NZ324511A

NZ324511A - Identification and isolation of glial cell line-derived neurotrophic factor receptors (gdnf receptors)

Info

Publication number: NZ324511A
Application number: NZ324511A
Authority: NZ
Inventors: Carlos Ibanez; Urmas Arumae; Hannu Sariola; Petro Suvanto; Miles Trupp; Mart Saarma
Original assignee: Carlos Ibanez; Urmas Arumae; Hannu Sariola; Petro Suvanto; Miles Trupp; Mart Saarma
Priority date: 1995-11-13
Filing date: 1996-11-13
Publication date: 1999-09-29
Also published as: EP0879247A4; AU718979B2; NO982141D0; WO1997018240A1; EP0879247A1; AU1158897A; KR19990067508A; JP2000501924A; NO982141L; CA2237641A1

Description

<div class="application article clearfix" id="description"> New Zealand No 324511 International No PCT/US96/18197 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates 13 11 1995,16 04 1996,27 06 19096,27 06 19 96,27 061996, Complete Specification Filed 1311 1996 Classification (6) C07K14/705 Publication date 29 September 1999 Journal No 1444 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention Glial cell line-derived neurotrophic factor receptors Name, address and nationality of applicant(s) as in international application form CARLOS IBANEZ, a Swedish citizen of Karolinska Institute, Department of Neuroscience -Mol Neuro, Stockholm 17 177 Sweden, URMAS ARUMAE, an Estonian citizen of Niittykatu 3 D 53, FIN-02200 Espoo, Finland, HANNU SARIOLA, a Finnish citizen of Oravapolku 15, FIN-00800 Helsinki, Finland, PETRO SUVANTO, a Finnish citizen of Ollaksentie 7 E 10, FIN-01670 Vantaa, Finland, MILES TRUPP, a Swedish citizen of c/-Karolinska Institute, Department of Neuroscience - Mol Neuro, Stockholm 17 177 Sweden, MART SAARMA, an Estonian citizen of Kulosaaren Puistotie 36 B 15, FIN-00570 Helsinki, Finland WO 97/18240 PCmiS9fi/l8107 GLIAL CELL LINE-DERIYED NEUROTROPHIC FACTOR RECEPTORS Pursuant to 35 U S C § 119(c), the present application claims priority 5 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 hereby incorporated by reference in their entireties 10 FIELD OF THE INVENTION The present invention relates to the identification of receptors for and functions of GDNF, and cell line1: expressing the receptors BACKGROUND OF THE INVENTION 15 Glial cell line-derived neurotrophic factor (GDNF) is a trophic polypeptide It is a disulfide bridge-linked homodimer of two 134 amino acids long glycosylated polypeptides, with a molecular weight of approximately 25-30 kD for each monomer Prior to the molecular clorung of GDNF in 1993, investigators sought a trophic polypeptide which would alleviate the neuronal loss associated with 20 Parkinson's disease, specifically dopaminergic neurons of the ventral mesencephalon The survival of this subpopulation of neurons has been known tor 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 dopamine uptake in primary 25 cultures prepared from embryonic ventral midbrain neurons (Lin et al , 260 Science 1120, 1993) Subsequently, GDNF was shown to promote survival of adult substantia nigra neurons in vivo following pharmacological treatments 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 30 specific for dopaminergic neurons, several other potent activities of this molecule have subsequently been demonstrated, including survival and phenotypic responses in facial and spinal motor neurons (Henderson et al , 266 Science 30 1062, 1994, Oppenheim et al , 373 Nature 344, 1995, Yan et al . 373 Nature 341, 1995), Printed from Mimosa 07 11 15 WO 97/18240 PCT/US96/I8197 -2- noradrenergic neurons of the locus coeruleus (Arenas et al , Neuron, in press, 1995), cerebellar Purkinjie cells (Mount et al , 92 PNAS 9092, 1995), sympathetic and sensory neurons m peripheral ganglia (Trupp et al , 130 J Cell Biol 137, 1995) and for populations of peripheral neurons with target-derived and paracrine 5 mode of action (Trapp, M et a! , / Cell Biol, 130, 137-148 (1995) Pitchcl, J Sanola, II , Hoffer, B &Westphal, H (unpublished observation), Buj-Bello, A Buchman, V L., Horton, A , Rosenthal, A & Davies, A M Neuron, 15, 821-828 (1995) As many of these neurons are affected in neurodegenerative diseases, GDNF may 10 have potent therapeutical applications Particularly, exogenous!) administered GDNF maintains dopaminergic neurons of the substantia nigra in experimentally induced Parkinsons disease in rodents (Beck et al (1995) Nature, 373, 339-341, Tomac et al (1995) Nature, 373, 335-339) and leads to functional recovery in Parkinsonian rhesus monkeys (Gash et al (1996) Nature, 380, 252-255) GDNF 15 treatment also rescues about half of the experimentally axotomized murine motoneurons (Oppenheim et al (1995) Nature, 373, 344-346, Li et al (1995) Proc Natl, Acad Set US A, 92 9771-9775) suggesting that GDNF may be used in treatment of motoneuronal diseases The studies of the mechanism of GDNF action in normal and pathogenic conditions have been, however, basically 20 hampered as its receptor was not known Based upon structural similarities (primarily seven conserved cysteine amino acid residues), GDNF appears to be a distant member of the transforming growth factor-beta (TGF-13) superfamily of multifunctional cytokines, which includes TGF-/3s, activins, bone-morphogenctic proteins (BMPs) and growth and 25 differentiation factors (GDFS) (Roberts et al , 327 Philos Trans R Soc Land 145,1990) TGF-/? and related ligands are known to suppress proliferation in epithelial and immune cells, to function as morphogens in early development, to induce cctopic expression of skeletal tissue, and to promote survival and differentiation of neurons TGF-/3 superfamily proteins interact with numerous 30 receptor subunits on the surface of responsive cells (Attisano et al , 1222 Mol Cell Res 71, 1994, Derynck, 19 Trends Biochem Sci 548, 1994) Different receptor types have been described based on the molecular weights of affinity labeled Printed from Mimosa 07 11 15 WO 97/1W-IO PCT/US9fi/lSI<)7 -3- complexes Among these are Ihe type I, type II and type III receptors, which represent binding proteins of 55kD, 70kD and 300kD, respectively Type III receptors are abundantly expressed transmembrane proteoglycan;, of approximately 300kD with a short cytoplasmic tail, and arc thought to function in recruitment of 5 ligand to an ohgomenc receptor complex (Lopez-Casilla1* et al , 67 Cell 785, 1991) Indeed, a type III receptor is required on some cell lines for TGF-/32 binding to the signaling receptors lype I and type II receptors are transmembrane proteins with an intracellular serine-threonine Kinase domain and can therefore transmit downstream signals upon ligand binding (Attisano ct al , 75 Cell 671, 10 1993, Derynck, 1994 supra) Type II receptors are constitutively activated kinases which upon ligand binding recruit type I receptors to a signaling complex In this complex, type I receptors are phosphorylated by type II receptors on a juxtamembrane domain rich in serine residues, this phosphorylation is thought to result in the activation of the ser-thr kinase activity of type I receptors and in 15 downstream signaling (Wrana et al , 370 Nature 341, 1994) According to this model, TGF-/3 superfamily proteins can not bind to type I receptors in the absence of type II receptors, although in some cases, type I receptors are necessary for efficient binding to lype II receptors (Letsou et al , 80 Cell S99, 1995) Multiple cDNA clones of type I, II and III rcceptors for TGF-/3S, activins and BMPs have 20 been isolated by either expression or homology cloning, including seven mammalian type I receptors, four type II receptors and one type III betaglycan receptor Additional membrane proteins binding different members of this family include glycosylphospliatidyl inositol (GPS)-hnked 150kD and 180kD proteins of unknown structure and function (MacKay and Danielpour, 266 J Biol Chem 25 9907, 1991), and endoghn, a 180kD disulphide linked duner which binds TGF-|31 but not TGF-/32 The isolation and characterization of GDNF receptors is a prerequisite for the understanding of the full range of biological actions of GDNF and the signaling events that take place upon GDNF binding to responsive cells Until now, 30 progress in this area has been hampered by the lack of cell lines responsive to GDNF, that is, cell lines comprising GDNP receptors Printed from Mimosa 07 11 15 WO 97/1S240 PCT/US96/18197 -4" r~ ~ 1 1 I « SUMMARY OF THE INVENTION Receptors for GDNF are disclosed herein, as are cell lines expressing the same Methods for identifying and isolating these receptors are also disclosed 5 In one aspect, the present invention relates to isolated receptors which bind GDNF In another aspect, the present invention relates a method for determining compounds or compositions which bind GDNF receptors Divisional specification no NZ relates to 10 methods for identifying homologs of GDNF by screening for compounds or compositions which have similar biological effects, such as tyrosine phosphorylation, increase m c-fos mRNA, and increases m cell survival, by using cells that express c-RET receptors In still another aspect, the invention of divisional specification no NZ relates to method for identifying anologs of GDNF by screening for compounds or compositions which are antagonistic for the biological effects of GDNF, such as are listed above,by using cells that express c-RET receptors 20 BRIEF DESCRIPTION OF THE DRAWINGS Fig 1 Binding of ,25I-GDNF to receptors on chick sympathetic neurons (a) Saturation steady-state binding of 125I-GDNF to E10 embryonic chuck sympathetic neurons Data are expressed as