CA2237641A1 - Glial cell line-derived neurotrophic factor receptors - Google Patents

Abstract

Receptors for Glial Cell Line-Derived Neurotrophic Factor (GDNF), their cellular expression, isolation, and biochemical characterization are disclosed. C-RET is disclosed as one receptor for GDNF; additional novel receptors are also disclosed. The preparation of monoclonal antibodies directed against GDNF is also disclosed.

Description

GLIAL CELL LINE-DERIVED NEUROTROPHIC FACTOR RECEPIORS

P~ AIII to 35 U.S.C. 119(e), the present applirAtiQn claims priority benefit of provisional application serial numbers 60/006,619, filed November 13,1995; 60/015,767, filed April 16, 1996; 60/021,965, filed June 27, 1996;
60/020,638, filed June 27, 1996; and 20/020,639, filed June 27, 1996, all herebyincorporated by ~rel~ce in their entireties.

EIELD OF THE D~VENTION
The present invention relates to the t(lentifi~Afion of receptors for and functions of GDNF, and cell lines ~A~ Ssi~lg the l~C~)~Ol~.

BACKGROUND OF THE INVENTION
Glial cell line-derived ~u,ol~oL~hic factor (GDNF) is a trophic polypeptide.
It is a ~ lfi~e bridge-linked homodimer of two 134-amino acids long glycosylatedpolypeptides, with a molecular weight of approximately 25-30 kD for each monomer. Prior to the molecular cloning of GDNF in 1993, investigAt >rs sought atrophic polypeptide which would alleviate the l~u~ollal loss associated with Palhil SOll'S disease, specifically dopaminergic neurons of the ventral mPsenrephalon. The survival of this subpopulation of ll~UlOlls has been known for some time to be promoted by soluble factors present in the conditioned media of glial cell lines. It was from one of these cell lines that the GDNF protein was initially isolated based upon its ability to promote do~A l-i,le uptake in pli~llaly cultures ~ aled from ~llll~lyollic venkal midbrain l~ulo~ Lin et al., 260 Science 1120, 1993). Subsequently, GDNF was shown to promote survival of adult sub~lia nigra l~urolls in vivo following ~hA- ".Af~ological treAtmP~t~ and lesions that mimic Parkinsonian syndromes (Beck et al., 377 Nature 339, 1995; Tomac et al., 373 Nature 335, 1995) Although GDNF was originally reported to be highly specific for d~ u--inel~,ic neurons, several other potent activities of this molecule have subsequently been demonstrated, including survival and phenotypic responsesin facial and spinal motor neurons (~e~(lPrson et al., 266 Science 30 1062, 1994;
Oppenheim et al., 373 Nature 344, 1995; Yan et al., 373 Nature 341, 1995), W O 97/18240 PCT~US96/18197 nora~ ic neu~ ls of the locus coeruleus (Arenas et al., Neuron, in press, 1995), cerebellar pllrkinjie cells (Mount et al., 92 PNAS 9092, 1995), ~y~ ic and sensory neurons in peripheral ganglia (Trupp et al., 130 J. Cell Biol. 137, 1995) and for populations of ~eli~hel~ ulolls with target-derived and paracrine mode of action (Trapp, M. et. al., J. Cell Biol., 130, 137-148 (1995); Pitchel, J., Sariola, H., Hoffer, B. & We~tI h~l, H. (unpublished obse. ~lion); Buj-Bello, A., R~ m~n, V. L., Horton, A., Rnsçnth~l, A.& Davies, A. M. Neuron, 15, 821-828 (1995).
As many of these n~ulolls are affected in neurode~ell~ldliv~ ~li.cP:lcps~ GDNF may have potent therapeutical applications. Particularly, exogenously ~lmini~t~red GDNF m~int~in~ do~ rgic neurons of the substantia nigra in experimPnt~lly in~ re(l Palhh~sOlls disease in rodents (Beck et al. (1995) Nature, 373, 339-3~1;
Tomac et al. (1995) Nature, 373, 335-339) and leads to functional recovery in ralhh~soniall rhesus monkeys (Gash et al. (1996) Nature, 380, 252-255). GDNF
treatment also rescues about half of the ek~cli"-Pnt~lly axolollli~ed murine motoneurons (Oppenheim et al. (1995) Nature, 373, 344-346; Li et al. (1995) Proc. Natl, Acad. Sci. U.S.A., 92, 9771-9775) suggesting that GDNF may be used in tre~tmPnt of motoll~ulollal ~ ç~Ps. The studies of the mPch~ni~m of GDNF
action in normal and pathogenic conditions have been, however, basically hampered as its receptor was not ~own.
Based upon structural similarities (primarily seven conserved cysteine amino acid residues), GDNF apye~ to be a distant member of the transforming growth factor-beta (TGF-13) superfamily of multi~l;Lional cytokines, which includes TGF-,~s, activins, bone-morphogenetic proteins (BMPs) and growth and dir~r~nliation factors (GDFS) (Roberts et al., 327 Philos.Trans.R.Soc.Land.
145,1990). TGF-~B and related ligands are known to ~u~)~Less proliferation in epithelial and immlm~ cells, to function as morphogens in early development, to induce ectopic e~ ssion of skeletal tissue, and to promote survival and dir~lell~iaLion of ~ Ul~llS. TGF-,~ superfamily plot~ s interact with numerous receptor subunits on the surface of responsive cells (Attisano et al., 1222 Mol.Cell Res. 71, 1994; Derynck, 19 Trends Biochem. Sci. 548~ 1994). Dirre~ L receptor types have been described based on the molecular weights of affinity labeled W O 97/18240 PCT~US96/18197 complexes. Among these are the type I, type II and type III receptors, which sellL binding ~lvLeills of 55kD, 70kD and 300kD, respectively. Type III
leceplols are abundalllly ~ulc:ssed tr~n~m~mhrane proteoglycans of apprnxim:~tely - 300kD with a short cytoplasmic tail, and are t~ought to function in recruitment of ligand to an oligomeric receptor complex (Lopez-Casillas et al., 67 Cell 785, 1991). Tn-leerl, a type III l~c~Lur is required on some cell lines for TGF-~2 binding to the ~ign~lin~ l~ceplo,s. Type I and type II receptors are tr~n~m~Thrane ulo~ s with an intracellular serine-threonine kinase d-)m~in and can Lllcl~,ful~transmit duwl~Ll~dlll signals upon ligand binding ~Attisano et al., 75 Cell 671,1993; Derynck, 1994 supra). Type II lecepl~ are col~liLuLively activated kinaseswhich upon ligand binding recruit type I receptors to a ~ign~ling complex. In this complex, type I receptors are phosphorylated by type II receptors on a rt~m~mhrane domain rich in serine residues, this phosphorylation is thought to result in the activation of the ser-thr kinase activity of type I rec~tol~ and in dowl~Ll~;alll sign~lTng (Wrana et al., 370 Nature 341, 1994). Accordil.g to thismodel, TGF-,B ~u~-elr~llilylJl'O~illS can not bind to type I l~ce~Lol~ in the absence of type II receptors, although in some cases, type I lccepLuls are ntocess~ry for efficient binding to type II ,ec~lols (Letsou et al., 80 Cell 899, 1995). Multiple cDNA clones of type I, II and III receptors for TGF-,13S, activins and BMPs havebeen isolated by either expression or homology cloning, including seven m~rnm~ n type I receptors, four type II receptors and one type III betaglycan receptor. Additional membrane proteins binding dirrt;l~l~L members of this family include glycosylphosphatidyl inositol (GPS)-linked 150kD and 180kD pluLeills of unknown structure and function (MacKay and Danielpour, 266 J. Biol. Chem.
9907, 1991), and endoglin, a 180kD disulphide linked dimer which binds TGF-~l but not TGF-,~2.
The isolation and charac~eli~alion of GDNF receptûrs is a prerequisite for the underst~n~Ting of the full range of biological actions of GDNF and the sign~ling events that take place upon GDNF binding to responsive cells. Until now, progress in this area has been hampered by the lack of cell lines responsive to GDNF, that is, cell lines Culll~liSillg GDNF receptors.

SUMMARY OF THE INVENTION
Receptors for GDNF are disclosed herein, as are cell lines e~ ,s~ing the same. Methods for idel.liryhlg and isolating these ,cce~ r~ are also disclosed.

In one aspect, the present invention relates to isolated rece~lol~ which bind GDNF.
In another aspect, the present invention relates a method for ~lel.. i~ g collll.oullds or compositions which bind GDNF l~ce~
In yet anolller aspect, the present invention relates to methods for idelllifyill~ homologs of GDNF by SC1Ct;l~11g for colll~,u~ds or compositions which have similar biological effects, such as tyrosine phosphorylation, increase in c-fos mRNA, and and increases in cell survival.
In still another aspect, the present invt;lllioll relates to method for id~ irying analogs of GDNF by screening for colll~ ds or compositions which are antagonistic for the biological effects of GDNF, such as are listed above.

BR~EF DESCRIPTION OF T~; DRAVVINGS
Fig. 1. Binding of l2sI-GDNF to receptors on chick symp~th~tic neurons. (a) Saturation steady-state binding of l25I-GDNF to E10 embryonic chick symp~th~ti~
neurons. Data are e~iessed as mean ~SD of triplicate ~lel~.,.~ ions. (b) .Sc~tch~rd lldl~Çollllation of the data plotted in (a). (c) Hill L,al~rollualion of the data p}otted in (a) nH: Hill coefficient.

Fig.2. Affinity l~h~ling of GDNF leceplol~ on chick ~y"~ neurons. l2sI-GDNF was cross-linked to E10 embryonic chick ~ylll~ ~ulvns, receptor complexes were fractionated by SDS/PAGE and vi~ by gel autoradiography (middle lane). A doublet at 100hD and a 300hD complex are in~lic~tPd by arrows.
Excess cold GDNF prevented cross-linking of l2sI-GDNF (right lane3. For comparison, cros~linking of l2sI-TGF-,~ to mink lung epithelial cells MvILu is also shown (left lane). Molecular weight lll&,he,~ are intlirat~l in kD.

Fig.3. Affinity labeling of GDNF receptors on cell lines. l2sI-GDNF was cross-linked to C6 ~liomA, RN33B raphe nucleus, L6 myoblast and MN-l motor neuron cell lines with either DSS or EDAC as crosslinker agents. Receptor complexes were fractionated by SDS/PAGE and vi~ 7f d by gel autoradiography. Excess cold GDNF pL~ve~ d cross-linking of l25I-GDNF (cold). Molecular weight alh~,ls are in~jc~te~ in kD.

Fig. 4. Individual col.x~ pnt ~ffiniti~e of GDNF receptor subunits in RN33B and MN-l cells. (a) Sizes of dirr~ lL GDNF receptor complexes on RN33B and MN-l cells after cross linking with EDAC or DSS. (b) and (c) l25I-GDNF was cross-linked to RN33B (b) or MN-1 (c) cells in the ~l~,sell~e of h~ ,asillg concentrations of cold GDNF. The l!el~ ge of ~25I-GDNF binding to the inAic~tPA recel)lor subunit is plotted as a function of the concentration of cold GDNF used during binding.
Fig. 5. Expression of GDNF mRNA in cell lines ~ lg GDNF receptors. (a) Autoradiogram of an RNAse protection assay using equal amounts of total RNA
from the inAi(~t~A cell lines. Kidney post natal day 1 and yeast tRNA were used as positive and negative controls, respectively. ~b) Qll~ntifir~tion of the level of GDNF rnRNA in different cell lines relative to the level in PI kidney. undiff RN33B, un~lirrert;llliated RN33B cells; diff RN33B, di~rel~llLial~d RN33B cells,diff RN338+GDNF, RN33B cells dirr~ At~d in the presence of GDNF.

Fig. 6. Expression of mRNA for c-ret in dirrel~llL cell lines.
Fig. 7. GDNF stimlllAtinn of tyrosine phosphorylation of ERKs in RN33B and MN-1 cells. RN33B (a) or MN-1 (b) cell monolayers were e~posed to 50 ng/ml GDNF during the in-lir~ted periods of time (in minlltes), cell lysates were fractionated by SDS/PAGE and Western blots probed with an anti-phosphotyrosine antibody (aP-Tyr). The blots were stripped and l~l~.bed with an anti ERK2 antibody (a-ERK2) that recognizes both p42'rk 12 and p44erkl (arrows to the right).
Molecular weight markers are in~liç~te(l in kD.
-W O 97/18240 PCT~US96/18197 Fig. 8. GDNF stim~ tion of c-fos mRNA ~ ssion in RN33B and MN-1 cells.
RN33B (a) or MN-1 (b) cell monolayers were exposed to 50 ng/rnl GDNF during the inllir~tP~l periods of times, total RNA was extracted and fractionated in agarose gels and Northern blots probed with a 32P-labeled rat c-fos probe. Shown are x-ray 5 autoradiograms of filters washed at high ~llhlgelley.

Fig. 9. GDNF increased the surv}val of RN46A cells. RN46A cells were dir~ lLia~d in media i 0-50 ng/ml GDNF for 8 days. The data ~ ,selll ~e means ~ SEM of three independent experiments (1,500-3,000 cells counted per 10 condition). ANOVA in(~ic~t~cl that the GDNF had a ~i~nifir~nt effect on survival at all concentrations c~ al~,d to media alone (overall ANOVA: df=6,203; F=l 1.39, p,0.001; lln~qu~l N LSD post hoc test, pØ001).

Fig. 10 a-c. Biological and bioch~mi~l responses of MN-1 to GDNF. (a) GDNF
15 stimlll~t~s survival of serum-deprived MN-1 cells. (b)GDNF stimlll~tPs rapid and transient tyrosine phosphorylation of several proLeills (asterisks) in MN-1 cells.
Tirne of GDNF tre~tmlont (in mimltes), and molecular weight Illalhels are in~1ic~t~-1. (C) Rapid and sll~s~in~d ERE~I and ERK2 tyrosine phosphorylation stimlll~t~ by GDNF in MN-l cells.
Fig.11 â-b. c-RET is a signal transducing receptor for GDNF. (a) Tll...ll....~pl~ iLation analysis of GDNF-receptor complexes in MN-1 cells.
GDNF-labeled binding ~l- leins could be plc~ ed with lectin Sepharose beads, or antibodies against GDNF, phospho-tyrosine (P-Tyr) and c-RET. Control P1G;IIIIIIIII~ antibodies did not imml~noprecipitate GDNF receptor complexes. (b) GDNF induces tyrosine phosphorylation of c-RET in MN-l cells. c-R~T tyrosine phosphorylation was det~ctecl already 5 ~--i--..~s after addition of GDNF (upperpanel). Saturation was observed at 30 ng/ml GDNF (lower panel).

Pig. 12 a-b. c-ret expression is sufflcient to mt~ t~ binding and biological responses to GDNF in fibroblasts. ~a) Iodinated GDNF could be cross linked to 3T3 cells stably transfected with MEN2a-ret or wild type c-ret e~Lession W O 97/18240 PCT~US96/18197 plasmids. Untransfected 3T3 cells (3T3) did not bind GDNF. The specificity of the binding was demolL~Lla~d by displacement of the labeling with 50X
excess cold GDNF. (b) GDNF ~lulllol~s survival and growth responses in 3T3 ~ fibroblasts stably transfected with a c-ret expression plasmid. UllLldl~r~cted cells did not respond to GDNF.

