cells.
• administration of soluble TrnR2 may act to sequester the growth factor in the plasma and possibly facilitate their excretion, thereby limiting the effects of the growth factor in the body.
• TrnR2 antisense oligonucleotides can be made and a method utilized for diminishing the level of expression of TrnR2 protein by a cell comprising administering one or more TrnR2 antisense oligonucleotides.
• TrnR2 antisense oligonucleotides reference is made to oligonucleotides that have a nucleotide sequence that interacts through base pairing with a specific complementary nucleic acid sequence involved in the expression of TrnR2 such that the expression of TrnR2 is reduced.
• 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 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 sequence as to allow hybridization to that sequence in a cell, i.e., under physiological conditions.
• the TrnR2 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 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.. Par asitology 10: 92-97 , 1994 which are incorporated by reference).
• modified internucleoside linkages modified nucleic acid bases 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
• 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 acid analogue and dithioate repeating units.
• 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 include the ex vivo treatment of cells.
• 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 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, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that 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 antibody to the transferrin receptor.
• CSF cerebrospinal fluid
• Co-administration may comprise physically coupling any known blood-brain penetrating agent to TrnR2.
• TrnR2 polypeptide can also be linked or conjugated with agents that provide other desirable pharmaceutical or pharmacodynamic properties.
• a TrnR2 polypeptide can be stably linked to a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties .
• a polymer such as polyethylene glycol
• 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 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 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 biologically compatible matrix which can be implanted into tissues requiring treatment.
• 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 formulation.
• the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
• excipients are those substances usually and customarily 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 the pharmacokinetic parameters of the dosage formulation and the route of administration used.
• formulations containing TrnR2 are to be administered orally.
• Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
• suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, 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 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 formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, 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 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 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.
• 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, 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.
• 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 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 could result from preferential activation of different receptors by different family members as is known to occur with members of the NGF family of neurotrophic factors and Trk receptors (Tuszynski and Gage, supra).
• TrnRl GDNFR- ⁇
• TrnR2 GDNFR- ⁇
• 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 and primers made available from the present work make possible this powerful new approach which can now successfully identify other gene family members.
• one may screen for genes related to TrnRl and TmR2 in amino acid sequence homology by preparing DNA or RNA probes based upon the conserved regions in the TrnRl and TrnR2 proteins.
• 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 members of the TrnR gene family.
• 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- 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 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.
• an inosine base, or another "universal" 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 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 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 determining stringency conditions are well known in the art (for example, see Sambrook et al., Molecular Cloning, 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 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.
• 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 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.
• TrnR family members may be 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 NO:9, SEQ ID NO: 10 and SEQ ID NO: 11.
• a polypeptide containing a TrnR conserved region e.g., an amino acid sequence selected from the group consisting of SEQ ID 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 .
• Example 1 This example illustrates that either TrnRl or TrnR2 can mediate the signaling of NTN or GDNF through the Ret protein tyrosine kinase.
• 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 . 43:233-245, 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). 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.
• 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 pBluescript KS (Stratagene, La Jolla, CA, sequenced and subcloned into the Hindlll and BamHI sites of pCMV-Neo (Brewer, 1994).
• TrnR2 expression construct the coding region of the long form of human TrnR2 cDNA was amplified from the same Marathon RACE human brain cDNA 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.
• 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. Louis, MO). TrnRl- and TrnR2-expressing clones were confirmed by western (Ret) and northern (TrnRl or TrnR2) blotting.
• a synthetic gene for the mature mouse NTN coding sequence was prepared from four partially overlapping oligonucleotides containing the Eschericia coli (E. coli ) 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 GAC GCA GAC GAC GCA GAC GAC GCA GGT CGT AGA TAC GGA TAG CAG CTT CGC ATG CAC CAG CGC AGT AAC GGA ACA GAA CAG TTT CGT- 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 G
• 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 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 vector pET30a(+) (Novagen, Madison, WI).
• a histidine tag followed by a enterokinase site was placed at the amino terminus of the NTN sequence by inserting oligonucleotide linkers A (5' -TAT GCA CCA TCA TCA TCA TCA TCA TGA CGA CGA CAA GGC-3')(SEQ ID NO:22) and B ( 5 ' -TAG CCT TGT CGT CGT CGT CAT GAT 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 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.