mean ±SD of triplicate determinations (b) Scatchard transformation of the data plotted m (a) (c) Hill transformation of the 25 data plotted m (a) nH Hill coefficient Fig 2 Affinity labeling of GDNF receptors on chick sympathetic neurons 125I-GDNF was cross-linked to E10 embryonic chick sympathetic neurons, receptor complexes were fractionated by SDS/PAGE and visualized by gel autoradiography 30 (middle lane) A doublet at lOOkD and a 300kD complex are indicated by arrows Excess cold GDNF prevented cross-linking of 125I-GDNF (right lane) For comparison, crosslinking of 125I-TGF-/3 to mink lung epithelial cells MvILu is also shown (left lane) Molecular weight markers are indicated m kD Fig 3 Affinity labeling of GDNF receptors on cell lmes 125I-GDNF was cross- WO 97/J 82-10 I'C 17US96/18197 -5- I inked to C6 glioma, RN33B raphe nucleus, L6 myoblast arid MN-1 motor neuron cell lines with either DSS or EDAC as crosslinker agents Receptor complexes were fractionated by SDS/PAGE and visualized by gel autoradiography Excess 5 told GDNF prevented cross-linking of 125I-GDNF (cold) Molecular weight markers are indicated in kD Fig 4 Individual constituent affinities of GDNF receptor subunits in RN33B and MN-1 cells (a) Sizes of different GDNF receptor complexes on RN33B and MN-1 10 cells after cross linking with EDAC or DSS (b) and (c) '"I-GDNF was cross- linkcd to RN33B (b) or MN-1 (c) cells in the presence of increasing concentrations of cold GDNF The percentage of '"I-GDNF binding to the indicated reccptor subunit is plotted as a function of the concentration of mid GDNF used during binding 15 Fig 5 Expression of GDNF mRNA in cell lines expressing GDNF receptors (a) Autoradiogram of an RNAse protection assay using equal amounts of total RNA from the indicated ccll lines Kidney post natal day 1 and yeast tRNA were used as positive and negative controls, respectively (b) Quantification of the level of 20 GDNF mRNA in different cell lines relative to the level m PI kidney undiff RN33B, undifferentiated RN33B cells, diff RN33B, differentiated RN33B cells, diff RN338+GDNF, RN33B cells differentiated m the presence of GDNF Fig 6 Expression of mRNA for c-ret in different cell lines 25 Fig 7 GDNF stimulation of tyrosine phosphorylation of ERKs in RN33B and MN-1 cells RN33B (a) or MN-1 (b) cell monolayers were exposed to 50 ng/ml GDNF during the indicated periods of time (in minutes), cell lysatcs were fractionated by SDS/PAGE and Western blots probed with an anti-phosphoiyrosine 30 antibody (aP-Tyr) The blots were stripped and reprobed with an anti ERK2 antibody (a-ERK2) that recognizes both p42CTk 12 and p44crkl (arrows to the right) Molecular weight markers are indicated in kD Printed from Mimosa 07 11 15 WO 97/18240 PCT/US96/18I97 -6- Fig 8 GDNF stimulation of c-fos mRNA expression in RN33B and MN-1 cells RN33B (a) or MN-1 (b) cell monolayers were exposed to 50 ng/ml GDNF during the indicated periods of timLS, 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 stringency Fig 9 GDNF increased the survival of RN46A cells RN46A cells were differentiated in media ± 0-50 ng/ml GDNF for 8 days The data represent the means ± SEM of three independent experiments (1,500-3,000 cells counted per 10 condition) ANOVA indicated that the GDNF had a significant effcct on survival at all concentrations compared to media alone (overall ANOVA df=6,203, F=1 1 39, p,0 001, unequal N LSD post hoc lest, p 0 001) Fig 10 a-c Biological and biochemical responses of MN-1 to GDNF (a) GDNT 15 stimulates survival of serum-deprived MN-1 cells (b)GDNF stimulates rapid and transient tyrosine phosphorylation of several proteins (asterisks) m MN-1 cells Time of GDNF treatment (in minutes), and molecular weight markers are indicated (c) Rapid and sustained ERK1 and ERK2 tyrosine phosphorylation stimulated by GDNF in MN-1 cells 20 Fig 11 a-b c-RET is a signal transducing receptor lor GDNF (a) Immunopreeipitation analysis of GDNF-rcceptor complexes in MN-1 cells GDNF-labeled binding proteins could be precipitated with lectin Sepharose beads, or antibodies against GDNF, phospho-tyrosint (P-Tyr) and c-RET Control 25 preimmune antibodies did not immunoprecipitate GDNF receptor complexes (b) GDNF induces tyrosine phosphorylation of c-RET m MN-1 cclls c-RET tyrosine phosphorylation was detected already 5 minutes after addition of GDNF (upper panel) Saturation was observed at 30 ng/ml GDNF (lower panel) 30 Fig 12 a-b c-rei expression is sufficient to mediate 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 expression Printed from Mimosa 07 11 15 WO 97/18240 PCT/US»6/18197 -7- plasrnids Untransfcctcd 3T3 cells (3T3) did not bind GDNF The specificity of the binding was demonstrated by displacement of the labeling with 50X excess cold GDNF (b) GDNF promote1, survival and growth responses in 3 H fibroblasts stably transfected with a c-ret expression plasmid Untransfected cells 5 did not respond to GDNF Fig 13 a c c-ret mRNA expression in adult bram and in developing substantia nigra (a) Ribonuclease protection analysis (RPA) of c-ret mRNA expression in different regioas of the adult rat brain (b) RPA of c-ret mRNA expression during 10 development of the rat ventral mesencephalon (nigra), and of GDNF mRNA expression in the developing striatum (c) mRN^ expression is indicated in arbitrary units where 100 corresponds to the level of expression in the respective regionj. in newborn animals 15 Fig 14 a-h c-RET is expressed in GDNF-responsive substantia nigra dopaminergic neuron (a) Dark field autoradiogram of c-ret mRNA expression analyzed by in situ hybridization in the adult substantia nigra Scale bar, 40 pim (b) Bright field autoradiogram showing substantia nigra neurons containing c-ret mRNA Scale bar, 7 5 fj.m (c) Immunohistochenucal analysis of u-RET protein expression in the 20 adult substantia nigra Scale bar, 27 (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 m the medial forebrain bundle ensures that only cells which actively take up and retrogradely transport dopamine will be compromised Note the disappearance of the labeling for c-ret 25 mRNA in the lesioned substantia nigra (arrowhead) 1 day and 5 days following th lesion (e)Immunohistochemical analysis of c-RET protein expression in the adult substantia 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 /im (f) Grafting of GDNF-expressmg fibroblasts 30 rescues c-RET-LI Note c-RET positive fibers surrounding and entering the GDNF producing graft (arrows) Same magnification as m (e) (g) Immunohistochcmical analysis of cRET protein expression in the adult locus coeruleus after lesion with Printed from Mimosa 07 11 15 WO 97/18240 PC WUS96/I8197 -8- 6-OHDA and grafting of mock transfected fibroblasts (control graft) Scale bar, 23 (h) Rescue of cell bodies expressing c-RCT-Ll by GDNF in of 6-OHDA lesioned locus cocruleus Same magnification as m (g) Graft is on the right in (c) and (f), and above in (g) and (h) 5 Fig 15 a-c. PC 12 and NB2/a cells respond to GDNF and bind GDNF (a) GDNF promotes survival of serum-deprived PC12 cells (b) GDNF increases the number of NB2/a cells (c) 1251-GDNF binds to PC 12 and NB2/a cells in the absence (open column) or presence (filled column) of 50-fold unlabeled GDNF 10 Fi£ 16 Affinity crosslinking of iyI-GDNF to cell lines 1J5I-GDNF was crosslinked to PC12 cells (lane 1), SY5Y cells (lane 2), E20 rat kidney cells (lane 3) and NB2/a cells (lane 4), and the resulting complexes were precipitated from detergent lysates by anti-GDNF antibodies (Santa Cruz) 15 Fig 17 a-b GDNF specifically binds to c-RET ( a) 12<I-GDNF was crosslinked to NB2/a cells in the presence (+) or absence (-) of 1 POQ-fold excess ot unlabeled GDNF (PeproTech EC Ltd ), and the resulting complexes were precipitated from detergent lysates by cocktail of monoclonal and polyclonal (Santa Cruz) anti-c-RET 20 antibodies recognizing the extracellular and intracellular domain of cRET, respectively Lysates were also precipitated by monoclonal anti-neurofilament antibodies 13AA8 (lane 3), by Protein A-Sepharose (lane 4) and by WGA-Agarose (lane 5) (b) 123I-GDNF binds to COS cells transiently expressing c-RET, but not to mock-translected (with pBK-CNV plasmid) COS cells Open column represents 25 binding in the presence, and filled column in the absence of 50-fold excess of unlabeled GDNF Fig 18 GDNF increases tyrosine phosphorylation of c-RET in transfected COS cells c-RET was lmmunoprecipitated from detergent lysates of GDNF-treated ( + ) 30 (lane 1) or untreated (-) (lane 2) COS cells transfected (lane 3) with c-ret cDNA or mock-transfected with PBK-CMV plasmid (a) immunoblot probed with anti-c-RET antibodies (Santa Cruz) (b) the same filter reprobed with anti-phosphotyrosine Printed from Mimosa 07 11 15 WO 97/18240 PCT/US%/I8197 -9- antibodies Fig 19 a-h GDNF binds m situ to c-ret-positive developing enteric neurons (a, b) Darkfield (a) and corresponding bright-field (b) microphotographs of GDNF 5 antisense cRNA hybridization to paraffin sections through E15 rat gut (c, d) Dark-field (c) and corresponding bright-field (d) micropholograph of in situ binding ol ,2,I-GDNF to E15 rat gut explants (e) c-ret antisense cRNA hybridization to a cryosection through El 5 rat gut (f) Immunostaining of E15 rat gut cryosection with anti-penpherin antibodies (g) GDNF sease cRNA hybridization to E15 rat gut 10 section (h) In situ binding of l25I-GDNF to El5 rat gut explants in the presence of 250-fold excess of unlabeled GDNF —, muscle layer, n, intestinal nerve plexus Bar, lOOfim Fig 20 a - b Crosslinked GDNF-c-RET-complexes