Fig. 13 a-c. c-ret mRNA expression in adult brain and in developing substantia nigra. (a) Ribonl~clç 3~e ~lo~;Lion analysis (RPA) of c-ret mRNA ~ ,s~ion in dirr~Glll regions of the adult rat brain. (b) RPA of c-ret rnRNA expression during development of the rat ventral mPsPn~~ephalon (nigra), and of GDNF mRNA
expression in the developing sLli~Lulll. (c) mRNA ~ cs~ion is inrlic~tPrl in a~ y units where 100 corresponds to the level of expression in the respective regions in newborn ~nim~

Fig. 14 a-h. c-RET is e~l~i,sed in GDNF--c~onsi~le substantia nigra dop~~ clgic neuron. (a) Dark field autoradiogram of c-ret mRNA expression analyzed by in situ hybridization in the adult substantia nigra. Scale bar, 40 ,um. (b) Bright fieldautoradiogram showing substantia nigra neurons cont~ining c-ret mRNA. Scale bar, 7.5 ,um. (c) Tmml-nohi.ctochPmir~l analysis of c-RET protein expression in the adult ~ub~L~ ia nigra. Scale bar, 27 }bm. (d) autoradiogram showing in situ hybridization for c-ret mRNA in the adult rat brain after a unilateral lesion with 6-OHDA. The injection of this toxic dopamine analogue in the medial forebrain bundle ensures that only cells which actively take up and retrogradely llal~3olLd~a.llhle will be colll~lolllised. Note the disappeal~lce of the labeling for c-ret mRNA in the lesioned substantia nigra (arrowhead) 1 day and 5 days following the lesion. (e)Tmmlln-hi~toc-h~ l analysis of c-RET protein ~ ,ression in the adult sub~ ia nigra after lesion with 6-OHDA and grafting of mock transfected fibroblasts (control graft). Note the nearly complete absence of c-RET-LI caused~ by the lesion. Scale bar, 20 ,um. (f) Grafting of GDNF-e~~ lg fibroblasts rescues c-RET-LI. Note c-RET positive fibers ~u~ ulldmg and entering the GDNF
producing graft (arrows). Same m~gnifi(~tion as in (e). (g) Tmmlln~histochemicalanalysis of cRET protein expression in the adult locus coeruleus after lesion with 6-OHDA and grafting of mock transfected fibroblasts (control graft). Scale bar, 23 ,um. (h) Rescue of cell bodies e~ essillg c-RET-LI by GDNF in of 6-OHDA
lesioned locus coeruleus. Same m~ni~lr~tion as in (g). Graft is on the right in (e) and (f), and above in (g) and (h).
s Fig. 15 a-c. PC12 and NB2/a cells respond to GDNF and bind GDNF. (a) GDNF
promotes survival of serum-deprived PC12 cells. (b) (~DNF i~ ,ases the number of NB2/a cells. (c) l25I-GDNF binds to PC12 and NB2/a cells in the absence (opencolllmn) or presence (filled column) of 50-fold nnT~bele~l GDNF.
Fig. 16. ~ffinity cro~linkin~ of l2sI-GDNF to cell lines. I25I-GDNF was crocslinkf d to PC12 cells (lane 1), SY5Y cells (lane 2), E20 rat kidney cells (lane 3) and NB2/a cells (lane 4), and the reSllltîn~ complexes were pleeipiLated fromdc;l~ lysates by anti-GDNF antibodies (Santa Cruz).
Fig. 17 a-b. GDNF specifically binds to c-RET. ( a) '25I-GDNF was cr~-s~link~od to NB2/a cells in the l)r~sel~ce (~) or absence (-) of 1000-fold excess of unlabeled GDNF (PeproTech EC Ltd.), and the resllltin~ complexes were precipitated from dc~l~enl lysates by cocktail of monoclonal and polyclonal (Santa Cruz) anti-c-RET
antibodies recognizing the extracellular and intracellular domain of cRET, r~ ecli~ely. Lysates were also precipitated by monoclonal anti-neurofil~m.orlt antibodies 13AA8 (lane 3), by Protein A-Sepharose (lane 4) and by WGA-Agarose (lane 5). (b) ~ GDNF binds to COS cells tr:~n~iently e~ .,ssillg c-RET, but not to mock-~l~l~,rt;cled (with pBK-CNV plasmid) COS cells. Open column lc~ sellL~, binding in the presence, and filled column in tne absence of 50-fold excess of unlabeled GDNF.

Fig. 18. GDNF illir~ases tyrosine phosphorylation of c-RET in transfected COS
cells. c-RET was immlln~,pleci~ ted from d~ ll lysates of GDNF-treated (~) (lane 1) or ullllcated (-) (lane 2) COS cells transfected (lane 3) with c-ret cDNA or mock-transfected with PBK-CMV plasmid. (a) immllnnblot probed with anti-c-RET
antibodies (Santa Cruz). (b) the same filter reprobed with anti-phosphoLylosille antibodies.

Fig. 19 a-h. GDNF binds in situ to c-ret-positive developing enteric n~ulons. (a, b) Darkfield (a) and corresponding bright-field (b) microphotographs of GDNF
antisense cRNA hybridization to ~ldrrhl sections through E15 rat gut. (c, d) Dark-field (c) and coll-,spulllillg bright-field (d) microphotograph of in situ binding of l25I-GDNF to E15 rat gut explants. (e) c-ret ~nti~en~e cRNA hybridization to a cryosection through ElS rat gut. (f) Tmm~lnl)st~inin~ of E15 rat gut cryosectionwith anti-periphlorin antibodies. (g) GDNF sense cRNA hybridization to E15 rat gut section. (h) In situ binding of l25I-GDNF to E15 rat gut explants in the presellce of 250-fold excess of unlabeled GDNF. ---, muscle layer; n, i..~ l nerve plexus.
Bar, lOO,um.

Fig. 20 a - b. Cro~clink~l GDNF-c-RET-complexes are obtained from GDNF-responsive cell lines and from c-ret-transfected cells (a) l25I-GDNF was cro~linkloc7 with EDAC to PC12 cells, NB2/a cells, dissociated E20 rat kidney cells, and COS
cells, and the reslllting cnmpl~rt?s were precipitated by anti-GDNF antibodies. (b) EDAC-cros~link~d l25I-GDNF-c-RET complexes were imml-n(~pr~ci~ d with anti-c-RET antibodies from the extracts of PC12 cells, stably c-ret-transfected (Ret.-3T3) or mock-transfected (mock-3T3) 3T3 cells, as well as from dissociatedE15 kidney cells in the p.~sellce (+) or absence (-) of 500-fold excess of unlabeled GDNF or TGF-~l. The ~SOK bands in all gels are the cro~link~cl dimers of GDNF.

Fig. 21 a - b. GDNF increases c-RET autophosphorylation in stably transfected 3T3 cell line. (a) GDNF dose-dependently increases tyrosine phosphorylation of 160 kD isoform of c-RET in c-ret-transfected (ret-3T3) but not in mock-transfected (mock) cells. (b) GDNF time-dep~-n-l.ontly increases tyrosine phosphorylation of 160 ~ kD isoform of cRET in c-ret-transfected 3T3 cells. Upper panels (Ret.-PTyr) are the immlm--blots stained with anti-phosphotyrosine antibodies, and lower panels (Ret.) show the reprobing of the corresponding filters with anti-c-RET antibodies.
.

Fig. 22. GDNF increases the number of trkC-3T3 fibroblasts tr~n~iently expressing c-RET (open squares), but not mock-lldl~ire~it~d cells (filled squares).
c-ret and mock-l~ Ç~cted cells in five parallels were treated with rat GDNF at inllirat~-l concentrations, or with NT-3, for five days. Cell llul~ l, quantified with Abacus~ Cell Proliferation Kit (Clontech), is e~L~r~s~ed as a percent of the control cells without growth factors. ~, p<0.001 compared to mock transfected cells.

Fig. 23 a-b. Purification of ,~c~yLor from L6 myeloblast cells. (a) Plasmon resonance analysis of fractions obtained from anion ex~h~n~e cllr~,llatography of L6 cell Iysates. Total protein of fractions is also depicted. (b) Further pnrifir~tion of lM fraction obtained from (a) by hydrophobic interaction chrolnaLugraphy.

Fig. 23. Autoradiographic film of the ligand blot ~ GDNF with ~lUl~ilLC7 from adult rat brain (lane 2) and liver (lane 3). 50-fold excess of llnlQ~eled GDNF (lane 1) ~ignifir~ntly reduces the binding.

DETAILED DESC~ION
A prereq~ ite for the unde,~ .p of the full range and merh~ni~m~ of action of GDNF is the char~rle~ iQn of GDNF receptors and their .~ign~ling y~. Although receptors fûr ûther members of the TGF-~ superfamily are well characterized, GDNF receptors r~m~in~d undefined until this disclosure.
Disclosed herein is the bioçh~mic~l charauLeli~aLion of GDNF receptûrs and theirduwl~ ull responses in symp~th~o~ir ntulo~ls and responsive cell lines. Using affinity labeling, multiple GDNF binding subunits that m~ te cooperative bindingof GDNF to embryonic symp~th~ n~ulolls are i~1entifiecl Screening of over thirty cell lines initially revealed high ~ ,e~,~,ion of GDNF binding proteins of 55 kD, 70 lcD, 135 lcD and 300 kD in conditionally immortalized ll~u,ollal precursors from the raphe nucleus. As the data demonstrate, GDNF receptors were highly in~ ce(l after nc~ulollal dirrelcllLialioll of these cells, which then became sensitive to the survival-promoting effects of GDNF. Dirrer~ combinations of these subunits were also seen in glioma, myoblast and Sertoli cells. A different receptor pattern W O 97/18240 PCT~US96/18197 was found in a motor neuron hybrid cell line, where the predo~ l component was a CPI-anchored protein of 155kD.
Despite the striking similarity with the ~ccc~ pattern of other TGF~
~u~cl~llily members, immlm- precipitation experiments in~ te~l that GDNF
receptor subunits of 55kD, 70kD, 135kD, and 300kD are novel proteins. The 155kD subunit was subseqllPntly ~let~.rmin~d to be the product of the c-ret proto-oncogene, c-RET, a receptor tyrosine kinase crucial for the development of parts of the excretory and nervous systems. GDNF stimn~ ERK tyrosine phosphorylation and c-fos mRNA expression with dir~lclll time-courses in raphe nucleus and motor neuron cell lines, suggcs~ g that dirrclcl.~ complements of GDNF receptor subunits can form distinct .~i~n~ling complexes.
Concomitantly, c-RET was identified as receptor for GDNF on additional cell lines. GDN~ rescues c-RET-positive do~ P-)gic and norad,~ gic lleulons in lesion models of Parkinson's disease, sugge~Li.,g that cRET may mPrli~te the anti-P~ lsonidn effects of GDNF in the adult brain.
c-ret proto-oncogene (T~k~h~hi et al. (1985) Cell, 42, 581-588) encodes a protein that is structurally related to receptor Lylosille kinases (T~k~h~hi et al.
(1988) Oncogene, 3,571-578). Its extracellular part contains an llm~ l cadherin-like domain and also a cysteine-rich domain, the biological roles for which are not understood. By ~ e splicing, several isoforms of c-ret mRNA have been described (Tahira et al. (1990) Oncogene, 5, 97-102; Myers et al., (1995) Oncogene, 11, 2039-2045;Lorenzo et al. (1995) Oncogene, 10, 1377-1383), but their biological mP~ning is ~;u~ ly not understood. In several cell lines, c-ret-encoded ~roleills with molecular weights of 160 kD and 140 kD are described, lcpl~es~ g the fully and partially glycosylated isoforms of 120 kD core protein,respectively (T~k~h~hi et al., 1988). As with other lcc~Lor tyrosine kin~es7 c-RET is activated by homodimerization followed by phosphorylation of its tyrosineresidues.
In the excretory system, c-ret is expressed in the nephric duct, the ureteric bud and the growing tips of the collecting ducts (Pachnis et al., (1993), supra).
Mice homozygous for a null mutation in the c-ret gene die soon after birth, withkidneys either absent or rudimentary and displaying severe defects in the enteric W O 97/18240 PCTrUS96/18197 nervous system (Sch~ rdt et al., Nature 367, 380-3 (1994). Based on this evidence, it had been proposed that the cognate c-re~ ligand may be a growth factor important for morphogenesis and neurogenesis.
During murine embryogenesis, c-ret mRNA is expressed primarily in the S nervous and excretory systems. c-ret mRNA is found in dorsal root, ~y~pi~lh~tic~
enteric and cranial ganglia (Pachnis et al., Development 119, 1005-17 (1993), aswell as in post migratory neural crest cells and in various tumors of neural crest origin, including pheochromocytoma, m~ ry thyroid calcillol~la and neurobl~tom~ (Ikeda, I., et al. Oncogene 5, 1291-6 (1990); Santoro, M., et al.
Oncogene 5, 1595-1598 (1990). In the developing central nervous system, sites ofc-ret expression include the ventral portion of the neural tube, the retina and motor neurons in spinal cord and hindbrain (Pachnis et al., (1993), supra). However, the pattern of expression of c-ret in the adult nervous system has not previously been reported.
The absence of a known ligand for c-RET has basically hampered the studies of intracellular y~Lllw~y~ that c-RET can m~ te. C~ ve analysis of the growth-promoting activity of the epit~ l growth factor receptor/c-RET
chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of epidermal growth factor receptor (Santoro et al. (1994) Mol. CeU. Biol. 14, 663-675). We disclose herein that both NGF and GDNF promote survival of PC12 cells, whereas only NGF induces their dirr~rt:llliation, suggesting only a partial overlap in the si~n~ling palllw~lys of c-RET and trkA, a receptor for NGF. Binding of an adaptor protein Grb2 to oncogenic forms of c-RET has been d~ d (Borrello et al. (1994) Oncogene, 9, 1661-1668). However, the details of the yalllw~ys are completely unknown. Now, having GDNF as a ligand, it is possible to address the intracellular sign~lin~ of c-RET upon GDNF binding.
Like c-RET, GDNF is abundantly e~yl~ssed in the muscle layer of the ~s~u;.~ l tract and in the con~en~ing m--sen~hyme of the kidney (Suvanto et al. (1996) Eur. J. Neurosci., 8, 816-822). Further, as disclosed herein, GDNF
specifically binds c-RET-positive cells in developing gut, GDNF can be cr ).~link~d to c-RET in several cell lines and in developing kidney, GDNF specifically induces W O 97/18240 PCT~US96/18197 lylosille phosphorylation of c-RET, and ectopical expression of c-RET in 3T3 cells confers a biological response of these cells to GDNF. Thus, c-RET is activated by GDNF and m~ t~s its functions.
The product of the c-ret proto-oncogene plays hnpoll~L roles in human disease. Realldl~ ents and mutations in the c-ret gene are associated with several tumors e.g. fs~mili~l m~ ry thyroid cal~;hlollla, multiple endocrine neoplasia type 2, etc., but also with Hi.~cl~ llg disease, a disorder that is characterized by the absence of enteric ll~ulvlls in the hinrl~lt r~slllting in ohstir~tion and megacolon in infants and adults (reviewed in Mak, Y. F. and Ponder, B.A. J. (1996)Curr. Op. Genet. Dev., 6, 82-86). T(1PI~I; r~ n of GDNF
as a ligand for c-RET further enables the analysis of the molecular basis of these ~liee~e~s Particularly, the mutations in GDNF gene can now be studied as possible cause for the ~Iirschsprung disease in the cases where c-ret locus is not mllt~te(l The phrases "GDNF receptor" and "lcc~Lol for GDNF" as used herein each refer to a single subunit which binds GDNF as well as combinations of the receptor ~ubu~ which bind GDNF.
The term "effect~ as used herein means an alteration or change. An effect can be positive, such as c~lleing an increase in some material, or negative, e.g., antagonistic or inhibiting.
The term "homolog" as used herein refers to a compound or composition having a similar biological effects as GDNF, such as are disclosed herein.
The term "analog" as used herein refers to a compound or composition having an antagonistic effect on the biological effects of GDNF.
The term "isolated" as used herein in reference to a GDNF receptor means a compound which has been sepal~led from its native ellvi~ llltll~ or, if recombhldllLly expressed, from its expression envil.,lllllent.
The phrase ''sub~ y pure" as used herein in lef~.~,.lce to a compound means an isolated compound which has been sep~r~ted from other components which naturally accolllpally it. Typically, a compound is ~ulJ.~I;...l;~lly pure when it is at least 75%, more preferably at least 90%, and most preferably 99% of the total material as measured, for example, by volume, by wet or dry weight, or by mole percent or mole fraction.