• NTN and GDNF pET30a(+) constructs were sequenced to confirm their authenticity and then transformed into E. coli strain BL21/DE3.
• the transformed bacteria were grown at 37° C in 2XYT medium (30 ⁇ g/ml kanamycin) with vigorous shaking.
• 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 HCl, 0.1 M Na phosphate, 10 mM Tris HCl, pH 8.0) at 1/lOth volume of the original culture volume and rocked overnight. Lysate was centrifuged at 10,000 x g for 15 min at 4° C.
• Buffer A 6 M guanidine HCl, 0.1 M Na phosphate, 10 mM Tris HCl, pH 8.0
• the supernatant was exposed to 4 ml of Nickel-NTA (nitrilo-tri-acetic acid) resin (Qiagen, 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 HCl, pH 8.0), and 5-10 volumes of Buffer C (8 M urea, 0.1 M Na phosphate, 10 mM Tris HCl, pH 6.3) until the A280 was ⁇ 0.01.
• Nickel-NTA nitrilo-tri-acetic acid
• 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/ ⁇ l in Buffer B and dialyzed against 4 M Renaturation Buffer (4 M urea, 5 mM cysteine, 0.02% Tween 20, 10% glycerol, 10 mM Tris HCl, 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.
• 4 M Renaturation Buffer 4 M urea, 5 mM cysteine, 0.02% Tween 20, 10% glycerol, 10 mM Tris HCl, 150 mM NaCl, 100 mM Na phosphate, pH 8.3, under argon
• 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 removed and assayed for total Ret expression by western blot analysis using an anti-Ret antibody. The remaining lysates were immunoprecipitated with an an i- phosphotyrosine antibody and analyzed by western blot using the anti-Ret antibody. The results are shown in Figure 3A.
• Anti-Ret immunoblot analysis for total Ret shows 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 left untreated ( - ) ( Total, Fig . 3A) .
• 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 ) .
• TmR2 a putative GPI-linked protein, can form a 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 ) .
• Example 2 This example illustrates that the short splice 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 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 either GDNF, neurturin or persephin at 100 ng/ml for 10 minutes and then lysed.
• the lysates were immunoprecipitated using an anti-phosphotyrosine antibody, and then analyzed by western blot using an anti-Ret antibody as described in Example 1. The results are shown in Fig. 3D.
• TrnR2-SV 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.
• neurturin 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.
• TrnR2-LV/Ret complex has a preferential affinity for neurturin over GDNF.
• Example 2A This example illustrates that a soluble TrnR2 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 N0:2, i.e., nucleotides 36-1331 of SEQ ID N0:1, to a polynucleotide encoding the Fc region of human IgG x using the plgPlus vector system (Invitrogen, 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 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 and 50 ⁇ l of each solution was applied to separate
• 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. 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 ⁇ g) isolated from 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, Annal. Biochem 162 : 156-159, 1987, incorporated herein by reference). To verify that 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 32 P-labeled fragment of TrnR2 cDNA.
• 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 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).
• TrnR2 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.
• TrnR2 tissue distribution 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 Comparison of TrnR2 and TrnRl expression was also investigated in known sites of GDNF and/or NTN action by 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 results of in situ hybridization of these fresh frozen tissue samples with antisense 33 P-labeled RNA probes transcribed from fragments of TrnRl , TrnR2 and Ret cDNAs are shown in Figure 5.
• TrnR2 In situ hybridization analysis showed only low-level 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 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 TrnRl (Fig.
• TrnRl and TrnR2 were expressed in the exiting nerve root (r) (Fig. 5D).
• r exiting nerve root
• Fig. 5c the developing kidney (k) and gut (g)
• TrnR2 Fig. 5c
• SCG superior cervical ganglion
• TrnRl and TrnR2 are partially overlapping expression pattern for TrnRl and TrnR2 in embryonic and adult central and peripheral nervous tissue.
• GDNF action including nigral and motor neurons, high levels of TrnRl and Ret are expressed, with only low or undetectable levels of TrnR2 expression.
• TrnR2 expression is largely limited to neuronal tissue in both embryo and adult, with highest levels of expression in sensory and sympathetic neuronal populations .