are obtained from GDNF-15 responsive tell lines and from r-re/-transfected cells (a) '"1-GDNF was crosslinkcd with EDAC to PC12 cells, NB2/a cells, dissociated E20 rai kidney cells, and COS cells, and the resulting complexes were precipitated by anti-GDNF antibodies (b) EDAC-crosslinked l24I-GDNF-c-RET complexes were immunoprecipitated with anti-c-RET antibodies from the extracts of PC12 cells, subly c-w-transfected 20 (Ret -3T3) or mock-transfected (mock-3T3) 3T3 cells, as well as from dissociated E15 kidney cells in the presence (+) or absence (-) of 500-fold excess of unlabeled GDNF or TGF-/J1 The ~50K bands in all gels are the crosslinked dimcrs of GDNF 25 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-rer-transfected (ret-3T3) but not in mock-transfected (mock) cells (b) GDNF time-dependently increases tyrosine phosphorylation of 160 kD isoform of cRET in c-rez-transfected 3T3 cells Upper panels (Ret -PTyr) are 30 the lmmunoblots stained with anti-phosphotyrosme antibodies, and lower panels (Ret ) show the reprobmg of the corresponding filters with anti-c-RET antibodies Printed from Mimosa 07 11 15 WO 97/1824(1 PCT/US96/1H197 -10- Fig 22 GDNF increases the number of trkC-3T3 fibroblasts transiently expressing c-RET (open squares), but not mock-transfected cells (filled squares) c-ret an'1 transfected cells in five parallels were treated with rat GDNF at indicated concentrations, or with NT-3, for five days Cell number, quantified 5 with Abacus™ Cell Proliferation Kit (Clontech), is expressed as a percent of the control celts without growth factors *, p<0 001 compared to mock transfected cells Fig 23 a-b Purification of receptor from L6 myeloblast cells (a) Plasmon 10 resonance analysis of fractions obtained from anion exchange chromatography of L6 cell lysates Total protein of fractions is also depleted (b) Further purification of 1M fraction obtained from (a) by hydrophobic interaction chromatography Fig 23 Autoradiographic film of the ligand blot 125I-GDNF with proteins from 15 adult rat brain (lane 2) and liver (lane 3) 50-fold excess of unlabeled GDNF (lane 1) significantly reduces the binding DETAILED DESCRIPTION A prerequisite for the understanding of the full range and mechanisms of 20 action of GDNF is the characterization of GDNF receptors and their signaling pathways Although receptors for other members of the TGF-/3 superfamily are well characterized, GDNF receptors remained undefined until this disclosure Disclosed herein is the biochemical characterization of GDNF receptors and their downstream responses in sympathetic neurons and responsive cell lines Using 25 affinity labeling, multiple GDNF binding subunits that mediate cooperative binding of GDNF to embryonic sympathetic neurons are identified Screening of over thirty cell lines initially revealed high expression of GDNF binding proteins of 55 kD, 70 kD, 135 kD and 300 kD in conditionally immortalized neuronal precursors from the raphe nucleus As the data demonstrate, GDNF receptors were highly 30 induced after neuronal differentiation of these cells, which then becamc sensitive to the survival-promoting cffccts of GDNT Different combinations of these subunits were also seen in glioma, myoblast and Sertoli cells A different receptor pattern Printed from Mimosa 07 11 15 WO 97/18240 |'CT/US96n 8197 -11- was found in a motor neuron hybrid cell line, where the predominant component was a CPI-anchored protein of 155kD Despite the striking similarity with the receptor pattern of other TGF-/J superfamily members, immunoprecipitation experiments indicated that GDNF 5 receptor subunits of 55kD, 70kD, 135kD, and 300kD arc novel proteins The 155kD subunit was subsequently determined to be the product of the c-ret proto-oncogene, c-RET, a receptor tyrosine kinase crucial for the development of pans of the excretory and nervous systems GDNF stimulated ERK tyrosine phosphorylation and c-fos mRNA expression with different time-courses in raphe 10 nucleus and motor neuron cell lines, suggesting that different compliments of GDNF receptor subunits can form distinct signaling complexes Conuomitantly, c-RET was identified as receptor for GDNF on additional cell lines GDNF rescues c-RET-positive dopaminergic and noradrenergic neurons in lesion models of Parkinson's disease, suggesting that cRET may mediate the 15 anti-Parkmsonian effects of GDNF in the adult brain c-ret proto-oncogene (Takahashi et al (1985) Cell, 42, 581-588) encodes a protein that is structurally related to receptor tyrosine kinases (Takahashi el al (1988) Oncogene, 3,571-578) Its extracellular part contains an unusual cadherin-likc domain and also a cysteine-rich domain, the biological roles for which are not 20 understood By alternative splicing, several lsoforms 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), bul their biological meaning is currently not understood In several cell lines, c-rct-encoded proteins with molecular weights of 160 kD and 140 kD are described, 25 representing the fully and partially glycosylated isoforms of 120 kD core protein, respectively (Takahashi ei al, 1988) As with other receptor tyrosine kinases, c-RET is activated by homodimerization followed by phosphorylation of us tyrosine residues In the excretory system, c-ret is expressed in the nephric duct, the ureteric 30 bud and the growing tips of the collecting ducts (Pachrus et al , (1993), supra) Mice homozygous for a null mutation in the c-ret gene die soon after birth, with kidneys either absent or rudimentary and displaying severe defects in the enteric Printed from Mimosa 07 11 15 WO 97/18240 PCI A.IS96/18197 -12- nervous system (Schuchardt et al , Nature 367, 380-3 (1994) Based on tins evidence, it had been proposed that the cognate c-rct ligand may be a growth factor important for morphogenesis and neurogenesis During murine enibryogenesis, c-ret mRNA is expressed primarily iri the 5 nervous and excretory systems c-ret mRNA is found in dorsal root, sympathetic, enteric and cranial ganglia (Pachnis et al , Development 119, 1005-17 (1991), as well as in post migratory neural crest cells and in various tumors of neural crest origin, including pheochromocytoma, medullary thyroid carcinoma and neuroblastoma (Ikeda, I , et al Oncogene 5, 1291-6 (1990), Santoro, M , et al 10 Oncogene 5, 1595-1598 (1990) In the developing central nervous system, sites of c-ret expression include the ventral portion ot the neural tube, the retina and motor neurons in spinal cord and hindbrain (Pachnis et al , (1993), supra) However, the pattern ot expression of c-ret m the adult nervous system has not previously been reported 15 The absence of a known ligand for c-RET has basically hampered the studies of intracellular pathways that c-RET can mediate Comparative analysis of the growth-promoting activity of the epidermal growth factor receptor/c-RET chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of epidermal growth factor reccptor 20 (Santoro et al (1994) Mol Cell Biol 14,663-675) We disclose herein that both NGF and GDNF promote survival of PC12 cells, whereas only NGF induces their differentiation, suggesting only a partial overlap in the signaling pathways of c-RET and trkA, a receptor for NGF Binding of an adaptor protein Grb2 to oncogenic forms of c-RET has been demonstrated (Borrello et al (1994) 25 Oncogene, 9, 1661-1668) However, the details of the pathways are completely unknown Now, having GDNF as a ligand, it is possible to address the intracellular signaling of c-RFT upon GDNF binding Like c-RET, GDNF is abundantly expressed in the muscle layei of the gastrointestinal tract and in the condensing mesenchyme of the kidney (Suvanto et 30 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 crosslinked to c-RET in several cell lines and in developing kidney, GDNF specifically induces Printed from Mimosa 07 11 15 WO 97/1H240 PCT/US96/18I97 -13- tyrosine phosphorylation of c-RET, and ectopical expression of c-RET in 3T3 cells confers a biological response of these t_ells to GDNF Thus, c-RET is activated by GDNF and mediates its functions 'Ihe product of the c-ret proto oncogene plays import,i rt roles m human 5 disease Rearrangements and mutations in the c-ret gene are associated with several tumors e g familial medullary thyroid carcinoma, multiple endocrine neoplasia type 2, etc , but also with Hirschsprung disease, a disorder that is characterized by the absence of enteric neurons in the hindgut, resulting in obstipation and megacolon in infants and adults (reviewed in Mak, Y F and 10 Ponder, B A J (1996)Cm/v* Op Genet Dev , 6, 82-86) Identification of GDNF as a ligand for c-RET further enables the analysis of the molecular basis of these diseases Particularly, the mutations in GDNF gene can now be studied as possible cause for the Hirschsprung disease in the cases where c-ret locus is not mutated The phrases "GDNF receptor" and "receptor for GDNF" as used herein 15 each refer to a single subunit which binds GDNF as well as combinations of the receptor subunits which bind GDNF The term "effect" as used herein means an alteration or change An effect can be positive, such as causing an increase in some material, or negative e g , antagonistic or inhibiting 20 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 25 a compound which has been separated from its native environment or, if recombmantly expressed, from its expression environment The phrase "substantially pure" as used herein in reference to a compound means an isolated compound which has been separated from other