blank page CA 0223764l l998-05-l2 W O 97/18240 PCTAUS96/~8197 subpopulations of n~u~ s, in particular d~a~ lel~ic and noradlcllel~ ic central llCU~UllS, as well as spinal and facial motor ll~u~ s. Given the activities of GDNF
in various mono~min~-rgic n~un~lls, the discovery of GDNF receptors in cell lines derived from the mlorh~ ry raphe in~ te that serotonergic lleul~ s may also respond to GDNF in vivo. The endogenous ~ sion of GDNF by these cells suggests that this factor may act in a paracrine/autocrine fashion with~n the raphe nucleus. Expression of GDNF receptors in Sertoli TM4 cells Sllg~St~ non-l~ul'ollal roles for GDNF in developing testis. In vivo, the temporal e~ s~ion of GDNF mRNA in- testis correlates with the expansion of the Sertoli cell population (Trupp et al., supra) which, together with the discovery of GDNP lece~Lol~ on the TM4 cell line, suggest an auloclille action of GDNF during Sertoli cell lllalu.dlion.
Similarly, the presence of GDNF r~ce~Lol~ in rat myoblast L6 cells, together with the ~les~ion of in developing muscle in vivo (Henderson et al., supra; Trupp et al., supra), inr1ie~tes a potential paracrine role of GDNF during myogenesis.
Despite the presence of receptors and biological activities of GDNF on embryonic~y~ ic neurons, PC12 cells which had been dirr~ .l into symp~th~ti~-like neurons with NGP did not express GDNF l~c~kll~ under initial ~el.,nental conditions. As ~ cn~se~ below, however, GDNF l~cepLo,~ were llltim~t~ly i~lentifi~ on PC12 cells.
GDNF receptors are absent in the pons noradrell~,~ic cell line CATH.a.
Given the robust effects of GDNF on adult central noradl~e~ ,ic ll~u~ from the locus coeruleus, the absence of GDNF receptors in CATH.a is intri~ling.
Recently, however, Gong et al. reported that GDNF can plc:v~lll the degenerationof CATH.a cells in-l~7cetl by 6-OH-dopa~ e lle~i..e..~ (Gong et al., 21 Abs. Soc.
Neurosci., 1789, 1995~, suggesting that GDNF receptors may be inf1~ e-l in thesecells after 6-OH-dopamine lesion. Indeed, in vivo studies have shown that GDNF
elicits a more profound induction of the phenotype of noradrenergic neurons following 6-OH-dopamine injection than in the non-lesioned locus coeruleus.
~ Fur~er, Treanor et al. recently reported upregulation of GDNF binding in sections of the substantia nigra after medical forebrain bundle transaction (Treanor et al., 21 Abs. Soc. Neurosci. 1301, 1995~, suggesting that the receptor upregulation may be a general mech~ni~m of control of GDNF responsiveness in the central nervous n~h~
-W O 97/18240 PCT~US96/18197 system.
GDNF rece~lol upregulation was also observed during in vitro .lirr~ LiaLion of raphe nucleus cells. These lines have recently been shown to retain the ability to respond to local microenvironm~nt~l signals after transplantation into the adult brain, where they dirr~lcllLial~ in a direction that is con.~i~tent with that of endogenous l~ lOlls in the transplantation site (Shihabuddin et al., 15 J. Neurosci. 6666, 1995). In vitro, ho~,v.,., a shift to the non-pel.~lissive ~ll~ ature dirr~ ,lial~s them along default ~d~1lw~ys into glllt~m~tPrgic (RN33B) or s~rolollc.~ic (RN46A) phe"ulyl,es, 1 ;;~e~;lively.
Dirr~l~J,liation in culture has also been shown to upregulate e~ Gssion of receptors for other trophic factors in these cells, including the llcuroL~ophin lect~
p75LNGFR and trkB (Whittemore and White, 615 British l~es. 27, 1993). Although they can give rise to different ll~uro~lal types depending upon the site of transplantation, RN33B cells are not able to ~lel~le glial elements, suggesting these cells represent n~ulunally le~ led multipotent precursors (Shihabuddin et al., supra). In this respect, it is il~Lele~lillg to note the absence of GDNF receptors in two plul;~oL~ ulollal stem cell types (Renfranz et al., 66 Cell 713, 1991;
Snyder et al., 30 68 Cell 33, 1992) suggesting that these cells are less restricted than the raphe nucleus cell lines. Taken together, these obse. v~Lions suggest that GDNF ,cceplor ~ ,ion may initially appear in newly dir~r~ A post-mitotic neurons and increase progressively during neuronal nlaluldlion.

2. M7~ 1e GDNFreceptor subunits The data demo~ te that novel GDNF receptor is composed of multiple subunits which cooperate to achieve high affinity binding. The coop~lalive binding of GDNF to embryonic symp~th~tir llt;ur~lls may thus be an indication of a multi-step mto~h~ni~l.. of receptor assembly. Rec~llse billding assays were performed at 4~C, binding coop~"aliviLy is unlikely to have resulted from substantial lateralmobility of tr~n~m~mhrane receptor plvL~ills, suggesting that GDNF binding 30 in~ ces colIfoln,ational changes on receptor complexes that are partially p~ ,ed on the membrane. The nearly iclentir~ ffiniti~s of the dirr....l~ GDNF receptor subunits obtained by crosclinking also support the notion of coo~dLiv~ binding of GDNF to a partially pre-assembled receptor complex.
The ~llu~;luldl similarities b~lweell GDNF and members of the TGF-,B
superfamily suggest that receptors for GDNF might conform to some of the y~es described for receptors of members of the TGF-,B family. Indeed, the S pattern of GDNF binding prol~ins described herein is strongly l~ ce"L of type I, type II and type m TGF-,B receptors.
Despite the overall similarities between GDNF and TGF-,B ~u~ r~ ily receptors, no GDNF receptors could be ~letecte~1 in several cell lines known to express various TGF-,B and activin ~eceplo~ subunits, including the mink lung epithelial cell line MvILu. In agreement with this obscl~dlion~ no binding of GDNF has been ~let~ctecl in COS cells ll~l~cl~d with dirr~,e"l combinations of known type I and type II TGF-~ supeAamily rec~lol~i (Ibanez, C., unpublished; P.ten Dijke, personal co--..l--~ tiQn)~ including the recently isolated type II l~c~to for BMPs (Rost;l~weig et al., 92 PNAS U~A 7632, 1995) and a novel brain-speciffc type I ,~c~lcr (Ryden et al., 21 Abs. Soc. Neurosci 1754, 1995).
Moreover, no GDNF receptor complexes could be recovered after op~ ion with ~llipel~ide anLis~ against any of the cloned TGF-~B
~upt;~ralllily ~ecepL~l~, in-lir~tin~ that GDNF receptor components are novel 3. c-RET is a receptor for GDNF
GDNF receptors were found in a motor neuron-neurobl~tom~ hybrid cell line, but not in a basal fo,~,~hl cell which was also a hybrid with the same neurobl~ctoln~, sugge~Lhlg that the receptors ~letecte(l on MN-1 cells IGl)r~senphysiologically relevant motor neuron GDNF lece~lol~. In contrast to raphe nucleus cells, GDNF e~pl~ssion could not be ~lete&t~o(l in the motor neuron cellline, con~i~tent with a target-derived mode of action for muscle-derived GDNF invivo (Henderson et al., 266 Science 1062, 1994; Trupp et al., supra). GDNF
binds to and induces tyrosine phosphorylation of the these receptor which were identified as the product of c-ret. c-ret was also able to m.otli~t~ GDNF binding and survival/growth responses to GDNF upon transfection into naive fibroblasts.
Moreover, dopaminergic neurons of the adult ~ul~nlia nigra were found to W O 97/18240 PCT~US96/18197 express high levels of c-ret mRNA, and c-RET e~lessi,lg dop~,li,~.~ic and noradlc;~ ic l~urolls in the CNS responded to the ~lotecLive effects of exogenous GDNF in vivo. Together, these data in~ tP that the product of the c-ret proto-oncogene encodes a functional receptor for GDNF which may mediate the ll~uloLl~ ic effects of this factor on dopA.. i.~ gic, norad,~"~l~ic and motor neurons.
The results disclosed herein in(~ te that the c-RET lece~Lol Lyl~ e kinase is a signal transducing receptor for GDNP. This finding is surprising, given ~atall lcc~Lol~ for members of the TGF-~ superfamily ch~rarteri7P(I so far are receptor serine-threonine kinases (Derynck, R. Trends Biochem Sci 19, S48-553 (1994); Attisano et al., J.Bba-Mol Cell Res,222, 71-80 (1994)). GDNF is in fact a very di~,.,r~ell~ mPmher of the TGF-~ ~upelr~llily, with which it shares primarily the spacing between conserved cysteine residues in the amino acid sequencer. Itsability to interact with a receptor tyrosine kinase in~ t~Ps a further functional 15 divel~ ellce from other members. of the TGF-~ superfamily. Conversely, these ri"~lj.,~s could suggest that other TGF-~B ~u~elralllily members may also utilize receptor tyrosine kinases.
The following results disclosed herein also implicate the c-ret proto-oncogene product as a functional receptor for GDNF:
i) GDNF binds to COS cells ectopically e~,essillg the c-ret proto-oncogene;
ii) GDNF can be chPrni~lly croc~linkPA to the product of the c-ret proto-oncogene ectopically expressed in COS cells or from NB2/a and PC12 cells;
iii) the c-ret proto-oncogene product ectopically ~ ssed in COS
cells, but also in NB2/a cells, becomes rapidly phosphorylated on tyrosine residues upon GDNF binding;
iv) GDNF promotes biological effects i.e. mitogenic or trophic in cel1s e~lc;ssing c-ret proto-oncogenic products.
-W O 97/18240 PCT~US96/18197 GDNF specifically binds to RET-e~r~ g (Figure l9 c, d, h) enteric neurons and the tips of ureteric buds in developing kidney. These tissues were absent or severely reduced in c-ret-deficient mice (Sc-hllr1l~rdt et al. (1994) Nature, 367,380-383; Durbec et al. (1996) Development, 122, 349-358). The data S disclosed herein further demon~ le GDNF-c-RET complexes from GDNF-responsive and c-ret-lld~lsrecl~d cells and from embryonic kidney cells. Finally, GDNF time and dose-dependently activates c-RET, and introduction of c-ret into GDNF-nonresponsive cells results in GDNP-responsiveness.