• Example 4 This example illustrates that both NTN and GDNF promote the survival of newborn rat SCG neurons in 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 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 8: 573-587, 1992, incorporated herein by reference ⁇ . After 2-4 h, this medium was replaced with medium containing 50 ng/ml NTN, GDNF, or NGF.
• NTN-induced Ret activation was similarly affected. SCG cultures were grown in serum-free N2 medium to maximize the activity of 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 mediated by a GPI-linked protein.
• 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.
• TrnRl is expressed diffusely and does not appear to be localized to neurons.
• TrnRl, TrnR2, Ret mRNA in neuronal cultures was assessed using semiquantitative reverse transcription-PCR (RT-PCR) as described in Freeman et al., supra and Estus et al., supra.
• RT-PCR semiquantitative reverse transcription-PCR
• Half of the poly-A RNA was converted to cDNA by reverse transcription with Moloney murine leukemia virus reverse transcriptase with random hexamers (16 ⁇ M) as primers.
• cDNA from approximately 150 cells was used in a 50 ⁇ l PCR reaction using the following primer sets: mouse Ret forward 5'- 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 forward 5 ' -AGCCGACGGTGTGGCTCTGCTGG-3 ' (SEQ ID NO: 30) and reverse 5 ' -CCAGTGTCATCACCACCTGCACG-3 ' ( SEQ ID NO: 31 ) .
• TrnR2 messages decreased as the neurons died, in a manner similar to neuron-specific enolase (NSE). In contrast, TrnRl levels remained constant, similar to the Schwann cell marker S-100.
• TrnRl is predominantly expressed in the non-neuronal population, consistent with its previously observed expression in Schwann cells ( reanor et al. , supra) .
• Example 5 This example illustrates that TrnRl, but not TrnR2, is up-regulated in distal sciatic nerve after nerve injury.
• GDNF is a well characterized trophic factor for both 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.Sci.USA 92:9771-9775, 1995, all are incorporated herein by reference ) .
• 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 .
• TrnRl is expressed by Schwann cells ( Treanor et al . , supra )
• TrnRl and TrnR2 were examined to determine if one or both of the TRN co-receptors is up- regulated after transection.
• TrnRl 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 equal loading of total RNA samples.
• TrnRl 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 ( ata not shown) . These results 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 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 adult mouse.
• Riboprobes were synthesized from plasmids containing mouse cDNA sequences of neurturin (nucleotides 293—598, 441-675 of GenBank accession number U78109), GDNF
• GFR ⁇ -1 (nucleotides 256-935 of GenBank accession number D88264), GFR ⁇ -1 (TrnRl, nucleotides 574-1069 of GenBank accession number U59486), GFR ⁇ -2 (TrnR2, nucleotides 1-569, 1002-1417 of GenBank accession number AF002701 ) and Ret (nucleotides 207-611 of GenBank accession number X67812). Both GFR ⁇ -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 variants. Plasmids were linearized with appropriate restriction enzymes and transcribed in vitro with 50 pCi of [ 33 P] 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 observed for the different mRNA. Two different sense probes were also used to ensure specificity further. Preparation of Tissue
• Sections were pretreated for hybridization as follows: 3x5 minutes in PBS; 3 minutes in 0.2% glycine in PBS; 5 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. 33 P-labeled RNA probes were diluted in hybridization buffer (50% formamide, 50mM
• 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, lmM EDTA) at 37°C; 30 minutes in 20 ⁇ g/ml RNase A in RNase buffer at 37°C; 2x15 minutes in 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 65 ⁇ C. Slides were then dehydrated and dipped in Kodak NTB2 emulsion.
• RNase buffer 0.5M NaCl, lOmM Tris-HCl, pH 8, lmM EDTA
• Labeling was considered specific if it was above the level of background.
• Background label is defined as 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 labeling; low—easily detected but low level of intensity; barely detected— ery low level of labeling in few or scattered cells; or, not detected— o labeling above background.
• Abbreviations are according to Paxinos and Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press Inc., 1986; and Franklin and Paxinos, The Mouse Brain in Stereotaxic Coordinates, Academic Press Inc., 1997.