components which naturally accompany it Typically, a compound is substantially pure when it 30 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 Printed from Mimosa 07 11 15 WO 97/182-10 PCT/US96/I8I97 -14- The phrase "nan-permissive culture conditions" as used herein rt.fi.rs to conditions which do not normally support survival of the cells being cultured in vitro, t g , temperature, media components, etc The phrase "an excess" as used in reference to the addition of labeled 5 GDNF in a competitive assay refers to an amount of labeled GDNFsufficient to facilitate the detection of a competing compound — for example, an amount of labeled GDNF which is twice the amount of the compound to be tested The term "bind" as used herein refers to the interaction between the GDNP ligand and its receptor, the binding being of a sufficient strength and for a 10 sufficient time to allow the detection of said binding under the conditions of the assays disclosed herein The term "about" in reference to a numerical value means ±10% of the numerical value, more preferably ±5%, most preferably ±2% 15 The specific examples presented below demonstrate that 1) GDNF receptor is present in multiple neuronal and non-neuronal cell types, 2) GDNF receptor is composed of multiple subunits which cooperate to achieve high affinity binding, 20 3) members of the ERK/MAP kinase family are components of the GDNF signaling mechanism, and 4) c-RET is a functional receptor for GDNr 1 Novel GDNF receptor expression in multiple neuronal and non-neuronal cell 25 types Heretofore unidentified receptors are identified herein as GDNF receptors These novel GDNF receptors were found in cells lines of different origins, although they appeared to be most abundant in neuronal cells Preferably, the cell lines are selected from the group consisting of RN33B, RN46A, and C6 (see Table 30 I), with RN33B being most preferred The identification of GDNF receptors in many of these cell types suggests novel cellular populations responsive to GDNF in vivo GDNF has been shown to promote survival and phenotype of distinct Printed from Mimosa 07 11 15 PCT/US 96/1819 jpEWUS 2 2 JAN '9E -15- subpopulations of neurons, in particular dopaminergic and noradrenergic central neurons, as well as spinal and facial motor neurons Given the activities of GDNF in various monoaminergic neurons, the discovery of GDNF receptors m cell lines derived from the medullary raphe indicate that serotonergic neurons may also respond to GDNF m vivo The endogenous expression of GDNF by these cells suggests that this factor may act in a paracrine/autocrine fashion within the raphe nucleus Expression of GDNF receptors m Sertoli TM4 cells suggests non-neuronal roles for GDNF in developing testis In vivo, the temporal expression of GDNF rnRNA in testis correlates with the expansion of the Sertoli cell population (Trupp et al , supra) which, together with the discoveiy of GDNF receptors on the TM4 cell line, suggest an autocrine action of GDNF during Sertoli cell maturation Similarly, the presence of GDNF receptors in rat myoblast L6 cells, together with the expression of m developing muscle in vivo (Henderson et al , supra, Trupp et al , supra), indicates a potential paracrine role of GDNF durmg myogenesis. Despite the presence of receptors and biological activities of GDNF on embryonic sympathetic neurons, PC12 cells which had been differentiated into sympathetic-like neurons with NGF did not express GDNF receptors under initial experimental conditions As discussed below, however, GDNF receptors were ultimately identified on PC 12 cells GDNF receptors are absent in the pons noradrenergic cell line CATH a Given the robust effects of GDNF on adult central noradrenergic neurons from the locus coeruleus, the absence of GDNF receptors in CATH a is intriguing Recendy, however, Gong et al reported that GDNF can prevent the degeneration of CATH.a cells induced by 6-OH-dopamme treatment (Gong et al , 21 Abs Soc Neurosci , 1789, 1995), suggesting that GDNF receptors may be induced in these cells after 6-OH-dopamme lesion Indeed, in vivo studies have shown that GDNF elicits a more profound induction of the phenotype of noradrenergic neurons following 6-OH-dopamme injection than in the non-lesioned locus coeruleus Further, Treanor et al recently reported upregulation of GDNF binding in sections of the substantia nigra after medial forebram bundle transection (Treanor et al ,21 Abs Soc Neurosci 1301, 1995), suggesting that the receptor upregulation may be a general mechanism of control of GDNF responsiveness in the central nervous k LiCklftCIt CLICET W0«>7/18240 rcmis^/i 81 <>7 -16- system GDNF receptor upregulation was also observed during in vitro differentiation of raphe nucleus cells These lines have recently been shown to retain the ability to respond to local microenvironmental signals after 5 transplantation into the adult brain, where they differentiate in a direction that is consistent with that of endogenous neurons in the transplantation site (Shthabuddm et al , 15 J Neurosci 6666, 1995) In vitro, however, a shift to the non-pernussive temperature differentiates them along default pathways into glutamatergic (RN33B) or serotonergic (RN46A) phenotypes, respectively 10 Differentiation in culture has also been shown to upregiilate expression of receptors tor other trophic factors in these cells, including the neurotrophin receptors p75Lvc.FR an(j tr|j} (Whittemore and White, 615 British Res 27, 1993) Although they can give rise to different neuronal types depending upon the site of transplantation, RN33B cells are not able to generate glial elements suggesting 15 these cells represent neuronally restricted multipotent precursors (Shihabuddin ct al , supra) In this respect, it is interesting to note the absence of GDNF receptors in two plunpotent neuronal stem cell types (Renfranz et al , 66 Cell 713, 1991, Snyder el al , 30 68 Cell 33 1992) suggesting that these cells are less restricted than the raphe nucleus cell lines Taken together, these observations suggest that 20 GDNF receptor expression may initially appear in newly differentiated post-nutotic neurons and increase progressively during neuronal maturation 2 Multiple GDNF receptor subunits The data demonstrate that novel GDNF receptor is composed of multiple 25 subunits which cooperate to achieve high affinity binding The cooperative binding of GDNF to embryonic sympathetic neurons may thus be an indication of a multi-step mechanism of receptor assembly Because binding assays were performed at 4"C, binding cooperativity is unlikely to have resulted from substantial lateral mobility of transmembrane receptor proteins suggesting that GDNF binding 30 induces conformational changes on receptor complexes that are partially preformed on the membrane The nearly identical affinities of the different GDNF receptor subunits obtained by crosslinking also support the notion of cooperative binding of Printed from Mimosa 07 11 15 WO 97/18240 1'CT/US!>6/18197 -17- GDNF to a partially prc-asseinbled receptor complex The structural similarities between GDNF anil members oi the TGF-/3 superfamily suggest that receptors for GDNF might conform to some of the prototypes described for receptors of members of the TGF-18 family Indeed, the 5 pattern of GDNF binding proteins described herein is strongly reminiscent of type I, type II and type III TGF-/3 receptors Despite the overall similarities between GDNF and TGF-/3 superfamily receptors, no GDNF receptors could be detected in several cell lines known to express various TGF-/3 and activin receptor subunits, including the mink lung 10 epithelial cell line MvILu In agreement with this observation, no binding of GDNF has been detected in COS cells transfected with diffeant combinations of known type I and type II TGF-/3 superfamily receptors (Ibanez, C , unpublished, P ten Dyke, personal communication), including the recently isolated type II receptor for BMPs (Rosenzweig et al , 92 PNAS USA 7632, 1995) and a novel brain-15 specific type 1 receptor (Ryden et al , 21 Abs Soc Neurosci 1754, 1995) Moreover, no GDNF receptor complexes could be recovered after immunoprecipiution with antipeptide antiscra against any of the cloned TGF-/3 superfamily receptors indicating that GDNF receptor components are novel proteins 20 3 c-RET ii a receptor for GDNF GDNF receptors were found in a motor neuron-neuroblastoma hybrid cell line, but not in a basal forebrain cell which was also a hybrid with the same neuroblastoma, suggesting that the receptors detected on MN-1 cells represent 25 physiologically relevant motor neuron GDNF receptors In contrast to raphe nucleus cells, GDNF expression could not be detected in the motor neuron cell line, consistent with a target-derived mode of action for muscle-derived GDNF in vivo (Henderson et al , 266 Science 1062, 1994, Trupp et al , supra) GDNF binds to and induces tyrosine phosphorylation of the these receptor which were 30 identified as the product of c-ret c-ret was also able to mediate GDNF binding and survival/growth responses to GDNF upon transfection into naive fibroblasts Moreover, dopaminergic neurons of the adult substantia nigra were found to Printed from Mimosa 07 11 15 WO 97/18240 PCT/IJS96/1R197 -18- exprLss high levels of c-ret mRNA, and c-RET expressing dopaminergic and noradrenergic neurons in the CNS responded to the protective cffccts of exogenous GDNF in vivo Together, these data indicate that the product of the c-ret proto-oncogene encodes a functional receptor for GDNF