4. Downstream signalingpa~*ways a~;liv~led by GDNF/tc~l)t~r Investigation of GDNF signal tr~n~d~lcin~ m~cllAoi~.~.e in raphe nucleus and motor neuron cell lines has been conducted. The dow~ am responses elicited by GDNF in these cells demo~ that the GDNF binding ~loL~ills identified herein lc~l~;sellL functional GDNF receptors. The initial biochemical chal~leli~lion ofGDNF signal trAn~dllrtion pathways has identified lll~lllbc-~ of the ERK/MAP
kinase family as components of the GDNF ~ign~lin~ m~ch~ni~m ERK/MAP kinase activation by phosphorylation is the final step in a ç~cade of kinases that is set in motion after activation of the Ras pa~way by various growth factors, including TGF-,~ (Yan et al., 269 J. Biol. Chem. 13231, 1994; Hartsough and Mulder, 270 J. Biol. Chem. 7117, 1995) and nerve growth factor (Thomas et al., 68 Cell 1031,1992; Wood et al., 68 Cell 10 II, 1992). More recently, ERK2 has also been shown to form part of the signal tr~n~du~tion p~Lllw~y activated by several cytokines, such as in~~ lls and interleukins, which are not known to activate Ras (David et al., 269 Science 1721, 1995). Whether or not Ras activation is oneof the steps in the sign~lin~ tr~n~ lction m~ A~ .. of GDNF is an area of further interest.
Interesting dirr~ ces were found between the patterns of ERK
phosphorylation in~111cerl by GDNF in raphe nucleus RN33B cells and in motor neuron MN-1 cells. GDNF tre~tm~nt stimnl~t~ very rapid (m~jmllm at 5 min~
and lldl~i~ mtl-otloct~hle after 60 min) tyrosine phosphorylation of ERKI and ERK2 in RN33B cells, but relatively slower (m~im-lm at 15 min) and more s lst~in~ocl (still det~ct~hle after 120 min) phosphorylation of ERK2, but not ERKI, in MN-1 cells. That these dirrerences may have functional significance is suggested by recent obselv~lions made in PC12 cells treated with ~1irr~lellL growth factors.
Exposure of PC12 cells to NGF or fibroblast growth factor (FGF) results in neuronal dirr~lc~llialion and in ~ t~in~l elevation of Ras activity and ERK tyrosine phosphorylation (Qiu and Green, 7 Neuron 977, 1991). In contrast, 1~ e.~l with epi~P-rm~l growth factor, which stimlll~t~S DNA synthesis and prolir~ lion of PC12 cells, results in only transient ( ~ 1 hr) activation of Ras and ERKs (Qiu and Green, 1991). Thus, dirren lll time-courses of ERK activation underlie dirr~lcllL
biological responses in PC12 cells. Taken together, the dirr~lc~ll palLell,s of GDNF
receptors and GDNF-in~ ced ERK phosphorylation in RN33B and MN-1 cells suggest that dirrere~ll GDNF receptor ~ub~llliL~ can cooperate to assemble distinct ~ign~lin~ complexes in dirrt;l~;llL cell types. Whether dirLl~lll GDNF signal tr~ns(l~ction paLLv~ays lln(lerlie the dirrc~l~"l biological effects of GDNF is an area of fur~er interest.
Upon activation, ERKs translocate to the nucleus where they phosphorylate and thereby regulate the activity of l~ sc~i~lion factors which, in turn, control gene ex~les~ion. Phosphorylation of p67SRF and p62TCF l~a,lsc~ ion factors recruits them to the serum response element (SRE) in the c-fos gene promoter andstimlll~trs c-fos gene l~ scl;plion (Gille et al., 358 Nature 414, 1992).
Tl~ns~ "ion of c-fos is rapidly and llansienlly inr1llred after various stimllli, inrlll-ling exposure of PC12 cells to NGF (Millbrandt, 83 PNAS USA 4789, 1986) and of osteoblastic cells to TGF-,~ (Machwate et al., 9 Mol.Endocnn. 187, 1995).c-fos forms part of the AP~ scli~lion factor complex, which is thought to be involved in the regulation of multiple genes, including growth factor, n~urope~ide and ~l~urol~ er synth~si~ing enzyme genes (Gizang-Ginsberg and Ziff, 4 Genes Dev. 477, 1990); Hengerer et al., 87 PNAS USA 3899, 1990; Jalava and Mai, 9 Oncogene 2369, 1994). The stim~ tion of c-fos ll~nscli~lion by GDNF in~1ir?trs a role for AP-l complexes in GDNF-in~ ced gene expression.
Thus, c-fos could mediate the increase in the tyrosine hydroxylase (TH) expression observed upon GDNF tre~tn ~ont of central noradrenergic neurons, or the GDNF-intl~lced upregulation of vasoactive illl~~ peptide (VIP) and ~ rol~chykinin-A
(PPTA) mRNAs in cultured ~y...p~l.Ptic ll~uiolls from the superior cervical W O 97/18240 PCT~US96/18197 ganglion (Trupp et al., 130 J. Cell Biol. 137, 1995).
The effects of GDNF on the survival of dirre~ t~d scl.~lullelgic raphe n 7~ s cells in~1ir.~t~ that the G~NF receptors i-l~ntifi-o(l on these cells are able to elicit relevant biological responses. The fact that cessation of proliferation, S dirr~ ;;.lion, and GDNF respo,~.ivclle~ were cOllc~.. llil;.. l with hl~ ,ased GDNF
receptor e~çcssion in these cells, suggests tnat GDNF may be a sunival factor for developing serotonergic raphe ll~u~olls in vivo. The data of this patent disclosure suggest a role for ERKs and c-fos in GDNF-Ill~ rd neuron survival. This can be directly established using (~-~min~nt negatives or ~nti~e~e oligonucleotides.
The GDNF receptor subunits and complexes disclosed herein have wide-range applicability. The i~lentifi~ti~n and isolation of GDNF receptor facilitates rational drug design for drugs useful in treating, for example, nculullal disorders, particularly those involving neulollal cell death. As was ~ c -~sed previously, GDNF has been shown to prol-loLe survival of adult ~ub~.L~nlia nigra neurons in vivo following ph~rm~rological l~ and lesions that mimic Parkinsonian syndromes, as well as sunival ,c~ollses in other llcul~ al cell lines. The drugscan be tested for billlill~, affinity to gdnf receptor, and for their inflllçn~e on the dowl~Llcam effect of GDNF disclosed below -- i.e., the phosphorylation of ERK2 and ERKI. As GDNF ,~cc~Lor has also been idçntifi~od on m~lip;n~nt cell lines, design of drugs for use in cancer therapy is also evident. Further, considering structural similarity with BMP, the development of drugs to be used in treating bone-related ~ çs, i.e., osteoporosis, and for promoting the healing of fractures is also contemplated.
Acco,~ gly, isolated lecc~Lo,~ according to the present invention can be 2~ used, inter alia, to screen for compounds or compositions which are analogs and homologs of GDNF. The potential analogs and homologs can be screened initially in colll~cliLivc binding assays employing either isolated receptor or cell linescx.l~rcSSlllg the receptor -- i.e., NB2/a cells -- and l2~I-labeled GDNF. Methods such as those disclosed in Example 13 can be used. Analog or homolog activity can then be ascertained by further idcllLiryhlg those compounds or compositions which, for example, effect a decrease or hl~lcas~, respectively, in the tyrosine phosphorylation of the RET proto-oncogene. Methods such as those disclosed in W O 97/18240 PCT~US96/18197 Example 17 can be used. .Al~ liv~;ly, GDNF can be used to screen for and identify other receptors using the above~ ellce procedures, or variations thereof.
The isolation of GDNF receptor also facilitates the development of antibodies, both polyclonal and monoclonal, against the receptor. These antibodies S can be used to purify the receptors themselves, identify other cells ~ e;,~hlg GDNr;,~c~lor, lhc~y ~rul"~)t,l,g other thel~eulic applications, identify other Type I-hlt~,la;liYe receptors, as well as be used as drugs themselves. The antibodies can initially be produced using the ligand/l~c~Lol complexes disclosed herein as the il~ lllngens. Antibodies specific for the ligand can be eli--,in~
from the polyclonal serum by absorption with the ligand. Hybridomas for monoclonal production can be se~ectf~l on the basis of binding of ligand, with the expansion of only those clones which do not bind the ligand uncomplexed withthe receptor. The antibodies can be plc~aled by m.othof1s well known to those skilled in the art.
~ IIAI;V~IY~ monoclonal and polyclonal antibodies against GDNF
receptor and GDNF protGills can be used for the chara~;lel.,dlion and/or isolation of GDNF receptor molecular clones. Further, anti-GDNF antibodies can potentially be used in a screen for homologs, or in the production of anti-idiotype antibodies which mimic GDNF.
The isolation of GDNF receptor also facilitates the isolation and/or production of nucleic acids for the ~ Gssion of recombinant GDNF receptol-, both in vitro and in vivo, for ~ n~sti~ and Lheldl,culic applications. The term "nucleic acids" as used herein includes, for example, genomic DNA, mRNA, and cDNA. Upon sequencing at least a portion of the GDNF IGC~IO1, oligonucleotide l?lhllc;l~
for isolating genomic DNA for GDNF receptor and ,ccepLor mRNA can be developed.
cDNA can be pl~Jal~d from isolated mRNA. The isolation and production of nucleic acids can be accomplished utili7in~ methods well known to those skilled in the art using standard molecular biology t~r~ es such as areset forth in Maniatis et al., Molecular Clonin~: A Laboratory Manual, Cold Spring W O 97/18240 PCTAUS96tl8197 Harbor Laboratory, Cold Spring Harbor, New York, 1982, incorporated herein by ler~lellce. RecombhldllLly produced receptors can be used in crystallography studies for rational drug design. Recombinant extracellular domain can be produced and used as a drug in ligand sink applications, e.g., for ligands with antagonistic properties.
The nucleic acids as set forth above can be utilized for gene therapy, using both in vivo and ex vivo to-chni-lnes. The nucleic acids can also be used to clone other related receptors using, for example, low ~Llillgellcy screens and reversed scli~se PCR; and to produce cells over~ ,s~ g the receptors to screen for other ligands, e.g., by panning, and other m~tDri~ serving as receptor agonists,antagonists, or partial agonists and antagonists. .All~ vc;ly, r~colllbillallLlyproduced receptor itself can be used for the sclee~illg assays. Additionally, cells ~res~illg chimeric lcc~Lol~ can be produced using other TGF-~ receptor family members to elucidate signal pathways. Intr~eell~ r targets of GDNF receptor can be i(1entifi~l using, for example, the yeast two-hybrid system. (Chen, et al., 377 Nature 548, 1995, incorporated herein by rererell~e.) The nucleic acids set forth above can also be used to develop lldnsgel~ic and/or gene targeted ~nim~ . For example, transgenic anim~l~ can be developed for testing the effects of the o~ ssion of GDNF l~c~ur. Procedures can be utilized such as are described in Hogan et al., Manipulatin~ ~e Mouse Emblvo: A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986, and Capecchi, M.R., Trends Genet, 5: 70-76, 1989, both incorporated herein by lerel~.lce.
Alternatively, cell lines and transgenic ~nim~l~ unable to express GDNFreceptor can be plepaled to ascertain the effects of blocking sign~ling by GDNF. Procedures such as are set forth in Wurst et al., Gene Targeting Vol. 126,edited by A. L. Joyner, IRL Press, Oxford Univ~l~iLy Press, Oxford, Fngl~n~l, pp.
33-61, 1993, incorporated herein by reference, can be ~ltili7PCl Other applications and mo~ifi~ tions are within the spirit and scope of the invention as herein disclosed and will be readily ~alenL to those skilled in the art.

EXAMPLES

W O 97/18240 PCT~US96/18197 The following Examples are provided for purposes of elucidation and not limitation on the disclosure or claims.
Unless otherwise in-liç~l~cl, binding and biocll~mir~~l studies were carried out with recombinant rat GDNF produced in Sf~l insect cells using a baculovirus e~ ession system. The protein was produced and purified as previously described (Trupp et al., supra, incorporated herein by l~rele,lce). GDNF protein was ri~ after silver st~ining of SDSIPAGE gels using standard curves obtained with comlnercial samples of ~rv~ ls of molecular weight similar to that of GDNF.Purified human TGF-,~l was generously provided by ~un-ichi Koumegawa, Kirin ~ew~ly, Tokyo, Japan. Proteins were labeled with Na-l2sI by the chlGldlllille-T
method to a specific activity of approximately I x 108 cpm/~4g.
Unless otherwise in-~ir~tecl, bill(lil,g assays were l)elfoll,led as follows.
Cells were inr3lh~tP-l with ioflin~te~l GDNF in Dulbecco's phos~haLt;l,urrt:,Gd saline and 2 mg/ml bovine serum albumin (BSA) on Millipore Hydrophilic Durapore 96-well filtration plates. Following two hours of vigorous .ch~king at 4~C, ~e cells were washed twice with ice-cold binding buffer under ~d~;UUIll. Dried filters were liberated and bound l2sI-GDNF qn~ntifice~ in a gamma counter. Non-specific binding was d~LG"~ ed by addition of 500-fold excess of cold ligand to the binding llli~ules.
For affinity labeling, io-lin~te~ OLGillS were bound to monolayer cultures of ~linldly ll~:ulvns or cell lines. Prior to binding, dissociated chick sympatll~tic neurons were cultured for 48 hours in the presence of NGF on polyoi .~ilhi~ minin coated dishes. Plated cells were ;~ tl with 10 ng/ml l2sI-GDNF at 4~C in binding buffer as described above. Ligand/receptor complexes were ch~mir~lly cross-linked for thirty Illilll~lrs at room LGlll~JC~ Ult~ using either disuccinimidyl suberate (DSS) or l-E~yl-3(-3-dimethylamino~ro~yl)-carbodiimide hydrochloride (EDAC) (Pierce Ch~mi~l, Rockl~n~T~ IL). Following q~ nrhin~ of the cross-linking reactions, cells were washed twice with lOmM Tris/HCI
bur~L~d saline, 2 mM EDTA, 10% glycerol, 1% NP40, 1% Triton x-100, 10 ,ug/ml IGU~ 0 ,ug/ml ~ ., 50 ug/ml aprotinin, 100 ~g/ml be.. ~;.. ,.i-lin~?
hydrochloride, 10 ~bg/ml ~epsl;.li" and 1 mM PMSF(~roL~i~lase inhibitors from Sigma). Cleared Iysates were boiled for S min in SDS/~-lnel~ia~loethanol buffer, W O 97/18240 PCT~US96/18197 fractionated by SDS/PAGE on 4-20% ~r~(lient electrophoresis gels, and vi~ li7~ cl by autoradiography. Molecular weights in~ t~(1 were obtained by subtracting the weight of a GDNF ligand monoll-el, e.g., 25-30 kD, more preferably 23 kD, from the e~ lerl molecular weights of cross-linked complexes vi~ li7.o~1 by SDS/PAGE. For affinity lllcasu~ llGllL~, of cross-linked complexes, cells were int~ tP~ on plates as above in the presence of increasing amounts of unlabeled GDNF. These samples were fr~ction~tec~ by gradient SDS/PAGE, gels were then dried and specific bands excised according to molecular weights ~lel~ Pc~ from autoradiograms, and count in a gamma counter. For ~ yl~cipitation of affinity labeled l~,C~ylOl complexes, after binding and cross-linking with iodinated ligands, cell lysates were cleared and in~llh~t~cl overnight at 4~C with 5-10 ,ul of antipeptide rabbit antisera against dirrG,._nce type I, 11 and III TGF-,B ~,uyelr~ ily 1GCG~ (ten Dijke et al., 264 Sci~n~e 101, 1994) (provided by Peter ten Dijke, Ludwig Tn~tihlt~ for Cancer Research, Uppsala, Sweden). Tmmllnr~complexes were collected with Protein A-Sepharose (Ph~ ia, Sweden), washed in lysis buffer and boiled for 5 ,,,i...~les before SDS/PAGE and autoradiography as above.

Example 1 20 GDNF ~c~ on ~"~ nic ~y~ neurons GDNF promotes survival of cultured embryonic chicken ~,y~p~ lir 11GU1V11S with similar efficacy and dose response curve as nerve growth factor (NGF) (Trupp et al., supra). Chicken symp~th~otic nGU~ IS~ isolated and preparedas previously described (Trupp et al., supra). Saturation binding with iodinated2~ GDNF was carried out on 11GUrO1IS isolated from embryonic day 10 (ElO) chick yard~vcl~Gbral ~ylllp~ Ptic ganglia m~ch~nir~lly dissociated in the presence of trypsin. The yltp~ on was preplated for two hours on ullllGdlGd tissue culture plastic in order to enrich in nGU1'~11S and allow for re e2~lGs~ion of receptors. Plots of saturation binding data produced a sigmoidal curve from which a Kd of 400 pM
could be appro~im~t~-1 (Fig. la). In agreement with the sigmoidal behavior of this curve, Scatchard l,al~rol,llation of the data produced an inverted U-shaped curve indicative of cooperative binding (Fig. lb). The measure of cooperativity of W O 97tl8240 PCTAJS96/18197 billdillg can be ascertained from a Hill transformation which produced a positive slope of 1.63 (Fig. 1c), suggeslillg oligomerization of either ligand or receptor ~Ullul~i~ .
In order to identify GDNF binding components on the membrane of S ~Y~ n~ t;C 11GUI'O11S~ c.h~mir~l cross-linking of l2sI-GDNF to these cells was ili7e~1 followed by vi~ li7~tion of the reslllting complexes by SDS-PAGE.
Gradient gel electrophoresis resolved binding ~r~ s of 70 and 300kD (molecular weights of GDNF receptor ~ '~oll~d l}~arL~l were obtained by subtracting the weight of a GDNF ligand monomer, e.g., 23kD, from the ~o-stim~t~d molecular weights of cross-linked complexes vi~ li7~o.1 by SDS/PAGE), resllltin~ in a pattern of bands which }esembled that obtained after cross-linlcing of TGF-,~l to MvILu mink lung epithelial cells (Fig.2). This result suggested that, like TGF-,B receptors, GDNF binding ~loLt;ills may also form an oligomeric lec~lor system. A large excess of cold ligand displaced iodinated GDNF from the l~c~ ur complex inrlje~ting the specificity of the labeling.