• GFR ⁇ -1 (TrnRl), GFR ⁇ -2 (TrnR2), and Ret mRNAs were widely expressed in the septum and olfactory system ( Figures 8A-C; Table 2).
• GFR ⁇ -1 (TrnRl), GFR ⁇ -2 (TrnR2), and Ret mRNAs were expressed in the medial and lateral septal nuclei, the nucleus of the diagonal band of Broca, and the piriform cortex ( Figures 8A, 8B). In each of these areas, the receptors were expressed in cells morphologically consistent with neurons .
• GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2) mRNAs were expressed in 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).
• GFR ⁇ -1 (TrnRl) was expressed at high levels in scattered clusters of neurons in the ventral pallidum.
• GFR ⁇ -2 (TrnR2) was expressed in neurons in the ventral pallidum at much lower levels and in fewer cells than GFR ⁇ -1 (TrnRl).
• NTN NTN
• GDNF mRNAs were expressed in piriform cortex, in the hippocampus and, at low levels, in neocortex ( Figures 8D, 9D; Table 2).
• 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 ventral pallidum, the subiculum, and the striatum (Table 2).
• Olfactory Bulb Olfactory Bulb
• mRNAs for all three receptors were expressed in the glomerular layer and in the granule layer (Table 2).
• GFR ⁇ -1 (TrnRl) and Ret were expressed at moderate levels and GFR ⁇ -2 (TrnR2) was expressed at a lower level.
• GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2) were expressed at higher levels than Ret.
• both GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2) were highly expressed in the absence of ret.
• GFR ⁇ -1 (TrnRl) was expressed in the absence of Ret and GFR ⁇ -2 ( rnR2 ) in the internal plexiform layer and the external plexiform layer.
• 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 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 . Abstr. 23:248.12, 1997). Receptor expression in the substantia nigra was predominantly seen in the pars compacta with much sparser labeling in the pars reticulata.
• Ret and GFR ⁇ -1 were expressed at highest levels, while GFR ⁇ -2 (TrnR2) was expressed at substantially lower levels.
• All three receptor components were predominantly, but not 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 receptor components were also expressed in the large motor neurons of the oculomotor nucleus (3).
• GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2) were expressed in the absence of Ret in the superficial layers of the superior colliculus, in the interpeduncular nucleus, and in the cerebral cortex ( Figure 10).
• GFR ⁇ -1 (TrnRl) and Ret, but not GFR ⁇ -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 in any other areas of the midbrain. GDNF mRNA was expressed at very low levels in the substantia nigra pars compacta, in the superficial layers of the superior colliculus and in the interfascicular nucleus. Thalamus
• Receptor components were expressed in several nuclei 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).
• mRNAs for GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2) were expressed at high levels while Ret mRNA was expressed at moderate-to-low levels.
• GFR ⁇ -1 (TrnRl) was expressed at high levels and the other two receptors were expressed at lower levels, particularly in the lateral habenular nucleus. All three receptor components were also expressed at low levels in the medial geniculate nucleus ( Figure 10) .
• NTN was expressed at moderate levels in the anteromedial and anteroventral nuclei of the thalamus ( Figure 9D ) .
• GDNF mRNA was expressed at low levels in 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 ( Table 2 ) .
• GF ( TRN ) receptor components were prominently expressed in many areas ( Figure 9 ; Table 2 ) . All three receptor components were detected in the 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.
• GFR ⁇ -1 (TrnRl) and GFR ⁇ -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 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 ( 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 prepositus hypoglossal nucleus, the spinal trigeminal sensory nuclei, the vagal dorsal motor nucleus, the lateral and medial vestibular nuclei, and the dorsal and ventral cochlear nuclei.
• GFR ⁇ -1 (TrnRl) and Ret were also expressed, in the absence of GFR ⁇ -2, in the region of the inferior salvitory nucleus and nucleus retroambiguous, and in the hypoglossal nucleus, and in motor neurons in the trigeminal motor nucleus .
• Ret was expressed in the absence of GFR ⁇ -1 TrnRl) and GFR ⁇ -2 (TrnR2).
• the receptors were expressed in large motor neurons.
• 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).