which may mediate the 5 neurotrophic effects of this factor on dopaminergic, noradrenergic and motor neuroas The results disclosed herein indicate that the c-RET receptor tyrosine kinase is a signal transducing receptor for GDNF This finding is surprising, given that all receptors for members of the TGF-/3 superfamily characterized so far are 10 receptor serine-threonine kinases (Dcrynck, R Trends Biochem Set 19, 548-553 (1994), Attisano a al , J Bba-Mol Cell Res,222, 71-80 (1994)) GDNT is m fact a very divergent member of the TGF-/3 superfamily, with which it shares primarily the spacing between conserved cysteine residues in the amino acid sequencer Its ability to interact with a receptor tyrosine kinase indicates a further functional 15 divergence from other members of the TGF-/3 superfamily Conversely, these findings could suggest that other TGF-/3 superfamily 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 20 i) GDNF binds to COS cells ectopically expressing the c-ret proto-oncogene, n) GDNF can be chemically crosslinked to the product of the c-ret proto-oncogene ectopically expressed in COS cells or 25 from NB2/d and PC12 cells, m) the c-ret proto-oncogene product ectopically expressed in COS cells, but also in NB2/a cells, becomes rapidly phosphorylated 30 on tyrosine residues upon GDNF binding, iv) GDNF promotes biological effects i e autogenic or trophic in cells expressing c-ret proto-oncogenic products Printed from Mimosa 07 11 15 WO 97/18240 I'C r/USW/18197 -19- GDNF specifically binds to RET-expressing (Figure 19 l, d, h) enteric neurons and the tips of ureteric buds in developing kidney These tissues wurc absent or severely reduced in r-ref-deficient mice (Schuchardt et al (1994) Name, 367,380-383, Durbec et al (1996) Development, 122,349-358) The data 5 disclosed herein further demonstrate GDNF-c-RET complexes from GDNF- rcsponsive and c-rer-transfected cells and from embryonic kidney cells Finally, GDNr time and dose-dependently activates c-RET, and introduction of c-ret into GDNF-nonresponsive cells results in GDNF-responsiveness 10 4 Downstream signaling pathways activated by GDNF receptor Investigation of GDNF signal transducing mechanisms in raphe nucleus and motor neuron cell lines has been conducted The downstream responses clicited by GDNr in these cells demonstrate that the GDNF binding proteins identified herein represent functional GDNF receptors The initial biochemical characterization of 15 GDNr signal transduction pathways has identified members of the ERK/MAP kinase family as components of the GDNF signaling mechanism ERK/MAP kinase activation by phosphorylation is the final step in a cascade of kinases that is set in motion after activation of the Ras pathway by various growth factors, including TGF-/3 (Yan et al , 269 J Biol Chem 13231, 1994, Hartsough and Mulder, 270 20 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 <?lso been shown to form part of the signal transduction pathway activated by several cytokines, such as interferons and interleukins, which are not known to activate Ras (David et al , 269 Science 1721, 1995) Whether or not Ras activation is one 25 of the steps in the signaling transduction mechanism of GDNF is an area of further interest Interesting differences were found between the patterns of ERK phosphorylation induced by GDNF in raphe nucleus RN33B cells and in motor neuron MN-1 cells GDNF treatment stimulated very rapid (maximum at 5 min) 30 and transient (undetectable after 60 min) tyrosine phosphorylation of ERKI and ERK2 in RN33B cells, but relatively slower (maximum at 15 min) and more sustained (still detectable after 120 min) phosphorylation of ERK2, but not ERKI, Printed from Mimosa 07 11 15 WO 97/18240 I'CTAJS96/I8I97 -20- in MN-1 cells That these diflerences may have functional significance is suggested by recent observations made in PC12 cells treated with different growth factors Exposure of PC 12 cells to NGF or fibroblast growth factor (FGF) results m neuronal differentiation and in sustained elevation of Ras activity and ERK tyrosine 5 phosphorylation (Qiu and Green, 7 Neuron 977, 1991) In contrast, treatment with epidermal growth factor, which stimulates DNA synthesis and proliferation of PC12 cells, results in only transient (< 1 hr) activation of Ras and ERKs (Qiu and Green, 1991) Thus, different tune-courses of ERK activation underlie different biological responses m PC12 cells Talan together, tne different patterns of GDNr 10 receptors and GDNF-mduced ERK phosphorylation in RN33B and MN-1 cells suggest that dilferent GDNF receptor subunits can cooperate to assemble distinct signaling complexes m different cell types Whether different GDNF signal transduction pathways underlie the different biological effects of GDNF is an area of further interest 15 Upon activation, ERKs translocate to the nucleus where they phosphorylatc and thereby regulate the activity of transcription factors which, in turn, control gene expression Phosphorylation ol p67SRF and p62TCF transcription factors rccruits them to thL scrum response element (SRE) in Uil c-fos gene promoter and stimulates c-fos gene transcription (Gille et al , 358 Nature 414, 1992) 20 Transcription of c-fos is rapidly and transiently induced after various stimuli, including exposure of PC12 cells to NGF (Millbrandt, 83 PNAS USA 4789, 1986) and of osteoblastic cells to TGT-(i (Machwate et al , 9 Mol Endocrin 187 1995) c-fos forms part of the AP-1 transcription factor complex, which is thought to be involved m the regulation of multiple genes, including growth factor 25 neuropeptide and neurotransmitter synthesizing enzyme genes (Gizang-Ginsberg and Ziff, 4 Genes Dev All, 1990), Hengerer et al , 87 PNAS USA 3899, 1990, Jalava and Mai, 9 Oncogene 2369, 1994) The stimulation of c-fos transcription by GDNF indicates a role for AP-1 complexes in GDNF-induced gene expression Thus, c-fos could mediate the increase in the tyrosine hydroxylase (TH) expression 30 observed upon GDNF treatment of central noradrenergic neurons, or the GDNF-mduced upregulation of vasoactive intestinal peptide (VIP) and preprotachykinin-A (PPTA) mRNAs in cultured sympathetic neurons from the superior ce-vical Printed from Mimosa 07 11 15 WO 97/18240 PCT/US96/18197 -21- ganglion (Trupp et al , 130 J. Cell Biol 137, 1995) The effects of GDNF on the survival of differentiated serotonergic raphe nucleus cells indicate that (lie GDNF receptors identified on these cells are able to clicit relevant biological responses The fact that cessation of proliferation, 5 differentiation, and GDNF responsiveness were concomitant with increased GDNF receptor expression in these cells, suggests that GDNF may be a survival factor for developing serotonergic raphe neurons in vivo The data of this patent disclosure suggest a role for ERKs and c-fos in GDNF-mediated neuron survival This can be directly established using dominant negatives or antisense oligonucleotides 10 The GDNF receptor subunits and complexes disclosed herein have wide- range applicability The identification and isolation of GDNF receptor facilitates rational drug design for drugs useful in treating, for example, neuronal disorders, particularly those involving neuronal cell death As was discussed previously, GDNF has been shown to promote survival of adult substantia nigra neurons m 15 vivo following pharmacological treatments and lesions that mimic Parkinsonian syndromes, as well as survival responses in other neuronal cell lines The drugs can be tested for binding affinity to gdnf receptor, and for their influence on the downstream effect of GDNF disclosed below — i c , the phosphorylation of ERK2 and ERKI As GDNF receptor has also been identified on malignant cell lines, 20 design of drugs for use in cancer therapy is also evident Further considering structural similarity with BMP, the development of drugs to be used m treating bone-related diseases, i e , osteoporosis and for promoting the healing of fracturcs is also contemplated Accordingly, isolated receptors according to the present invention can be 25 used, inter alia, to screen for compounds or compositions which arc analogs and homologs of GDNF The potential analogs and homologs can be screened initially in competitive binding assays employing either isolated receptor or cell lines expressing the receptor - 1 e , NB2/a cells — and 125I-labeled GDNF Methods such as those disclosed m Example 13 can be used Analog or homolog activity can 30 then be ascertained by further identifying those compounds or compositioas which for example, effect a decrease or increase, respectively, in the tyrosine phosphorylation of the RET proto-oncogene Methods such as those disclosed in Printed from Mimosa 07 11 15 WO 97/18240 PCT/US96M8197 -22- Example 17 can be used Alternatively, GDNF can be used to screen for and identify other rectptors using the above-reference procedures, or variations thereof The isolation of GDNF receptor also facilitates the development ot antibodies, both polyclonal and monoclonal, against the receptor These antibodies 5 can be used to purify the receptors themselves, identify other cells expressing GDNFreceptor, thereby prompting other therapeutic applications, identify other Type I-interactive receptors, as well as be used as drugs themselves The antibodies can initially be produced using the ligand/receptor complexes disclosed herein as the immunogens Antibodies specific for the ligand can be eliminated 10 from the polyclonal serum by absorption with the ligand Hybridomas for monoclonal