FY~mp'~ 2 GDNF le~t~ s on cell lines Over thirty cell lines were screened for e~ ,ion of GDNF receptors using affinity labeling with iodin~t~d GDNF (Table I, zn~a). Except as otherwise noted, all cell lines used in this study are available from and described by theAlllclicdn Type Culture Coll~cti~-n, Rockville, MD. A875 human neuroblastoma was provided by Mart Saarma, Unive~iLy of Helsinki, Finland. CATH.A, a noradrenergic cell line isolated from a tumor in the pons of l~ gel~ic mice expressing SV40 T antigen under the ll~nsc~ iollal control of a tyrosine hydroxylase promoter (Suri et al., 1993), was g~ 1 and provided by Dona Chik~ldishi, Tufts UniveLsily School of Medicine, Boston, MA. The rat neural stem cell line C17-2 (Snyder et al., 68 Cell 33, 1992) was genel~ted and provided by Evan Snyder, HarvardMedical School, Boston, MA. LAN5 human neuroblastoma was provided by Sven P~hlm~n, Uppsala Uni~c~ily, Sweden. David ~mmond, University of Chicago, produced and provided SN6 cells, a hybrid of mouse basal forebrain cholinergic neurons and the mouse neuroblastoma N18TG2 W O 97/18240 PCT~S96/18197 (~mm~nd et al., 1986). Human neuroblastoma SY5Y was a provided by David Kaplan, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD. STlSA rat neural stem cell line was kindly provided by Ron McKay, National Tn~tihlted of Health, MD. The gen~aLion and S chara~L.,~ Lion of raphe nucleus cell lines RN33B and RN46A has been describedelsewhere (Whittemore and White, 1993). RN33B and RN46A cells were obtained from Dr. Scott Whillr....)lc of the Univt; ~ily of Miami. The motor neuron hybrid cell line 2FI.10.14 (referred to here as MN-1) has been previously described (Salazar-Grueso et al., 2 Neuroreport 505, 1991).
Multiple GDNF ~cceplol subunits were r1~t~cte~l in various glial, lleulunal and non-llcul~,llal cells (Table I). A large molecular weight band of 300hD
appeared to be the most prevalent species in several cell lines after cro~linking with disuccymidyl suberate (DSS), and it was the only l~c~Lor which appeared to bind ligand in the absence of all other receptors (Table I). A similar pattern was seen in rat C6 glioma, mouse Sertoli TM4 cells, and in two cell lines derived from embryonic llcu~ lal precursors of the rat raphe nucleus, which have previously been shown to express multiple neuronal lllalhcl~, including gll~t~m~tf~- (RN33B) and serotonin- (RN46A) ,yl~lh.os;~ g enzymes (Whittemore and White, 615 Brain Res. 27, 1993; White et al., 14 J. Neuro., 1994; Eaton et al., 170 Dev. Biol.
169,1995). The col~,el~us pattern in these cells after cross-linking with DSS
consisted of the large molecular weight band of 300kD, and two other receptor subunits with molecular weights at 50-55kD and 65-70kD, respectively (Fig. 3 andTable I). The 50-SSkD c~lllpollt,lL often ran as a doublet or triplet. The smeary appeala,lce and heterogeneous range of sizes displayed by the large molecular weight component suggests a post-translational mo-lific~tion, pl~ulllably glycosylation, and appears similar to that previously described for type III
bet~glycan TGF-,B receptors. This species was somewhat smaller in the cells derived from the raphe nucleus, which could in-1ir~t~ either a distinct core protein - or dirrclcllce levels of glycosylation.
GDNF receptors could not be rl~tecte~l in pheochromocytoma PC12 cells under the present assay conditions of 4~C, even after NGF-in~ re~l dirreLcntiation into a symp~thPtic neuron-like phenotype (Table I, and data not shown). No or W O 97/18240 PCT~US96/18197 very low GDNF lec~Lor expression could be seen in various neuroblastomas, and in two pluripotent neuronal stem cells (Table I).
-W O 97/18240 PCT~US96/18197 TAiBL~ I
CELL Ln~E DESCRUPTION 55 ~ 70k1~ 135kl~ 15SkD 300 5 A875 human melanoma _ _ _ _ +
Balb.SF M E mouse embryonic cell CATH.a rat pons noradrenergic MvlLu mink lung epithelia cell COs-7 monkey kidney r~ bla:lL
10 C2-C12 mouse myoblast C~; rat glioma + + + +
C17-2 rat CNS stem cell FR-3T3 rat r~ ,l;ld,L
HELA human cervical c~. ~ill.,ll.a LAN5 human neuroblastoma _ _ _ _ +
L6 rat myoblast _ _ + _ +
MN-l mouse motor neuron _ + _ + +
NB41A3 TH+ mouse _ _ _ _ _ neurob}astoma NRK-49F rat kidney rll,lublai,l _ _ _ _ _ PC12 rat pheochromocytoma Pl9 mouse embryo C~ ,illuma RN33B rat raphe nucleus + + + _ +
(glutamat) RN46A rat raphe nucleus + + + _ +
(seroton) ~ SK-N-M C human n~w~c~ elioma 25 SK-N-SH DRH+ mouse _ + _ _ +
rl~ ul)~ c m ~
SN6 mouse basal forebrain (cholin) STlSA rat CNS stem cell _ _ _ _ +
S W1353 human cl~o~ s~-,u.,-a SY5Y human neuroblastoma T M3 mouse Leydig cell T M4 mouse Sertoli cell + + + _ +
U138M G human glioblastoma Presence (+) or absence (-) of specific GDNF receptor complexes in the ~leeign~tt-cl cell line.
Affinity labeling using the cross-linker ethyl-dimethyl-aminopropyl carbo-liimi~le (EDAC) revealed the presence of an additional GDNF receptor c~ ol~llL of 120-135kD (Fig. 3), only seen after very long exposure of gels in DSS cross-linked complexes. Like DSS, EDAC also cross-linked GDNF to wo 9711824Q PCTAUS96/18197 receptors of SO-SSkD and 65-70kD; the high molecular weight subunit of 300kD
was, however, not as efficiently cross-linked by EDAC~ (Fig.3).
The raphe m~C!~3ls cell lines are only conditionally immortalized and do not show signs of kansfonn~tion At the non-~ is~ive temperature and in defined S mP~ m, they stop proliferating and dirr~lcllliat~ into po~ ni~olic neurons ~Vhitternore and White, 1993). GDNF binding was greatly increased in RN33B
and RN46A cells following dirr~ lion (not shown). The overall pattern and the relative amounts of GDNF recepLol components did not change after diLrt;le.lliation.
Analysis of GDNF binding prolei~s on the rat myoblast cell line L6 revealed a dirr~ t pattern of receptor subunits m~rkPrl by the a~alGlll absence of 50-SSkD and 65-70kD leceplols. Only the high molecular weight component of 200-400kD could be seen after cross-linking with DSS (Fig. 3). Cros~linking withEDAC, however, readily labeled the 120-135kD subunit previously seen in C6, TM4 and raphe nucleus cell lines (Fig. 3). As in these other cell lines, this component also run as doublet in L6 myoblasts.
A distinct receptor complex was found on an embryonic mouse spinal cord motor neuron hybrid cell (Fig. 3). This line was obtained by fusion of E14 mousespinal cord motor neurons and the N18TG2 mouse neuroblastoma, followed by selection of clones t;~res~illg high levels of choline acetyllldll~r~ldse activity (Salazar-Grueso et al., 2 Neuroreport 505, 1991). Importantly, SN6, a hybrid cell line of embryonic mouse basal fo~ldin cholinergic ~Ul~llS and t'ne same N18TG2 neuroblastoma (~mmond et al., 234 Science 1237, 1986), showed no GDNF
receptors (Table I), intlir~ting that the GDNF binding ~ tt;ills seen on the motor neuron cell (hereafter referred to as MN-l) are likely to represent GDNF receptor components present in spinal motor neurons. As with the L6 myoblasts, the pred(3mil~l" receptor in MN-l cells was pr~rer~lllially cross-linked with EDAC, although in these cells it was a larger protein of 155kD (Fig. 3). This was subsequently i~1~ntifie-1 to be a c-RET receptor (see Example 9 below). MN-l cells also expressed 65-70kD binding proleills and low amounts of the 300kD
receptor (Fig. 3 and Table 1).
In order to dissect the individual constituent ~ffiniti~os of GDNF receptor subunits, displacement binding assays were performed, followed by cross-linking and SDS-PAGE. Receptor-ligand complexes were viell~li7P(l by autoradiography, cut out from the gel and counted in a gamma counter. The restllting displacementcurves inf~ tPtl a Kd of approximately 0.2 nM for all components on RN33B and MN-1 cells (Fig. 4 a-c). These data at present do not clearly establish whether all GDNF l~,c~lor ~u~ s display similar binding ~ffinitiPs or, whether they are all required to assemble a high affinity receptor complex.

Example 3 Bio~~ ~~' characlt;~lion of GDNF lecep The overall .~imil~rity in the pattern of receptors beLw~ell GDNF and TGF-~
prclllpl~d an Px~min~ti-)n of whether any of the previously identified receptors for TGF-~B ~.u~elr~llily members was part of the GDNF receptor complex. Cross-linked l25I-GDNF-receptor complexes from dirr~ 1 RN33B cells were subjected to immllnoprecipiLaLion with different anti-peptide ~nti~Pr~ specific for all cloned TGF-f3 ~.upelralllily receptors, including type I receptors (ALK-l to ALK-6), type II receptors TBRII, ActRlI and BMPRII, the type III receptors betaglycan, and endoglin. In a parallel control experiment, l25I-TGF-,~l was cross-linked totype I, type II and type III receptors on the mink lung epithelial cell line MvILu followed by imml-nnpleci~iL~Lion with antisera against TBRI (ALK-5), TBRII and betaglycan, respectively. Altnough type I, type II and type III TGF-,B receptorswere recovered in the control ~ l-elll, none of the GDNF receptor components in differ~ r~l RN33B cells could be immunoprecipitated by any of the tested antisera (not shown). These data confirmP-~l that the ~DNF receptor subunits ~lcssed on these cells are novel ~lvl~hls.

Example 4 Endogenous GDNF ~I,.e~ion in cell lines e2~l-;e~ g GDNF reee,L3~o~
Traditional models for the action of n~uloLlo~hic factors have described them as target-derived polypeptides that promote survival and dirÇelellLiation of specific neuronal subpopulations. More recently, it has become evident that n~uloLlu~hic factors may also have paracrine and even autocrine modes of action W O 97/18240 PCTrUS96/18197 (Ernfors and Persson, 3 Eur. J. Neurosci. 953, 1991; Acheson et al., 374 Nature 450, 1995). ~xpression of GDNF mRNA in cell lines ~ ,ssillg GDNF receptors was ex~minp~l Cells were homogenized in gl~nir1inP isothiocyanate (GITC) and ~B-mercaptoethanol. RNA extraction and GDNF RNAse protection assay were as 5 previously described (Trupp et al., supra).
Unexpectedly, all cell lines, with the exception of the motor neuron line MN-l, e~lessed sub~ lial levels of GDNF mRNA as assayed by RNAse protection analysis (Fig. 5). The highest GDNF mRNA eA~l~s~ion was found in cells from raphe nucleus, which showed up to 5-fold higher c;~lc,,sion ~an 10 postnatal day 1 (Pl) kidney, one of the richest sources of GDNF mRNA in the developing rat (Trupp et al., supra). I~ gly, upon dirreLe~ ion of RN33B
cells, GDNF mRNA ~ ,s~ion decreased to about 30% of the level in undiffele..~;~t~i cells (Figure 5). GDNF tre~tmPnf of dirr~ (l RN33B cells did not alter the expression of GDNF mRNA (Fig. 5) or GDNF receptors (not shown).
Expression of c-ret mRNA was investig~t~(l in RN33B, L6, and MN-1 cells, using the RNAse protection assay. Ten micrograms of total RNA from the cell lines in~lir~tPd wasa analyzed using a riboprobe complementary to 400 nucleotides of coding sequence from the kinase domain of the mouse c-ret mRNA.
Although high e~lJression was seen in MN-1 cells, no c-ret mRNA was tlPt~ctpfl in either the RN33B or L6 cells (Fig. 6). These results infiir~t~ that a .cign~lingreceptor for GDNF other than c-RET must be present in these cells.

Example S
Activation of the ERK signal l.~ ct~ .dy in GDNF responsive cell lines Whether the GDNF binding proteins characterized in cell lines were able to form ligand-dependent .5ign~1ing complexes was also investi~tPc~ Cell monolayersin 10 cm plates were inrub~tet1 at 37~C in the presence of 50 ng/ml GDNF for thein-iir~tP(l time periods and imme~ tPly lysed with 1 ml of ice cold Iysis buffer (as above) with the addition of 1 rnM sodium othovanadate. Whole cell lysates were fractionated by SDS-PAGE (10% polyacrylamide) and blotted to nitrocellulose CA 02237641 1998-0~-12 wo 97/l8240 PCT/USg6/18197 filters. Western blots were probed with an anti-phosphoLylbsi.le antiserum (UBI,Lake Placed, NY), followed by horseradish peroxidase-conjugated goat anti-mouse IgG and developed with the ECL Western Detection System (~m~r.ch~m, UK). For reprobing, blots were first stripped by a 30 minute incubation at 50~C in 62.5 mM
Tris-HCI pH6.7, lOO mM ,B-mercaptoethanol, 2% sodium dodecyl slllph~t~. After removal of antibodies, blots were probed with a rabbit polyclonal antisera raised against recombinant rat ERK2 (a gift of Teri Boulton, Rege,~lc,.l Ph~rm~ce~ltir~1.c Inc., Tarrytown, NY) which recognizes both ERKI and ERK2, and developed as above using a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody.
Because of their distinct patterns of GDNF receptor subul~ , intracellular ~ign~lin~ responses were initially char~rt~-ri7e~1 in the raphe nucleus cell line RN33B and in the motor neuron cell line MN-l. Changes in the pattern of tyrosine-phosphorylated ~lO~illS elicited by GDNF tre~tm-ont of RN33B or MN-l cells were invçsf;g~tçcl Tyrosine phophorylation is a ~ ,al m~ch~ni~cm of regulation of intr~r~ r si~n~ling plvteins that is stim~ ted by numerous cytokines and growth factors. RN33B and MN-l monolayers were exposed to a ~hlr~tin~ conce"L,dlion of GDNF (5 ng/ml) for dirr~-el,~ periods of tine, and total cell lysates were analysed for tyrosine phosphorylation by SDS/PAGE and Western blotting with an anti-phosphotyrosine monoclonal antibody. Two ~ Le;hls with mobilities corresponding to 42kD and 44kD, respectively, were phosphorylated on tyrosine within 5 minutes of GDNF tre~ nt of RN33B cells (Fig. 7A). A similar result was obtained in dirÇel~-lLialed RN33B cells (not shown) of exposure to GDNF.
Based on comparison of their size with descriptions of growth factor-in~ cecl protein tyrosine phosphorylation el~wl~r~ (Qiu and Green, 9 Neuron 705,1992), the 42kD and 44kD species would appear to be, respectively, p42 ,k2 and p44 ,kI, two protein serine-threonine kinases members of the extracellular signal-regulated kinase (ERK, also termed microtubule-associated protein kinase) family(Boulton et al., 65 Cell 663, l99l). To confirm the identity of these protei- s as ERK2 and ERKI, respectively, protein blots which had been reacted with the anti-phosphotyrosine antibody were stripped and reprobed with a rabbit polyclonal W O 97/18240 PCT~US96/18197 antibody raised against recombinant ERK2 that recognizes both ERKI and ERK2 in protein blots. Comparison of autoradiograms of blots probed with the anti-phosphotyrosine antibody and the anti-ERK2 antibody i(1~ntifie-1 the p42 and p44proteil s as ERK2 and ERKI, respectively (Fig. 7a). Although GDNF tre~tm~nt of MN-l cells a~eal.,d to only stim~ te phosphorylation of ERK2, both ERKI and ERK2 were present in MN-l cell lysates (Fig. 7b). Thus, GDNF ~o~tmPnt stim~ t~ very rapid and transient tyrosine pho~l~l ylation of ERKI and ERK2 in RN33B cells, but relatively slower and more s~lst~in~l phosphorylation of ERK2 and MN-l cells.
Activation of the ERK pathway has previously been shown to induce rapid and transient increase in L~ sclil,lion of imme~ te early genes, inrlll-7in~ the c-fos protooncogene (Gille et al., 358 Nature 414, 1992). Accor~ ,ly, the ability of GDNF to induce c-fos mRNA in dirrelc.~ raphe nucleus RN33B cells and in motor neuron MN-l cells was investig~t~l. For analysis of c-fos mRNA
e~ ssion in cell lines, culture mf~(lillm was r.h~nge-l 90 mimltes prior to addition of lO0 ng/ml GDNF to cell monolayers. At the in-lir.slt~-l time intervals, media was removed, cells solubilized with gll~ni-linf~ isothiocyanate and ~-mercaptoethanol and RNA extracted as previously described (Trupp et al., supra). Twenty micrograms of total RNA was fractionated on 1% agarose gels co..l;~;..i.~g 0.7% form~l-le~yde and ll~ ,r~ d to Hybond-C membranes (Amersham, UK). Northern blots were hybridized with an a-32P-dCTP labeled rat c-fos gene fragment (Curran et al., 2 Oncogene 79, 1987), washed at high stringency and vi~ li7~1 by autoradiography on x-ray films.
Cell monolayers were exposed to salulaLillg concentrations of GDNF for dirrelellL periods of time and levels of c-fos mRNA were subsequently analyzed in Northern blots of total RNA (Fig. 8). This analysis revealed transient upregulation of c-fos mRNA 15 mimlt(?s after exposure of RN33B cells to GDNF, LeLulllillg to basal levels 45 mimlt~s after tr~trn~nt (Fig. 8a). c-fos mRNA was also upregulated in MN-l cells but not until 30 Il.ill..l~s of GDN~ LleaLlllellL (Fig. 8b). Elevated c-fos mRNA levels p~ ed for about an hour and returned to basal levels 120 ~-.i...~les after the initiation of treatment (Fig. 8b). Thus, like tyrosine phosphorylation of ERKS, c-fos mRNA upregulation in~ cecl by GDN~ treatment was very rapid and Llallsie.lL in RN33B cells, but sc,m~wllaL slower in MN-1 cells.