• GFR ⁇ -2 Low levels of GFR ⁇ -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 GFR ⁇ -2 (TrnR2) mRNA was expressed in a few scattered motor neurons. Cerebellum
• NTN NTN
• GDNF GDNF
• GF receptors 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 GFR ⁇ -1 (TrnRl) or GFR ⁇ -2 (TrnR2) mRNA are present. In some areas of the brain, most notably the cerebral cortex and the hippocampus, co-receptors GFR ⁇ -1 (TrnRl) and GFR ⁇ -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 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.
• TRN GF
• GDNF central neuronal populations respond to GDNF in the adult rodent including spinal motor neurons ( Li et al., Proc. Natl. Acad. Sci. 92:9771-9775, 1995), facial 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; Kearns and Gash, Brain Res . 672:104-111, 1995; Tomac et al., Nature 373:335-339, 1995; Cass, J. Neurosci.
• GDNF GF
• GDNF 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 GFR ⁇ -1 (TrnRl).
• GDNF Tomac et al . , supra
• MPTP 1-methyl-4-phenyl-1, 2, 3, 6- tetrahydropyridine
• GDNF treatment also protects cholinergic neurons of the septal/diagonal band nuclei from axotomy by fimbria/fornix section in adult rats (Williams et al., supra).
• 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.
• GDNF treatment protects noradrenergic neurons from death (Arenas et al . , supra ) .
• Neurons in the locus coeruleus expressed all three GF receptors.
• TRN GF
• GF (TRN) receptors were also expressed in areas of 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 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.
• 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, suggesting that NTN and/or GDNF may be involved in these processes.
• TRN GF
• NTN and GDNF were expressed in the targets of neurons that respond to these factors.
• 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.
• SNc substantia nigra compacta
• GDNF mRNA in the compacta region of the substantia nigra, where GDNF protein may support dopaminergic neurons through an autocrine or paracrine 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 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 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 GFR ⁇ -1 ( TrnRl ) and neurons in the locus coeruleus that express Ret and GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2).
• NTN and GDNF mRNA were also expressed in a number of areas of the hippocampal formation including all three fields of Ammon ' s horn ( CAl-3 ) within the hippocampus proper and in the dentate gyrus.
• GDNF mRNA was expressed in the subiculum.
• the hippocampus is a potential source of NTN and GDNF for neurons that project to this region of the brain and express GF receptors.
• the medial septal nucleus and the nucleus of diagonal band of Broca which also expressed GF (TRN) receptors, have large efferent projections to 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 hippocampus proper also receive a prominent projection from the locus coeruleus, which also expressed GF (TRN) receptors .
• NTN neuronal neural network
• TRN GF
• 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 GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2).
• Afferent 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.
• GF TRN
• 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
• TRN GF
• 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 GFR ⁇ -1 (TrnRl) and GFR ⁇ -2 (TrnR2). In other regions, most strikingly neocortex and hippocampus, GFR ⁇ -1 (TrnRl) or GFR ⁇ -2 (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.
• NTN and GDNF were also expressed in cerebral cortex. Therefore, thalamic neurons that express Ret may obtain NTN or GDNF as well as the ligand-binding receptor component from their target cortical neurons.
• GF (TRN) receptors are expressed in areas of the adult brain in which neurons that respond to these factors are located.
• GF (TRN) receptor expression in other areas suggests that additional NTN- or GDNF-responsive populations exist.
• the mRNA expression pattern 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.
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• 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 TAC ACC TAC CGC ATG CTC TTC TGC TCC TGC CAA GAC CAG GCG TGC GCT 725 Tyr Thr Tyr Arg Met Leu Phe Cys Ser Cys Gin Asp Gin Ala Cys Ala 195 200 205
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• AGT AAA AAG GTG ATC AAA CTT TAC TCA GGC TCC TGC AGA GCC AGA CTG 1344 Ser Lys Lys Val He Lys Leu Tyr Ser Gly Ser Cys Arg Ala Arg Leu 415 420 425
• MOLECULE TYPE protein
• HYPOTHETICAL YES
• SEQUENCE DESCRIPTION SEQ ID NO : 6 :
• MOLECULE TYPE protein
• HYPOTHETICAL NO
• MOLECULE TYPE protein
• HYPOTHETICAL NO

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