production can be selected on the basis of binding of ligand, with the expansion of only those clones which do not bind the ligand uncomplexed with the receptor The antibodies can be prepared by methods well known to those skilled in the art 15 Alternatively, monoclonal and polyclonal antibodies against GDNF receptor and GDNF proteins can be used for the characterization 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 antiidiotype antibodies which mimic GDNr 20 The isolation of GDNF receptor also facilitates the isolation and/or production of nucleic acids for the expression of recombinant GDNF receptor, both in vitro and in vivo, for diagnostic and therapeutic applications The term "nucleic acids" as used 25 herein includes, for example, genomic DNA, mRNA, and cDNA Upon sequencing at least a portion of the GDNF receptor, oligonucleotide primers for isolating genomic DNA for GDNF receptor and receptor mRNA can be developed cDNA can be prepared from isolated mRNA The isolation and 30 production of nucleic acids can be accomplished utilizing methods well known to those skilled in the an using standard molecular biology techniques such as are set forth in Maniatis et al , Molecular Cloning A Laboratory Manual, Cold Spring Printed from Mimosa 07 11 15 WO 07/18240 rcr/ns%/i8i97 -23 Harbor Laboratory, Cold Spring Harbor, New York, 1982, incorporated herein by reference Recombinantly 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 5 antagonistic properties The nucleic acids as set forth above can be utilized for gene therapy, using both in vivo and ex vivo techniques The nucleic acids can also be used to clone other related receptors using, for example, low stringency screcns and reversed transcriptase PCR, and to produce cells overexpressing Ihe receptors to screen for 10 other ligands, c g , by panning, and other materials serving as receptor agonists, antagonists, or partial agonists and antagonists Alternatively, recombinantly produced receptor itself can be used for the screening assays Additionally, cells expressing chimeric receptors can be produced using other TGF-/3 receptor family members to elucidate signal pathways Intracellular targets of GDNF receptor can 15 be identified using, for example, the yeast two-hybrid system (Chen, et al , 377 Nature 548, 1995, incorporated herein by reference ) The nucleic acids set forth above can also be used to develop transgenic and/or gene targeted animals For example, transgenic animals can be developed for testing the effects of the overcxpression of GDNF receptor Procedures can be 20 utilized such as are described in Hogan et al , Manipulating the Mouse Emhlvo 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 reference Alternatively, cell lines and transgenic animals unable to express 25 GDNFreceptor ean be prepared to ascertain the effects of blocking signaling by GDNF Procedures such as are set forth in Wurst et al , Gene Targeting Vol 126, edited by A L Joyner, IRL Press, Oxford University Press, Oxford England, pp 33-61, 1993. incorporated herein by reference, can be utilized Other applications and modifications are within the spirit and scope of the 30 invention as herein disclosed and will be readily apparent to those skilled in the art EXAMPLES Printed from Mimosa 07 11 15 WO **7/18240 l'CT/US96/18197 -24- The following Examples arc provided for purposes of elucidation and nut limitation on the disclosure or claims Unless otherwise indicated, binding and biochemical studies were carried out with recombinant rat GDNF produced in Sf21 insect cells using a baculovirus 5 expression system The protein was produced and purified as previously described (Trupp et al , supra, incorporated herein by reference) GDNF protein was quantified after silver staining of SDS/PAGE gels using standard curves obtained with commercial samples of proteins of molecular weight similar to that of GDNF Purified human TGF-/31 was generously provided by Jun-icln Koumegawa, Kirin 10 Brewery, Tokyo, Japan Proteins were labeled with Na-12,I by the chloramme-T method to a specific activity of approximately 1 x 10s cpm//ig Unless otherwise indicated, binding assays were performed as follows Cells were incubated with lodinated GDNF in Dulbecco's phosphatebuffered saline and 2 mg/ml bovine serum albumin (BSA) on Millipore Hydrophilic Durapore 96-15 well filtration plates Following two hours of vigorous shaking at 4°C, the cells were washed twice with ice-cold binding buffer under vacuum Dned filters were liberated and bound 1251-GDNF quantified in a gamma counter Non-specific binding was determined by addition of 500-fold excess of cold ligand to the binding mixtures 20 For affinity labelmg, lodinated proteins were bound to monolayer cultures of primary neurons or cell lines Prior to binding, dissociated chick sympathetic neurons were cultured for 48 hours in the presence of NGF on polyorruthine/lamirun coated dishes Plated cells were incubated with 10 ng/ml ,2<il-GDNF at 4°C in binding buffer as described above Ligand/receptor complexes 25 were chcmically cross-linked for thirty minutes at room temperature using either disuccinimidyl suberate (DSS) or l-Ethyl-3(-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC) (Pierce Chemical, Rockland, IL) Following quenching of the cross-linking reactions, cells were washed twice with lOmM Tris/HCI buffered saline, 2 mM EDTA, 10% glycerol, 1% NP-40, 1% Triton x-100, 30 10 fig/ml leupeptin, 10 /ig/ml antipain, 50 ug/ml aprotinin, 100 /ig/ml benzamidine hydrochloride, 10 fig/ml pepstatin and 1 mM PMSr(proteinasc inhibitors from Sigma) Cleared lysates were boiled for 5 min in SDS//3 mercaptoethanol buffer Printed from Mimosa 07 11 15 WO 97/18240 PCTA1S96/I8I97 -25- fractionated by SDS/PAGE on 4-20% gradient electrophoresis gels, and visualized by autoradiography Molecular weights indicated were obtained by subtracting the weight of a GDNF ligand monomer, e g , 25-30 kD, more preferably 23 kD, from the estimated molecular weights of cross-linked complexes visualized by 5 SDS/PAGE For affinity measurements of cross-linked complexes, cells were incubated on plates as above in the presence of increasing amounts of unlabeled GDNF These samples were fractionated by gradient SDS/PAGE, gels were then dried and specific bands excised according to molecular weights determined from autoradiograms, and count in a gamma counter For unmunoprecipitation of 10 affinity labeled receptor complexes, after binding and cross-linking with lodinated hgands, cell lysates were cleared and incubated overnight at 4 C with 5-10 /il of antipeptide rabbit antisera against difference type 1, 11 and III TGF-0 superfamily receptors (ten Dyke et al , 264 Science 101, 1994) (provided by Peter ten Di|ke, Ludwig Institute for Cancer Research, Uppsala, Sweden) Immunocomplexes were 15 collected with Protein A-Sepharose (Pharmacia Sweden), washed in lysis buffer and boiled for 5 minutes before SDS/PAGE and autoradiography as above Example I 20 GDNF receptors on embryonic sympathetic neurons GDNF promotes survival of cultured embryonic chicken sympathetic neurons with similar efficacy and dose response curve as nerve growth factor (NGF) (Trupp et al , supra) Chicken sympathetic neurons, isolated and prepared as previously described (Trupp et al , supra) Saturation binding with lodinated 25 GDNF was carried out on neurons isolated from embryonic day 10 (ElO) chick paravertebral sympathetic ganglia mechanically dissociated in the presence of trypsin The preparation was preplated for two hours on untreated tissue culture plastic in order to enrich in neurons and allow for re expression of receptors Plots of saturation binding data produced a sigmoidal curve from which a Kd of 400 pM 30 could be approximated (Fig la) In agreement with the sigmoidal behavior of this curve Scatchard transformation of the data produced an inverted U-shaped curve indicative of cooperative binding (Fig lb) The measure of cooperativity of Printed from Mimosa 07 11 15 WO 97/182-40 rCT/US9(i/|S197 -26- bmding can be ascertained from a Hill transformation which produced a positive slope of 1 63 (Fig lc), suggesting oligomenzation of either ligand or receptor subunits In order to identify GDNF binding components on the membrane of 5 sympathetic neurons, chemical cross-linking of lISI-GDNF to these cells was utilized, followed by visualization of the resulting complexes by SDS-PAGE Gradient gel electrophoresis resolved binding proteins of 70 and 300KD (molecular weights of GDNF receptor suhunits reported hereafter were obtained by subtracting the weight of a GDNF ligand monomer, e g , 23kD, from the estimated molecular 10 weights of cross-linked complexes visualized by SDS/PAGE), resulting in a pattern ol bands which resembled that obtained after cross-linking of TGF-/31 to MvILu mink lung epithelial cells (Fig 2) This result suggested that, like TGF (3 receptors, GDNF binding proteins may also form an oligomenc receptor system A large excess of cold ligand displaced lodinated GDNF from the receptor complex 15 indicating the specificity of the labeling Example 2 GDNF receptors on ccll lines Over thirty cell lines were screened for expression of GDNF receptors using 20 affinity labeling with lodinated GDNF (Table I, infra) Except as otherwise noted, all cell lines used in this study are available from and described by the American Type Culture Collection, Rockville, MD A875 human neuroblastoma was provided by Mart Saarma, University of Helsinki, Finland CATH A, a noradrenergic cell line isolated from a