5 F,Y~nrl~ 6 Surviva~ responses p~ l by GDNF in differentiated raphe nucleus cells Advantage was taken of the conditional nature of the irnmortalization of the raphe nucleus sel~ gic cell line RN46A by eX~mining whether GDNF
10 may be a survival factor for difr~r~ cl raphe nucleus neurons. Survival assays were performed as previously described (Eaton et al., 1995, supra). Briefly, 105RN cells were seeded to collagen/fibronectin coated 8-well glass slides and 1 at 33~C (growth ~clll~issive temperature) until 75-90% confl~ nt The slides were then shifted to 39~ (non-pellllissiv~ t~nl~ Lulc) and serum cont~ining mto~ m was replaced by B16 defined merlillm (Brewer and Cotrnan, 494 Brain Res. 65,1989) cont~ining 1% BSA, 1 ,ug/ml tldl~r~,l.ill, 5 ,ug/ml insulin, 100 mM
putrescine, and nM progesl~ e plus or minus 0-50 ng/ml rhGDNF
(Promega, Madison, WI). Media and GDNF were replaced every two days for 8 days after which the cells were fixed in 4% paraform~l~lehydet2%
glutaraldehyde, rinsed and coated with a glycerol mounting medium co~ i"g 1 mM bisben~:~mi~ (Hoechst dye 33342) to stain viable nuclei. Fields of cells werem~gnifi~l to 40x on a Zeiss Axiophot microscope, ~ i for fluorescen~ nuclei (at 355 nM exitation, 465 nM emission), the images video captured, and the cells counted with Imade In' software. For each condition, 10 fields of cells were counted from each of 3 independent exp~"nellL~.
RN46A cells were cultured at the non-permissive temperature in defined mP~ m in the pl~sellce of increasing concentrations of GDNF. Nine days after plating, ~ulvivillg cells were counted and compared with cultures established inthe absence of GDNF. A 3-fold increase in the number of surviving cells was observed in cultures grown in the presence of GDNF ~Fig. 9). The effect of GDNF on the survival of dirre~ led RN46A cells was dose dependent, with an EC50 at Sng/ml.

W O 97/18240 PCT~US96/18197, Example 7 Generation, cloning and ch - ~le. ~lion of anti-GDNF monoclonal antibodies Immunisation S Five young female mice were i~ A with 35 ug of insect cell-derived recombinant GDNF em~ ifi~l with complete Freund's adjuvant (FA). Second and third i.. ,.. ~;,;.lions were performed 2 and 4 weeks after ~e ~lrst one in incomplete FA. All the injections were given inlld~c~ eally (i.p.). Two weeks after the last T..~,.---.i~,.~ion, antibody titer in serum was chee~d by ELISA and Western Blotanalysis using ~ ldld mPthoA~. The mouse with the highest titer (more than 1:2000) was boosted i.p. with 3 ~4g of GDNF in incomplete FA 3 days before the cell fusion.
Cell fusion Cell fusion was done according to the method of Kohler and Milstein (1975), incorporated herein by rc;r.,rellce, with some moAifir~tions.
a) Day before fusion:
Viable cells from the Sp2/0 murine cell line were adjusted to 2xlOs cells /
ml with complete DMEM (10% fetal calf serum, 1% L-pl~ o, 100 U/ml penicillin and 100 ug/ ~ omycill sulphate).
Cells from a non~ e~ mouse were obtained from the ~clilolleal cavity by iniection of 0.34M sucrose solution. The cells were resuspended in complete DMEM CO~ : hypo~ lOO,uM; aminopterin 0.4M; and thymidine 16 ,uM, (HAT m~Aillm), to 1xlOs cells /ml. 100 ,ul of the cell suspension was added to the 60 inner wells of 96 well plates and inr~1b~ted overnight at 37~C in an atmosphere of 5% C02 in air. These cells were the source of growth factors.
b) Fusion Spleen cells from the mouse exhibiting the highest serum titer (see above) were homogenized in 10 ml DMEM removing surface fat and other adhering tissue in a sterile hood.
4.2x107 Sp2/0 cells were fused with 8.4 x 107 spleen cells in a solution of melted PEG (3000-3700, Hybri-Max, Sigma). The cells were then grown in HAT
m~ m at 37~ C in an atmosphere of 5% CO2 in air. After one week of culture, the wells were incpect.oc~. When hybrids cells covered 10 to ~0% of the surface area of the well, the culture ~u~e~ were assayed for antibody by ELISA.
For the ELISA, wells of microplates (Costar, EIA/RIA plate high binding) - were coated with 100 ul of 2ug/ ml of GDNF diluted in carbonate/ bicarbonate S buffer, pH 9.6. After an overnight inl~tlb~tion at 4 ~C, ~e wells were washed with 0.05M pho~srh~te ~urr.,l~d saline, pH 7.2, conts~ining 0.05% Tween (PBS-T).
Nonspecific binding was blocked with PBS-T cO~ p 3% non-fatty miLk and 1%
goat normal serum. Su~ samples were incubated 4 hours at room L~ eldLule. Peroxidase goat anti-mouse antibody was used and the ~ub~ Le was 10 o-phenylen~ minP dihydrochloride (OPD). Plates were read at 492 nm in an E~ISA reader. Negative controls included completed mPtlillm and normal mouse serum.
The hybrids were grown in HAT m~riinm up to two weeks after fusion.
Cells were subsequently grown in HT mP-linm until the completion of two cloning 15 procedures, using the limiting dilution method. After each step (when cells reached 10 to 50% confluence), assays for specific antibody in ~u~ li.nt~ were done by ELISA. Upon recloning, 5 positive hybridoma clones were chosen and the cells were m:~int~in.o(l in complete DMEM for 30 days.
Isotyping of monoclonal antibodies The class and subclass of the monoclonal antibodies were (l~l~ .. --il.Pd by FTIsA using a DAKO panel for isoLy~ g of mouse monoclonal antibodies. All five 5 monoclonal antibodies were characterized as IgG,.
Purification of monoclonal antibodies Monoclonal antibodies from culture sup~...,.l~l.l~ were purified by Protein G
Sepharose fast flow (Pharmacia, Biotech) according to m:lmlf~rh-rer's instructions.
Culture ~u~c.llala~ were concentrated and filtered through a 0.45 ~4m membrane (Schleicheer and, Schull, Germany) and then pumped overnight through the column previously equilibrated with 20mM sodium phosphate, pH 7Ø Ig was - eluted with 0.05M glycine buffer.
nrl~ 8 A motor neuron cell line showing biological and b;o~ l responses to W O 97/18240 PCT~US96/18197 GDNF
MN-l cell monolayers were exposed to hlcl~asillg co~lc~llLl~lions of GDNF
in serum-free m~ m and assayed 3 days later for cell survival and growth by measurement of acid phosphatase activity (Clontech). GDNF was produced and S purified from baculovirus infect~d insect cells as previously described (Trupp et al.,supra). GDNF l~ of serum deprived-MN-l monolayers increased cell number in a dose-dependent manner (Fig. lOa). The biological ~ ul~se of MN-l cells correlated with bior-llPmi~l and lldllsc,i~lional lt;bl~ullses to GDNF
tre~tm.ont MN-l cell monolayers were exposed to 50 ng/ml GDNF for increasing periods of time, cell lysates were fractionated by SDS/PAGE and Western blots probed with an anti-phospholyiosille antibody (UBI).
Several ~r~leills were seen to have ill~,~ased tyrosine phosphorylation after GDNF Lr~aLlllelll of MNl cells, including a protein with an electrophoretic mobility of 42K (Fig. lOb). Based on com~L~lison of its size with descriptions of growth factor-in~ ce~l protein tyrosine phosphorylation elsewhere (Boulton, T.G., et al.
Cell 65, 663-75 (1991), the 42K species would appear to be p42er~, a serine-threonine kinase member of the extracellular signal-regulated kinase (ERK) family.
The identity of this protein as EI~K2 was confirm~c~ after immllnoprecipitation with an antiERK2 polyclonal antiserum followed by analysis of tyrosine phosphorylation (Fig. lOc). Lysates of GDNF-stimlll~tpfl MN-l cells were immllnoprecipitated with an anti-ERK2 a,llise,u", (Santa Cruz) that also recognizes ERKI followed by antiphospholylosi,le Western blotting. This analysis further revealed that another member of the ERK family, p44erkl, was also phosphorylated on tyrosine after GDNF tre~tm~nt of MN-l cells (Fig. lOc). Activation of the ERK pathway has previously been shown to induce a rapid and transient increase in the Ll~lscli~Lion of imm.o(li~t~ early genes, including the c-fos proto-oncogene (Gille et al., Nature 358, 414-7 (1992).

Example 9 The product of the c-ret proto-onco~-~ne as a signal ~ cin~ r~ . for GDNF
GDNF receptor complexes from MN-1 cells could be recovered by W O 97/18240 PCTnJS96/18197 "..,l~cpl~ci~i~tion with anti-GDNF antibodies or by binding to lectin-Sepharose beads (Fig. lla). Unexpectedly, the 180kD receptor complex (i.e., c-RET; 180kD
23kD--157kD, which is approximately equal to the 155kD l~,c~tul i(le~ntifi~1 as c-RET -- see Exarnple 2, infra) could also be recovered by immlln~3l~ciyiLaLion S with anti-phosphulyl~.ine antibodies (Fig. lla), in~1ir~ting that the GDNF binding protein in this complex could be a l~c~Lo~ tyrosine kinase.
The product of the c-ret proto-oncogene is highly e~ylcssed in ~lhllal,y motor 11~U1O1~S (Pachnis et al., supra, and Tsuzuki, T., et al. Oncogene 10, 191-8 (1995) and is of similar molecular weight âS the major GDNF eec~Lor component IlPtect~-l in MN-1 cells (T~k~h~hi, M., et al. Oncogene 3, 571-578 (1988). We tested whether this species l'~ylese~L~d a C-RET-GDNF cross-linked complex by immlln..ylcciyila~ion with anti-c-RET antibodies.
GDNF was cross-linked to MN-1 cells using EDAC and receptor complexes were precipitated with antibodies against GDNF (Trupp et al., supraJ, lectin Sepharose beads (Formica), anti-phosphotyrosine antibodies (UBI), anti-c-RET antibodies (Santa Cruz) and control antibodies from non-tmml-nP rabbits. An antipeptide c-RET rabbit a~ltise.ull~ readily i....,.~ cpl~cipitated the major 180kD
ligand-receptor complex in MN-1 cells (Fig. lla), while a number of unrelated monoclonal and polyclonal antibodies used as controls failed to i~ uyl~ciyitate 20 this complex (Fig. 11 a and data not shown).
Because the product of the c-ret gene is a receptor tyrosine kinase, we inves~ig~te-l whether GDNF could stimlll~t~ tyrosine phosphorylation of the c-l~ET
protein in MN-1 cells. MN-1 cell monolayers were exposed to GDNF at different concentrations or for dirr~lcllL periods of time and cell lysates were i.,.. ,ll.. nprecipitated with anti-c-RET antibodies and analyzed by SDS/PAGE and Western blotting with antiphosphotyrosine antibodies as disclosed above. GDNF
treatenent stiml~ rapid c-RET tyrosine phosphorylation in MN-1 cells (Fig.
11b). Maximal phosphorylation was reached 5 ..-i-~ es after GDNF treatment and lasted for at least 60 minutes. A dose-response analysis of GDNF inflelce~ c-RETphosphorylation in MN-1 cells showed m~xim~l phosphorylation at 30 ng/ml of GDNF (Fig. 11b), which is similar to the response of both serum deprived MN-1 cells (Fig. lOa) and embryonic symp~th.otir neurons (Trupp et al., supra~ to W O 97/18240 PCT~US96/18197 GDNF. Taken together, these data in~lir~te that the c-RET receptor may be an important component in the signal tr~n~fh~,ction m~ch~ of GDNF.

Example 10 c-ret transfection reCol-ct;t~lt~, GDNF b~nd~ng and biolo~ activities to GDNF
E~elin~ ,l, were con~ tP~l to ~l~-lr~ ;llP whether e~les~,ion of the c-ret gene product could be sufficient to allow binding of GDNF to cells lacking GDNF
receptors. To this end, GDNF binding and cross-linking e~ le~, were performed in naive 3T3 fibroblasts, and 3T3 cells stably transfected with either a wild type c-ret or an oncogenic form of this gene found in MEN2a pz~ti~nt~
Tigs~n et al., supra~. For c-ret ~r~s~,ion in transfected cells, human wild typec-ret and MEN2a-ret cDNAs were su~cloned in pcDNA3 (I~lviLIogell). Cold GDNF
was used at ~Ox molar excess. For survival/growth assays, cells were cultured for 6 days in serum-free mP~ m supplemented with the im~ tPrl concentrations of GDNF; mPf~ m and GDNF were replaced every two days. Cell number was ua~l~ified by measurementof acid phosph~t~ce activity (Clontech).
After imm~m-precipitation with c-RET antibodies, GDNF-labeled receptor complexes of approximately 180K were riPt~cte~l in both MF.N2a-ret and c-ret transfected 3T3 fibroblasts, but not in ullLlal~7r~cted cells (Fig. 12a). The labeling could be displaced by excess cold GDNF, in~ ting that it represented specific GDNF binding (Fig. 12a).
E~,e~ lenl, were also con-lllctP~1 to clele~ P whether c-ret could mP/ii~tP
a biological response to GDNF upon llall~,recLion in non-responsive cells. Survival and growth responses to GDNF were investig~tP~l in ul-Llal~ected and c-ret Lldl~re~Led 3T3 fibroblasts cultured in serum-free mPrlillm. GDNF elicited a dose-dependent increase in cell number in c-ret transfected, but not in unLIdl~r~;L~d, 3T3 cells (Fig.12b) which was collli)dl~ble to the one previously observed in serum-deprived MN-lcells. Since naive 3T3 cells did not express any appreciable amount of GDNF receptors prior to l-dn~recLion (see Fig. 12a), it was concluded that c-ret expression was sufficient for m~oAi~tin~ a biological response to GDNF in these cells.

~,Y~mrl~ 11 c-ret ~~ .ion in adult brain and do~ ,;c ~ s of the ~ ~sl~Ldia nigra ~ Experirnents were con~ ct~1 to t~ whether the c-ret product may 5 m~ te the nt;u,vL~hic effects of GDNF in the brain by e~mining the ~ cs~ion of c-ret in dir~iellL regions of the rat central nervous system. A rat c-ret riboprobe was ge~ L~d using as template a cDNA fragment obtained by PCR with primers based on seq~le~res U22513 and U22514 (Genbank acces~.ioll llulllb~,ls). High levels of c-ret mRNA were found in MN-l cells and in rat spinal cord (data not shown).
10 High c-ret mRNA ~ ssion was also found in the adult pons, m~-lnll~, locus coeruleus and hypoth~l~mlls (Fig. 13a), as well as in th~l~mllc and cerebellum (data not shown). c-ret mRNA was expressed at barely ~llotpct~le levels in ~Lii~Lulll,hippocampus and cerebral cortex (Fig. 13a). In the ventral m~sçn~ephalon, cnnt~ining the cell bodies of GDNF-responsive dop~,..;..~.~ic neurons, c-ret rnRNA
levels hl;l~,ased proglessively during post-natal development (Fig. 13b). A peak of s~.ion was ~letectecl between post natal day 6 (P6) and P8, at which time axons of dopaminergic ~eulo,hs of the substantia nigra begin innervation of the striatum, and coin~i~lent with an increase in GDNF mRNA t~ ssion in this target region (Fig. 13b). For mRNA qll~ntif~ tion~ a glyceraldehyde-3-P dehydrogenase (GAPDH) riboprobe was included in the RPA, and values of relative mRNA
expression, obtained after de,~ilometric sc~nning of gel autoradiograms, were norrn~liee(l using the GAPDH signal of each RNA sample. RPA for GDNF mRNA
has been previously described (Trupp et al., supra).
In situ hybridisation and immnnohietoc~ mietry were ~ o""ed as previously described (Arenas, E. & Persson, H. Nature 367, 368-371 (1994);
Neveu, I & Arenas, E. J. CeU Biol. in press (1996). c-RET protein was ~letectt--l using a hamster monoclonal anti-mouse c-RET antibody which also recognises rat c-RET (Lo, supra) followed by fluorescein-conjugated rabbit anti-hal~lsLe~
secondary antibodies (Southern Biotechnologies). In situ hybridization on sections through the adult substantia nigra revealed strong labelling over neurons throughout this structure (Figs. 14 a-b). In addition, cells positive for c-RET-like imml1noreactivity (c-RET-LI) were found throughout the adult substantia nigra, W O 97/18240 PCT~US96/18197 with strong labelling over cell bodies (Fig. 14c).
In order to establish that c-ret e~ression in the adult ~ l ;A nigra was confined to dol a,ni~ ,ic neurons, these cells were selectively lesioned with a unilateral injection of 6-hydroxydopa~ e (6-OHDA); the cells were then analyzed S for c-ret m~RNA e~Ll,lession by in situ hybridisation. Lesions of d~l~ gic llc;urOllS of the substantia nigra we~ r ~ ed by stereotaxic injections of 8 pig 6-OHDA in the medial forebrain bundle at the following coor.l;..~l~s: 1.6 mm caudal to bregma, 1.3 mm lateral to mirllinf, and 8.4 mm under the dural surface with the incisor bar 5 mm over the hlL~ldulal line. Animals were ~ e~led with 25 mg/kg de~iplalllille (i.p.) 30 minlltPs prior to 6-OHDA in~ection. 0.75 x 106 GDNF-e~ ssillg fibroblast cells in 3 ,ul of m~ lm were injected supranigrally at the following cooldi"ates: 3,1 mm from illLel~-ulal line, 2 mm lateral to mi(1linP, and 7 mm under the dural surface, with tbe incisor bar at -3.3 mm. Lesion and grafting in the locus coeruleus were as previously described (Arenas et al., Neuron 15, 1465-1473 (1995). The ~en~.~Lioll and char~-lr~ nn of GDNF
~ressiilg fibroblasts have been described previously (Arenas et al., supra).
Five hours after the lesion, no dirr~l~,.lce could be seen between ipsi and contralateral sides in c-ret mRNA ~ ssion (Fig. 14d). However, a marked reduction in c-ret mRNA expression was seen in the lesioned substantia nigra already one day after 6-OHDA treatment, and was nearly absent 5 days after the lesion (Fig. 14d). c-ret mRNA expression in the side contralateral to the lesionwas, however, not affected (Fig. 14d). This result intliC~te~l that in the adultsnbst~nti~ nigra, c-ret mRNA ~ ssion was confined to d(Jp~ ,ic neurons.

Example 12 GDNF re~ s c-RET-positive dop~ ic and norad~ c ~I~.U ~lS
Experiments were cor~ cted to ~leterminf~ whether c-RET e~lessi,lg neurons of the adult substantia nigra and locus coeruleus responded to GDNF. Forthis, nigral dopaminergic neurons lesioned with 6-OHDA, and were then e~min~d to deL~ e whether grafts of GDNF ~ S~illg fibroblasts in~ cerl responses on c-RET imm~lnnreactive neurons. In lesioned ~nim~l~ that received a graft of control fibroblasts, no c-RET-Il could be dett~cted, intli~ting a depletion of c-ret-W O 97/18240 PCTrUS96/18197 expressing cells by selective lesion of d~--li--PJ~ic neurons in the aduit substantia nigra (Fig. 14e). However, c-RET-LI could be rescued by the GDNF-e~ g graft, where c-3~ET i-"----~.-. positive fibers could be seen SUllOU~ lg and penetrating the graft (Fig. 14i~. Similar results were obtained in the locus coeruleus, where lesion with 6-OHDA depleted c-RET-tmmllnnreactive cell bodies (Fig. 14g), which could be rescued by exogenous ~lmini~tr~tion of GDNF (Fig.
14h). In both brain regions, the rescue of c-RET-LI positive cells and ~lou~ g in the ~nim~l~ grafted with GDNF-expressing fibroblasts paralleled that of tyrosinehydroxylase immlmnreactivity (data not shown), demo~ g that c-RET-e~r~ss,llg adult dopaminergic and noradr~ne ~ic ll~UlVlls respond to GDNF.

e 13 T(l~ ;ri~ation of GDNF c-RET receptors PC12 cells and NB2/a cells were washed three times with serum free RPMI-1640 or DMEM, respectively, p}ated on non~o~te-l (NB2/a cells) or collagen-coated (PC12 cells) dishes (5000-6000 cells per dish) in the presence or absence of 50 ng/ml of GDNF (Peprotech EC Ltd.) and the number of cells was microscopically counted after 48 hours. PC12 and NB2/a cells were harvested (100,000 cells, five parallels), incubated with 10 ng/ml human lZ5I-GDNF
(iodinated by Chloramine T method, 100 ,uCi/~g) in the presence or absence of 50-fold unlabeled GDNF for 120-150 min on ice, the unbound factor was removed by ce~ irùgation through 30% sucrose cushion, and the cell-associated radioactivitycounted on 1271 RIAGAMMA counter (LKB Wallac).
Recombinant human GDNF promoted survival of about 20% of serum deprived rat pheochromocytoma PC12 cells at concentration of 50 ng/ml (Fig.
15a). Serum-deprived PC12 cells are also m~int~in~l by nerve growth factor (NGF). Upon tre~tm~nt with (NGF), PCI2 cells also stop dividing and dirr~ iate into ~y~ tic neuron-like cells with long neurites. Thus, GDNF is a survival-- promoting factor for PCI2 cells, although less potent than NGF, but it does not induce dirr~r~ iation of PC12 cells at the concentrations stn~liP~ presumably because of the differences in signal t~n~ ctinn of NGFactivated trkA receptors and GDNF receptors.

Human neuroblastoma NB2/a cells were plated in serum-free mPtlinm in the plt:SellCe or absence of 50 nglml of GDNF and the number of cells was counted after 48 hr of culture. GDNF ~i~nifir~ntly increased the number of NB2/a cells (Fig. l5b). Monkey COS cells, human SY5Y cells and mouse NIH 3T3 cells showed neither mitogenic nor survival response to GDNF (data not shown). Thus, GDNF exerts biological effects on rat PC12 cells and human NB2/a cells, in~ ting that both cell lines express functional GDNF receptors.
To ~etPrmin~, whether GDNF binds to the responsive cells, PC12 cells and NB2/a cells were inrub~t~P(l with ~25I-labeled human GDNF at 40~C as im~ te~l inthe legend to Fig. 15. As shown in Fig. lSc, both PC 12 and NB2/a cell lines bind GDNF 30 efficiently. More impo~ Lly, the binding of '~5I-labeled GDNF
could be competed with a 50-fold excess of unlabeled GDNF (Fig. lSb). Thus, the binding of GDNF to the receptors on PCI2 and NB2/a cells appears to be specific.
Example 14 T~ r~ of GDNF c-RET binding C~
PC12 cells, SYSY neurobl~ctom~ cells and NB2/a cells where chemically cross-linked to '25I-GDNF with EDC. 3-5 x 106 cells or mPçh~ni-~lly dissociated cells from 2 E20 rat kidneys were incubated with 10 ng/ml of l25I-GDNF for I hour on ice and cross linked with 30 mM EDAC (Pierce) for 30 ~ s on ice.
De~ e,.l Iysates were i~ u-~Qplccipitated, the precipiL~Les collectç~l by Protein A-Sepharose, separated on 7% SDS-PAGE, and vi.~ li7Pd by Phosphorimager SI
(Molecular Dynamics).
The resultin~ complexes were imm~lnnprecipitated with rabbit antibodies to GDNF, analyzed by SDS-PAGE and vi~ li7P-fl by autoradiography. Embryonic kidney cells were also studied as the source of putative GDNF receptor (Suvanto,P. et. al., Eur. J. Neurosci., 8, 101-107 (1996); Sainio, K. et. al., Nature, (1996) sllbmitte(l). Cross-linked complexes of 170 and 190 kD were obtained from the exLracL~ of PC12 cells, SY5Y cells and NB2/a cells and a 190 ~ complex from embryonic kidney extracts. (Fig. 16).
The molecular weights of the crosslink~d proteins minus GDNF of approximately 25-30 kDsubstantially, if not exactly, correspond to the molecular weights of c-RET protooncogene, an orphan receptor Iylosille kinase (T~k~h~hi, M., Ritz, J. & Cooper, G. M. Cell, 42, 581-588, 1985; T~k~h~hi, M. et.al., Oncogene, 3, 571-578 (1988)) (140 kD and 160 kD, ~ . 5~511~;11~ dil'r~ y glycosylated forms of c-RET., Tsuzuki, T., T~k~h~hi, M., Asai, N., lwashita, T.,Malbuyalna, M. & Asai, J. Oncogene, 10, 191-198 (1995).

-~np'e 15 Affinity Cross ~,inl~in~ of GDNF to c-RET
The cross linked complexes were ill~ ci~ led from the NB2/a cells with the cocktail of antibodies recognizing extracellular and intracellular part of the c-RET receptor. As shown in Fig. 17a (lane 1), the complexes of 170 kD and 190 kD were plecipilaled by anti-c-RET antibodies, which thus correspond to cross linked GDNFc-RET complexes. Binding of 'Z~I-GDNF to c-RET proteins was completely abolished by 500-fold excess of unlabeled GDNF (lane 2). No proteins were precipitated by monoclonal anti-neurofil~mPnt antibodies (lane 3) or by Protein A-Sepharose only (lane 4). No cross linked complexes were obtained from COS cells (not shown). Since c-ret proto-oncogene is a glycoproLeill~ l25I-labeled NB2/a cell extracts were also immlmoprecipitated with wheat ge~n aggll.~i..;..
Again, ~l~ Lt;ills of 170 and 190 kD were obtained (lane 5).
To establish further that GDNF specifically binds c-RET, the mouse c-ret cDNA was cloned into the m~mm~ n e~ ,s~ion vector PBK-CMV and tr~n~iently expressed in monkey COS cells. Mouse c-ret cDNA (Pachnis, V., Mankoo, B, &
Cost~ntini, F. Development, 119, 1005-1017 (1993)) in pbluescript SK' (Stratagene) was cleaved with Sacll and EcoRV and cloned into SacU and SmclI site of pBK-CNV vector (Strategene). COS cells were tr~n~i~ntly llal~re~;~d with c-ret cDNA or with empty plasmid by electroporation (Bio Rad) with - 30 % efficiency by fluorescen-~e of c~ re~ Red Shift Green Fluol.,;,cellL Protein in PEF-BOS
vector. 48 hours later, 10 x 10' transfected COS cells or 3-5 x 106 parental COScells or NB2/a cells were treated With l25I-GDNF, cross linked and analysed as specified in legends of Fig. 15 and Fig. 16.
First, the expression of c-RET protein by was e~:lmin.ocl Western blotting.
c-ret-transfected COS cells (Fig. 18a) and NB2/a cells (not shown) expressed cletect~hle amounts of the c-RET protein, whereas no c-RET protein was detected in mock-transfected (with PBK-CM~ plasmid) COS cells ~Fig. 18a). PC12 cells also express c-RET protein, albeit at considerably lower level than NB2/a cells or c-ret-transfected COS cells (not shown). COS cells, transiently e,.l,lcssil~ mouse c-ret proto-oncogene were inr~lb~tt?~ with l25I-GDNF. As shown in Fig. 17b, those cells bound GDNF, and binding of '2sI-GDNF can be competed with excess of unlabeled GDNF. In contrast, no signifirzlnt binding-of GDNF was observed in mock-transfected COS cells.

Example 16 Pho~l-h~ yl~tion of Iylosi~e resi~ues 10 X 106 transfected COS cells (48 hr after transfection) were treated with 50 ng/ml of GDNF (P1~1UL~Ch EC Ldt.) for 5 minllt~e in serum-free DMEM, or not treated, and then quickly washed with the same ~-.f~ --. NB2/a cells were similarly treated (results not shown). c-RET ~roLeills were immlln~lGci~i~t~d from d~lc~ lL extracts by corl~t~il of monoclonal (Lo, L. & Anderson, D. J.
Neuron, 15, ~27-539 (1995) and polyclonal (Santa Cruz) anti-c-ret antibodies, separated by 7% SDS-PAGE, l~ d to nitrocellulose, probed by anti-c-ret antibodies (Santa Cruz), stripped and reprobed by anti-phosphotyrosine antibodies (Sigma).
This lleat.llenl resulted in .5ignifir~nt h~ ,a~e in tyrosine phosphorylation of190 kD cRET proto-oncogene, the 170 kD form being less plo~ y phosphorylated (Fig. 18b). In both cell lines, relatively high c-R~T
phosphorylation was ~lçtect.o(l also in the absence of GDNF (Fig. 18), most probably via endogeneous GDNF secreted by these cells and/or ligand-independent receptor dimerization.

Example 17 '251-GDNF binds to c-ret-l,o~ilive enteric neurons '25I-GDNF was bound to developing rat tissue explants in situ. In situ binding of human l25I-GDNF (PeproTech. EC Ltd.), io~lin~t~l by Chloramine T

W O 97/18240 PCT~US96/18197 Method, was carried out essçnti~lly as clescrihe~ (P~ and Thesleff, 1987).
Briefly, explants of E15 rat gut were in~lb~t~d with 10 ng/ml of '25I-GDNF in Eagle's ~ l es~onti~l m~ m on the Nuclepore filter (Costar) for 90 min at - room l~lllpel~ture. 250-fold excess of unlabeled GDNF was applied as a competitor to control explants. After careful washing, the explants were fixed with 3.5% paraform~ yde in PBS, sectioned and exposed to NTB-2 emulsion (Kodak).
The ga~Llu~ tract was chosen as it ~Llungly e~ ,;,ses GDNF mRNA
(Suvanto et al., 1996); Figure 19 a and b) and c-RET-positive ll~ulU.lS are absent in the ga~L.ui,.~ l;,r~l tract in c-ret-deficient mice (~hllrh~rt et al., 1994; Durbec et al., 1996). l25I-GDNF binds to a group of cells within the muscle layer of embryonic day (E)15 rat gut (Figure l9 c and d). This binding was specific as itwas tûtally cnmret~d with 250-fold excess of unlabeled GDNF (Figure l9h). The cells that bind GDNF were the enteric neurons of the lllyelllelic plexus, as revealed by peripherin immnnr)reactivity (Figure l9f). Moreover, these llc~urolls also expressed c-ret mRNA, as demonstrated by in situ hybridization (Figure l9e).
Cloning of the GDNF cDNA and in situ hybridization with GDNF probe was p;;lroll-led exactly as described (Suvanto et al., 1996). A 646 bp long fragment of mouse c-ret cDNA (Pachnis et al., 1993) covering the 3'-region of the shorter form (T~k~h~chi et al., 1988) of c-ret was cloned into NotI-Xhol site ofpBSK+ vector (Stratagene). cRNAs in antisense and sense orientation were labeledwith digoxigenin-UTP (Boehringer-Mannheim), hybridized to cryosections through E15 rat gut and vi.c~1i7f!d with ~lk~lin.o phosphatase-conjugated anti-digoxigenin antibodies according to m~mlf~tllrers i~illu~;Lions. In both cases, only background labeling was obtained with hybridization of corresponding probes in sense orientation (Figure l9g). Polyclonal anti-peripherin antibodies (Bio-Rad) were applied to cryosections of E15 rat gut at a dilution of 1: 100 for 1 hr and Vi~:uz~1i7t~( by FITC-conjugated secondary antibodies (Jackson~. Thus, GD~F specifically - binds to c-RET-e~ressillg enteric neurons of developing rat.
Example 18 Affinity-clo~ ing of GDNF to c-RET