tumor in the pons of transgenic mice 25 expressing SV40 T antigen under the transcriptional control of a tyrosine hydroxylase promoter (Sun et al , 1993), was generated and provided by Dona Chikaraishi, Tufts University School of Medicine, Boston, MA The rat neural stem cell line CI7-2 (Snyder et al , 68 Cell 33, 1992) was generated and provided by Evan Snyder, HarvardMedical School, Boston, MA LANS human 30 neuroblastoma was provided by Svcn Pahlman, Uppsala University, Sweden David Hammond, University of Chicago, produced and provided SN6 cells a hybrid of mouse basal forcbrain cholinergic neurons and the mouse neuroblastoma N18TG2 Printed from Mimosa 07 11 15 WO 97/18240 l'CT/US96/18197 -27- (Hammond 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 ST15A rat neural stem cell line was kindly provided by Ron McKay, National Instituted of Health, MD The generation and 5 characterization of raphe nucleus tell lines RN33B and RN46A has been described elsewhere (Whittemore and White, 1993) RN33B and RN46A tells were obtained from Dr Scott Whittemore of the University of Miami The motor neuron hybrid cell line 2H 10 14 (referred to here as MN-1) has been previously described (Salazar-Grueso et al , 2 Neuroreport 505, 1991) 10 Multiple GDNF receptor subunits were detected in various glial, neuronal and non-neuronal cells (Tabic I) A large molecular weight band of 300kD appeared to be the most prevalent speucs in several cell lines after crosslinking with disuccymidyl suberate (DSS), and it was the only receptor which appeared to bind ligand in the absence of all other receptors (Table I) A similar pattern was 15 seen in rat C6 glioma, mouse Sertoli TM4 cells, and in two cell lines derived from embryonic neuronal precursors of the rat raphe nucleus, which have previously been shown to express multiple neuronal markers, including glutamate- (RN33B) and serotonin- (RN46A) synthesizing enzymes (Whittemore and White, 615 Brain Res 27, 1993, White et al , 14 J Neuro , 1994, Eaton et al , 170 Dev Biol 20 169,1995) The consensus 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 and Table I) The 50-55kD component often ran as a doublet or triplet The smeary appearance and heterogeneous range of sizes displayed by the large molecular 25 weight component suggests a post-translational modification, presumably glyeosylation, and appears similar to that previously described for type III betaglycan TGF-j3 receptors This species was somewhat smaller in the cells derived from the raphe nucleus, which could indicate either a distinct core protein or difference levels of glyeosylation 30 GDNF receptors could not be detected in pheochromocytoma PC12 cells under the present assay conditions of 4°C, even after NGF-induced differentiation into a sympathetic neuron-like phenotype (fable I, and data not shown) No or Printed from Mimosa 07 11 15 WO 97/18240 PCTA)S9fi/18197 -28- very low GDNF receptor expression could he seen in variuus nturoblastomas, and in two pluripotcnt neuronal stem cells (Table I) Printed from Mimosa 07 11 15 WO 97/18240 l'CT/l!S96/18197 10 15 20 25 30 CELL LINE DESCRIPTION A87S Bulb SFME CATH a MvlLu COS-7 C2-C12 C6 CI 7-2 FR-3T3 HELA LANS L6 MN-1 NB41A3 NRK-49r PC 12 P19 RN33B RN46A SK-N-MC SK-N-SH SN6 ST15A SW1353 SY5Y TM3 TM4 U138MG -29-TABLE I 55kD 70kD 135kD 155kD 30()kD huinun melanoni.i mouse embryonic cell rat pons noradrenergic mink lung cpithelm ct.ll monkey kidnt,v fibrublasi mouse myoblast rat glioma rat CNS stem cell rat fibroblast human cervical carcinoma luiman neuroblastoma rat myoblast mouse motor neuron TH+ mouse neuroblastoma rat kidney fibroblast rat pheochromocytoma mouse embryo carcinoma rat raphe nucleus (glutamat) rat raphe nuclcus (seroton) human neuroepithelioma DRH+ mouse neuroblastoma mouse basal forebrain (cholin) rat CNS stem cell human chondrosarcoma human neuroblastoma mouse Leydig cell mouse Sertoli cell human Glioblastoma + - 4 + +■ + + + + + 35 Presence (+) or absence (-) of specific GDNF receptor complexes in the designated cell line Affinity labeling using the cross-linker ethyl-dimcthyl-aminopropyl carbodnmide (EDAC) revealed the presence of an additional GDNF receptor 40 component 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 Printed from Mimosa 07 11 15 WO 97/1824(1 PCT/IJS9(i/l8197 -30- receptors of 50-55KD and 65-70kD, the high molecular weight subunu of 300KD was, however, not as efficiently cross-linked by EDAC (rig 3) The raphe nucleus cell lines are only conditionally immortalized and do not show signs of transformation At the non-permissive temperature and in defined 5 medium, they stop proliferating and differentiate into postmitotic neurons (Whittemore and White, 1993) GDNF binding was greatly increased in RN33B and RN46A cells following differentiation (not shown) The overall pattern and the relative amounts of GDNF receptor components did not change alter differentiation 10 Analysis of GDNF binding proteins on the rat myoblast cell line L6 revealed a different pattern of receptor subunits marked by the apparent absence of 50-55kD and 65-70kD receptors Only the high molecular weight component of 200-400kD could be seen after cross-linking with DSS (Fig 3) Crosshnking with CDAC, however, readily labeled the 120-135kD subunit previously seen in C6, 15 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 mouse spinal cord motor neurons and the N18TG2 mouse neuroblastoma, followed by 20 selection of clones expressing high levels of choline acetyltransferase activity (Salazar-Grueso et al 2 Neuroreport 505, 1991) Importantly, SN6, a hybrid cell line of embryonic mouse basal forebrain cholinergic neurons and the same N18TG2 neuroblastoma (Hammond ct al , 234 Science 1237, 1986), showed no GDNT receptors (Table I), indicating that the GDNF binding proteins seen on the motor 25 neuron cell (hereafter referred to as MN-1) are likely to represent GDNF receptor components present in spinal motor neurons As with the L6 myoblasts, the predominant receptor in MN-1 cells was preferentially cross-linked with EDAC, although in these cells it was a larger protein of 155kD (Fig 3) This was subsequently identified to be a c-RET receptor (see Example 9 below) MN-1 30 cells also expressed 65-70kD binding proteins and low amounts of the 300kD receptor (Fig 3 and Table 1) In order to dissect the individual constituent affinities of GDNr receptor Printed from Mimosa 07 11 15 9 WO 97/182-10 I'C I/US96/I8197 -31- subunits, displacement binding assays were performed, followed by cross-linking and SDS-PAGL Receplor-ligand complexes were visualized by autoradiography, cut out from the gel and counted in a gamma countcr The resulting displacement curves indicated a Xd of approximately 0 2 nM for all components on RN33B and 5 MN-1 cells (Fig 4 a-c) These data at present do not clearly establish whether all GDNF receptor subunits display similar binding affinities or, whether they are all required to assemblt a high affinity receptor complex Example 3 10 Biochemical characterization of GDNF receptors The overall similarity in the pattern of receptors between GDNF and TGF-/3 prompted an examination of whether any of the previously identified receptors for TGF-/8 superfamily members was part of the GDNF reccptor complex Cross-linked 12SI-GDNF-receptor complexes from differentiated RN33B cells were 15 subjected to immunoprecipitation with different anti-peptidc antisera specific tor all cloned TGF-f3 superfamily receptors, including type I receptors (ALK-1 to ALK-6), type II receptors TBR1I, ActRII and BMPRII the type III receptors betaglycan, and endoglin In a parallel control experiment, ll5I-TGF-01 was cross-linked to type I, type II and type III receptors on the mink lung epithelial cell line MvILu 20 followed by immunoprecipitation with antisera against TBRI (ALK-5) TBRII and betaglycan, respectively Although type I, type II and type III TGF-/9 receptors were recovered in the control experiment none of the GDNF receptor components in differentiated RN33B cells could be immunoprecipitated by any of the tested antiiera (not shown) These data confirmed that the GDNF receptor subunits 25 expressed on these cells are novel proteins Example 4 Endogenous GDNF expression in cell lines expressing GDNF receptors Traditional models for the action of neurotrophic factors have described 30 them as target-derived polypeptides that promote survival and differentiation of specific neuronal subpopulations More recently, it has become evident that neurotrophic factors may also have paracrine and even autocrine modes of action Printed from Mimosa 07 11 15 WO 97/18240 I'C I/US96/18197 -32- (Ernfors and Persson, 3 Eur J Ncurosci 953, 1991, Acheson ei al , 374 Nature 450, 1995) Expression of GDNF mRNA in cell lines expressing GDNF receptors was exnmined Cells were homogenized in guanidine isotluocyanate (GITC) and (}-intrcaptoethiinol R.NA 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-1, expressed substantial levels of GDNF mRNA as assayed by RNAse protection analysis (Fig 5) The highest GDNF mRNA expression was found in cells from raphe nucleus, which showed up to 5-fold higher expression than 10 postnatal day 1 (PI) kidney, one of the richest sources of GDNF mRNA in the developing rat (Tnipp et al . supra) Interestingly, upon differentiation of RN33B cells, GDNF mRNA expression decreased to about 30% of the level in undifferentiated cells (Figure 5) GDNF treatment of differentiated RN33B cells did not alter the expression of GDNF inRNA (Fig 5) or GDNF receptors (not shown) 15 Expression