W O 97/18240 PCT~US96/18197 PC12 cells and NB2/a cells were washed three times with serum-free RPMI-1640 or DMEM, respectively, plated on lm~o~te~ (NB2/a cells) or collagen-coated (PC12 cells) dishes (5000-6000 cells per dish in triplicate) in the presence or absence of 50 ng/ml of GDNF (PeproTech EC Ltd.), and the number of cells microscopically counted after 48 h.
For c-RET expression in transfected cells, the shorter form (T~k~h~chi el al., 1988) of human wild-type c-ret cDNA was subcloned in pcDNA3 (lnvitrogen).
3T3 fibroblasts were stably transfected with c-ret ~ s~ion plasmid or with emptyvector (mock-Lla~ ;Led cells) and positive cells lines sel~ctçd with G418.
Transient Il~lLsre~;Lion of trkC 3T3 fibroblasts (Ip et al. (1993) Neuron, 10,137-1~9) with human c-ret cDNA in pcDNA3 vector or with empty vector was p~,lrolllled by the lipofectin method (Gibco-BRI.). c-ret and mock-llal~ L~d cells (10.000 - 15.000 cells per well) in five parallels were treated with rat GDNF (Trupp et al. (1995) J. Cell. Biol. 130, 137-148) at il,t~
collce~ tions for five days. NT-3 was used as positive control at 30 ng/ml. Cellnumber was q~l~ntifi--d by measurement of acid phosphatase activity using AbacusTM
Cell Proliferation Kit (Clontech).
3-5 x 106 PC12 cells, NB2/a cells, COS cells or c-ret-3T3 as well as mock-3T3 cells or mf~çh~ni-~lly dissociated cells from two F20 or from 17 E15 rat kidneys were inr.uk~cl with 10 ng/ml of l25I-GDNF (human GDNF from PeproTech EC Ltd. or rat GDNF from C. ~. Ibanez) (Trupp et al., 1995), iodinated by Chloramine T method, for 1 hour on ice. 250-fold excess of unlabeled GDNF (PeproTech EC) or TGF-~1 (kindly provided by Dr. M. Laiho) was applied to control sample. l2sI-GDNF was then croselink~-~l to the cells with 30 mM of ethyl-dimethylaminopropyl carbodiimide (EDAC) (Pierce) for 30 lnilluL~s on ice.
DeL~rl3el1L Iysates of the cells were immllnnpleci~ d with polyclonal anti-GDNF
antibodies (Santa Cruz) or with the cocktail of monoclonal (kindly provided by Dr.
D. Anderson, Lo and Anderson, 1995) and polyclonal (Santa Cruz) anti-c-RET
antibodies to neurofil~m~ont ~lvl~ s (a gift of Dr. I. Virtanen) were used as control antibodies. The precipitates were collected by Protein A-Sepharose (Pharmacia) or by WGA-agarose (a gift from Dr. O Renkonen), s~al~l~d on 7% SDS-PACE, and vi.e~ d with a Phosphorimager SI (Molecular Dyn~nT ;~e).

W O 97/18240 PCT~US96/18197 First, ~ GDNF was cro.cclink~or1 to PC12 cells, NB2/a cells and COS cells with ethyl-d~ e~lyial~ ~ropyl carbodiimide (EDAC), and the complexes were preci~?ilaL~d with anti-GDNF antibodies. As shown on Figure 20a, complexes with molecular weight of 190 kD and 170 kD were obtained from PC12 and NB2/A
5 cells, but not from COS cells. The molecu}ar weights of the cross linked plot~ills (minus GDNF monomer of ~25K) correspond to those of c-RET, (140 kD and 160 kD, l~r~se~ partially and fully glycosylated isoforms of c-RET, respectively) (T~k~h~Chi el al., 1988).
Next, l2sI-GDNF was cros~linke~l to PC12 and NB2/a cells by EDAC and 10 immnno~ ci~iL~ted formed complexes with anti-c-RET antibodies. The bands withmolecular weight of 190 kD were obtained from both cell lines (Figure 20 a and b). Formation of the complexes was abolished by S00-fold excess of unlabeled GDNF. The reason why both fully and partially glycosylated forms of c-RET were precipitated by anti-GDNF antibodies, but only the larger isofc l.ll by anti-c-RET
antibodies, is unclear. The same complexes, although much weaker, were also obtained when dithiobis(succinimi-lylpl-Jl)ionate) was used as a crosslinker (data not shown).
The EDAC-crosslink approach was also used to reveal GDNF-c-RET
complexes from E15 embryonic kidney cells, where c-ret mRNA is strongly expressed in the tips of growing ureter branches. With both anti-GDNF and anti-c-RET antibodies, a band of 190 kD was obtained (Figure 20 a and b) that was competed with excess of unlabeled GDNF. Thus, only the fully glycosylated form of c-R~T is ~ ssed in embryonic kidney cells.
Croc.clink~ 25I-GDNF-c-RET complexes from the cells ectopically t;~ ,SSillg c-ret were also demol~LlaLt;d. 3T3 cells were Llal~recl~d with c-retcDNA or with empty plasmid, and established stable transfected cell lines (c-ret-3T3 cells or mock-3T3 cells). Cross linking of '25I-GDNF to these cells followedby anti-RET-~l~,cipiL~Iion revealed a 190 kD band that was abolished with 250-fold excess of unl~helefl GDNF (Figure 20b). As GDNF is a distant member of TGF-,~
family, we also used a 250-fold excess of TGF-~1 as a co~eLil~Jl. No competition was observed with TGF-~Bl (Figure 20b) Taken together, these data show that GDNF directly and specifically binds to c-RET.

WO 97/18240 PCT~US96/18197 .Y~-nr e 19 GDNli spe-~ific~lly i.~ as~s tyrosine phosphorylation of c-RET
c-ret-3T3 cells and mock-3T3 cells were treated with C~DNF and the proteins from these cells were irnmuno~le~i~iLal~d with anti-c-RET antibodies.
5 The precipitated plo~ins were then analyzed by Western blotting with anti-phosphotyrosine antibodies. 10 x 106 c-ret-3T3 cells were treated with different doses of GDNF (PeproTech LC Ltd. or from C. F. Ibanez) (Trupp et al., 1995) for 5 min, or with 50 ng~ml of GDNF for inrli~t~d times in serum-free Dulbecco's modified Eagle's m~dillm cont~ining 1 mM Na3VO4 and then quickly 10 washed with the same m~ m c-RET ~lol~ills were i~ p~ ipilaL~d from delc;lgellt extracts, co-.li.i..;.~g 1 mM Na3VO4 by cocktail of monoclonal (Lo, L. and Anderson, D.J. (1995) Neuron, 15 527-539) and polyclonal (Santa Cruz) anti-c-RET antibodies, s~l-alated by 7% SDS-PAGE and llal~rc;~;L~d to nitrocellulose which were probed by anti-phosph~lyl-,sille antibodies to nikocellulose with wasprobed by anti-phosph~lylo~ e antibodies PY20 (Tr~n~dlletion Labol~c,lie~), thenstripped and reprobed by anti-c-RET antibodies (Santa Cruz).
As shown on Figure 21a, a short trç~.tm~nt of c-ret-3T3 cells with GDNF
dose-dependently ~beginning at 25 ng/ml) hlcr,_ased tyrosine phosphorylation of the 160 kD c-RET isoform, whereas the phosphorylation of the 140 kD isoform rem~ined unrh~nged. An increase in c-RET phosphorylation was evident at 25 ng/ml of GDNF and above it. No c-RET proteins were detected in mock-3T3 cells.
c-ret-3T3 cells were also treated with GDNF (50 ng/ml) for different times. An increase in c-RET tyrosine phosphorylation was evident after S min-ltes of tre~.tm-ont and continued at least for one hour (Figure 21b). With prolonged exposition, the increase in phosphorylation of lower c-RET isoforrn also became evident. A basal level of c-RET phosphorylation was ~l~tecte~'. in the absence of GDNF, possibly via a ligand-hld~;pelldent dirnerization of that It:Ce~tOl. To reveal the arnounts of c-RET protein in these experiments, the filters were stripped from antibodies and reprobed with anti-c-RET antibodies. The level of c-RET protein was not changed by GDNF tre~7tment in c-ret-3T3 cells (Figure 21a and b, lower panels). The finding that GDNF specifically activates c-RET in~ tes that c-RET
is a ~i~n~ling receptor for GDNF.

.

CA 0223764l l998-05-l2 W O 97/18240 PCT~US96/18197 Example 20 c~ T ~ ~ion confers GDNF-respon~ O,, to 3T3 cells Mouse 3T3 fibroblast cell line expressing trkC (trkC-3T3) (Ip et al, 1993) ~ were tr~n~iently transformed with c-ret ~ ssion plasmid. trkC-3T3 cells die S within 2-3 days in serum-free m~ m in the ~hsPn~e of trkC ligand ll~ulollu~hin-3 (NT-3~ (Ip et al., 1993) and do not express ~let~Pct~hle amounts of c-ret. GDNF
dose-dependently increased the l~ bel of c-ret-llal~recL~d but not of mock-transfected trkC-3T3 cells (Figure ~), which was comparAble to the response elicited by NT-3. Whether this is a proliferative or survival-promoting r~s~ollse could not be distinguished based upon the data. Thus, introduction of c-RET to GDNF-nonresponsive cells is sufficient to bring about the biological response toGDNF.

li',Yqmrl~ 21 t; ,n of GDNF receptor L6 myoblast cells were Iysed with 1% NP40 and cell lysates were fractionated by anionic exrh~n~e on a Q-Sepharose column. Fractions eluted at dif~lcll~ ionic strength were dialyzed and assayed for binding to C~DNF
immolihlized on a chip in a Biacore device (ph~rm~ri~). A distinct binding component was (lP~tectp-fl in a fraction of L6 cell lystaes (Figure 23a). In theFigure, solid bars i~lir.~lt; total protein (as absolbhllce at 280 nm); h~t~hP~l bars in-lir~t~ GDNF finding (in resonance units). This fraction was not particularly rich in protein, inrlic~t;ng a substantial purification over the total protein llli~lUl~:. The equivalent fraction of a ~OS cell Iysate did not show binding under the same conditions (data not shown).
Further purification of the GDNF binding activity in the first lM salt ~ fraction was obtained after hydrophobic interaction chromatography (Figure 23b).
The data ~ selll~ the ratio between GDNF binding (in resonance units) and protein concentration (OD at 280 nm). Fractions were eluted with a step-wise gradient of ammonium sulfate.

W O 97/18240 PCT~US96/18197 All~lnalively, purification may be effected by crosslinking GDNF to cells in the presence of tracer amounts of radiolabeled ligand, and ligand/l.,ce~Lol complexes can by fractionated through ion exch~n~e cllr~llldLography followed byhydrophobic interaction chromatography and SDS/PAGE. Bands corresponding to S the molecular weights of GDNF-l.,c~lor complexes can by excised, dissociated, and then sequenced by mass ~e~l-u---eLly or Edman degral1~tion, de~elldil~g uponthe yield of recovery.

Example 22 10 A novel GDNF-binding ~ in brain By ligand blotting, we have identified another GDNF-binding protein from total brain extract. We bound l2sI-GDNF to the filters carrying protein from thetotal extracts of brain and liver (a ligand blot assay). A major band wi~ MW of about 50 kD was obtained from brain extract, but not from liver (Figure 24). This 15 bill.lil-g is ~eciric as ~ GDNF did not bind to other ~ s from total Iysates, it is not found in liver Iysates ( nor in some other tissues), and it can be co--lpeLt:d with excess of unlabeled GDNF. Binding of '25I-GDNF to c-RET was not revealed in the ligand blots. The reason for this may be the very low share of c-RET in total brain extract. All~ aLively, by analogy with other receptors for 20 GDNF, c-RET might not bind GDNF directly, but might first bind to another nonsign~lling receptor that thel~drLl presents the ligand to c-RET, a ~ign~ling receptor. A 50 lcD GDNF-binding protein is a good ç:~n~ te for the putative y~ S~ ,cc~lol .

~Y~np~ 23 r for i~ in~ novel si~l;n~ recept. ~ for GDNF
In the ~bsen~e of serum, 3T3 fibroblasts can be made ~lepe~(lent on a given exogenous growth factor provided applopli~l~ receptors are t~ cssed on the cell surface. An expression library can be made using RN33B cDNA, which can then be Ll,.. x~,;l~l into 3T3 fibroblasts by procedures well known in the art (Maniatis et al., supra). Stable transfectants can be selected in serum-free media supplement~d with GDNF. Fibroblast clones that express .cign~ling GDNF receptors will selectively grow in the presence of GDNF in serum-free media. The selection stepmay allow detection of even very reare clones due to their dirf~ llial growth advantage. Further analysis of the recovered clones in media with or without GDNF would help to distinguish GDNF-dependent from GDNF-independent S survival of clones.

All references cited herein are hereby inco.~ul~Led by lel~ ce in their entireties.

Claims (21)

WHAT IS CLAIMED IS:

1. An isolated receptor which binds glial cell line-derived neurototrophic factor (GDNF), said receptor comprising at least one polypeptide having a molecular weight selected from the group consisting of polypeptides of about 55kD, 70kD, 135kD, and 300kD molecular weight, as determined by SDS-PAGE on 4-20% gradient gels.

2. A competitive assay for identifying compounds which bind to GDNF
receptors comprising a) incubating said compounds with cells which express c-RET receptors in the presence of an excess of labeled GDNF;
b) measuring the amount of labeled GDNF bound to said cells; and c) comparing amount labeled GDNF bound to said cells to that of controls not incubated with said compounds.

3. The method of claim 2 wherein the cells are selected from the group consisting of NB2/a, MN-1, and PC12 cells.

4. The method of claim 2 wherein the labeled GDNF is 125I-GDNF.

5. A competitive assay for identifying compounds which bind to isolated GDNF receptors comprising a) incubating said compounds with isolated c-RET receptors in the presence of an excess of labeled GDNF;
b) measuring the amount of labeled GDNF bound to said receptors; and c) comparing amount labeled GDNF bound to said receptors to that of controls not incubated with said compounds.

6. The method of claim 5 wherein the receptors are polypeptides which bind GDNF selected from the group consisting of polypeptides about 55kD, 70kD, 135kD, 155kD, and 300kD molecular weight.

7. The method of claim 6 wherein the polypeptide is about 155kD molecular weight.

8. The method of claim 5 wherein the isolated receptor is c-RET.

9. The method of claim 5 wherein the labeled GDNF is l25I-GDNF.

10. A method for identifying compounds which are GDNF homologs comprising a) incubating said compounds with cells which express c-RET receptors;
and b) determining whether said compound effects tyrosine phosphorylation.

11. The method of claim 10 wherein said cells are selected from the group consisting of PC12 MN-1, and NB2/a.

12. A method for identifying compounds which are GDNF homologs comprising a) incubating said compounds with cells which express c-RET receptors;
and b) determining whether said compounds effect an increase in c-fos mRNA
levels.

13. The method of claim 12 wherein said cells are selected from the group consisting of PC12 MN-1, and NB2/a.

14. A method for identifying compounds which are GDNF homologs comprising a) incubating said compounds with cells which express c-RET receptors under non-permissive conditions for said cells; and b) determining the number of surviving cells as compared to controls not incubated with said compounds.

15. The method of claim 14 wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.

16. A method for identifying compounds which are GDNF analogs comprising a) incubating said compounds with cells which express c-RET receptors in the presence of concentrations of GDNF effective for phospholylating tyrosine; and b) determining whether said compounds effect a decrease in the tyrosine phosphorylation as compared with controls not incubated with said compounds.

17. The method of claim 16 wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.

18. A method for identifying compounds which are GDNF analogs comprising a) incubating said compounds with cells which express c-RET receptors in the presence of concentrations of GDNF effective for increasing c-fos mRNA
levels; and b) determining whether said compounds effect a decrease in c-fos mRNA
levels as compared with controls not incubated with said compounds.

19. The method of claim 18 wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.

20. A method for identifying compounds which are GDNF analogs comprising a) incubating said compounds with cells which express c-RET receptors under non-permissive conditions for said cells in the presence of amounts of GDNF
effective for cell survival; and b) determining the number of surviving cells as compared with controls not incubated with said compounds.

21. The method of claim 20 wherein said cells are selected from the group consisting of PC12, MN-1, and NB2/a.