of c-ret mRNA was investigated in RN33B, L6, and MN-1 cells, using the RNAse protection assay Ten micrograms ot total RNA from the cell lines indicated wasa analyzed using a nboprobe complementary to 400 nucleotides of coding sequence from the kinase domain of the mouse c-ret mRNA Although high expression was seen in MN-1 cells, no c-ret mRNA was detected m 20 cither the RN33B or L6 cells (Fig 6) These results indicate that a signaling receptor for GDNF other than c-RET must be present in these cells Example 5 25 Activation of the ERK signal transduction pathway in GDNF responsive cell lines Whether the GDNF binding proteins characterized in cell lines were able to form ligand-dependent signaling complexes was also investigated Cell monolayers in 10 cm plates were incubated at 37"C in the presence of 50 ng/ml GDNF for the 30 indicated time periods and immediately lysed with 1 ml of ice cold lysis buffer (as above) with the addition of 1 mM sodium othovanadate Whole cell lysates were fractionated by SDS-PAGE (10% polyacrylamide) and blotted to nitrocellulose Printed from Mimosa 07 11 15 WO *>7/1K240 PCT/US%/1H197 -31- filters Western hints were probed with an anti-phosphotyrostne antiserum (UBI, Lake Placed, NY), followed by horseradish peroxidase-conjugated goal ami-mouse IgG and developed with Ihe ECL Western Detection System (Amersham, UK) For reprobmg, blots were first stripped by a 30 minute incubation at 50CC in 62 5 mM 5 Tris-FICI pH6 7, 100 mM /J-mercaptoethanol, 2% sodium dodecyl sulphate After removal of antibodies, blots were probed with a rabbit polyclonal antisera raised against recombinant rat ERK2 (a gift of Ten Boulton, Regeneron Pharmaceuticals Inc , Tarrytown, NY) which recogni/ts both ERKI and ERK2, and developed as above using a horseradish peroxidase-conjugated goat anti-rabbit secondary 10 antibody Because of their distinct patterns of GDNF receptor subunits, intracellular signaling responses were initially characterized in the raphe nucleus tell line RN33B and in the motor neuron lcII line MN-1 Changes in the pattern of tyrosine-phosphorylatcd proteins elicited by GDNF treatment of RN33B or MN-1 1*) cells were investigated Tyrosine phophorylation is a universal mechanism of regulation of intracellular signaling proteins that is stimulated by numerous cytokines and growth factors RN33B and MN-1 monolayers were exposed to a saturating concentration ot GDNF (5 ng/ml) for different periods of tmc, and total cell lysates were analysed for tyrosine phosphorylation by SDS/PAGE and Western 20 blotting with an anti-phosphotyrosine monoclonal antibody Two proteins with mobilities corresponding to 42kD and 44kD, rtspcctively, were phosphorylated on tyrosine within 5 minutes of GDNI treatment of RN33B cells (Fig 7A) A similar result was obtained in differentiated RN33B cells (not shown) of exposure to GDNF 25 Based on comparison of their size with descriptions of growth factor- induced protein tyrosine phosphorylation elsewhere (Qiu and Green, 9 Neuron 705, 1992), the 42kD and 44kD species would appear to be, respectively, p42 k2 and p44 ,kl „ two protein serme-threomne kinases members of the extracellular signal-regulated kinase (ERK, also termed microtubule-associated protein kinase) family 30 (Boulton et al , 65 Cell 663, 1991) To confirm the identity of these proteins as ERK2 and ERKI, respectively, protein blots which had been reacted with the anti-phosphotyrosine antibody were stripped and reprobed with a rabbit polyclonal Printed from Mimosa 0711 15 WO "17/ 1X240 I'CT/IIS%/I8I07 -34- antibody raised against recombinant ERK2 that recognizes both ERKI and ERK2 in protein blots Comparison of autoradiograms of blots prohcd with the anti-phosphotyrosme antibody and the anti-HRK2 antibody identified the p42 and p44 proteins as ERK2 and ERKI, respectively (Fig 7a) Although GDNF treatment of 5 MN-1 cells appeared to only stimulate phosphorylation of ERK2, both ERKI and ERK2 were present tn MN-1 cell lysates (Fig 7b) Thus, GDNF treatment stimulated very rapid and transient tyrosine phosphorylation of ERKI and ERK2 in RN33B cells, but relatively slower and more sustained phosphorylation of FRK2 and MN-1 cells 10 Activation of the ERK pathway has previously been shown to induce rapid and transient increase in transcnption of immediate early genes, including the c-fos protooncogene (Gille et al , 358 Nature 414, 1992) Accordingly, the ability of GDNF to induce c-fos mRNA in differentiated raphe nucleus RN33B cells and in motor neuron MN-1 cells was investigated For analysis of c-fos mRNA 15 expression in ccll lines, culture medium was changed 90 minutes prior to addition of 100 ng/ml GDNF to cell monolayers At the indicated time intervals, media was removed, cells solubilized with guanidine isothiocyanate and 0-mercaptoethanol and RNA extracted as previously described (Trupp et al , supra) Twenty micrograms of total RNA was fractionated on 1% agarose gels containing 0 7% formaldehyde 20 and transferred 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 visualized by autoradiography on x-ray Films Cell monolayers were exposed to saturating concentrations of GDNF for 25 different periods of time and levels ot c-fos mRNA were subsequently analyzed m Northern blots of total RNA (Fig 8) This analysis revealed transient upregulation of c-fos mRNA 15 minutes after exposure of RN33B cells to GDNF, returning to basal levels 45 minutes after treatment (Fig 8a) c-fos mRNA was also upregulated in MN-1 cells but not until 30 minutes of GDNF treatment (Fig 8b) Elevated c-30 fos mRNA levels persisted for about an hour and returned to basal levels 120 minutes after the initiation of treatment (Tig 8b) Thus like tyrosine phosphorylation of ERKS c fos mRNA upregulation induced by GDNF treatment Printed from Mimosa 07 11 15 WO 97/18240 rCT/US96/18197 -35- was very rapid and transient in RN33U cells, but somewhat slower in MN-1 cells 5 Example 6 Survival responses promoted by GDNF in differentiated raphe nucleus cells Advantage was taken of the conditional nature of the immortalization of the raphe nucleus serotonergic cell line RN46A by examining whether GDNF 10 may be a survival factor for differentiated raphe nucleus neurons Survival assays were performed as previously described (Eaton et al , 1995, supra) Briefly, 105 RN cells were seeded to collagen/fibronectm coalid 8-wUI glass slides and incubatcd at 33"C (growth permissive temperature) until 75-90% confluent 'I he slides were then shifted to 39° (non-permissive temperature) and serum containing 15 medium was replaced by B16 detined medium (Brewer and Cotman, 494 Brain Res 65.1989) containing \% BSA, 1 /xg/ml transferrin, 5 /xg/ml insulin, 100 mM putrescine, and nM progesterone plus or minus 0-50 ng/ml rhGDNF (Promega, Madison, \VI) Media and GDNF were replaced every two days for 8 days alter which the cells were fixed in 4% paraformaldehyde/2% 20 glutaraldehyde, rinsed and coated with a glycerol mounting medium containing 1 mM bisbenzamide (Hoechst dye 33342) to stain viable nuclei Fields of cells were magnified to 40x on a Zeiss Axiophot microscope, examined for fluorescent nuclu (at 355 nM exitation, 465 nM emission), the images video capturcd, and the cells counted with Imade I™ software For each condition, 10 Fields of cells were 25 counted from each of 3 independent experiments RN46A cells were cultured at the non-permissive temperature in defined medium in the presence of increasing concentrations of GDNF Nine days after plating, surviving cells were counted and compared with cultures established in the absence of GDNF A 3-fold increase in the number of surviving cells was 30 observed in cultures grown in the presence of GDNF (Fig 9) The effect of GDNF on the survival of differentiated RN46A cells was dose dependent, with an EC50 at 5ng/ml Printed from Mimosa 07 11 15 WO 97/18240 PCT/US96/181Q7 -36- Example 7 Generation, cloning and characterization of anti-GDNF monoclonal antibodies Immunisation 5 Five young temalc mice were immunised with 35 ug of insect cell-derived recombinant GDNF emulsified with complete Freund's adjuvant (FA) Second and third immunizations were performed 2 and 4 weeks after the first one in incomplete TA All the injections were given mtrapentoneally (i p ) Two weeks after the last Immunisation, antibody titer in serum was checked by ELJSA and Western Blot 10 analysis ustng standard methods The mouse with the highest titer (more than 1 2000) was boosted i p with 3 fig of GDNF m incomplete FA 3 days before the cell fusion Cell fusion Cell fusion was done according to the method of Kohler and Milstem 15 (1975), incorporated herein by reference, with some modifications a) DdV before fusion Viable cells from the Sp2/0 murine cell line were adjusted to 2x10s cells / ml with complete DMEM (10% fetal calf serum, I % L-glutamme, 100 U/ml penicillin and 100 ug/ streptomycin sulphate) 20 Cells from a non-immunised mouse were obtained from the peritoneal cavity by injection of 0 34M sucrose solution The cells were resuspended in complete DMEM containing hypoxanthine lOO^M, dminoptenn 0 4M, and thymidine 16 /xM, (HAT medium), to 1x10s cells /ml 100 jk1 of the cell suspension was added to the 60 inner wells of 96 well plates and incubated overnight at 37°C in an 25 atmosphere of 5% C0t 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 30 4 2xl07 Sp2/0 cells were tused 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 medium at 37° C in an atmosphere of 5% C02 in air After one week of eulture, Printed from Mimosa 07 11 15 WO 97/182-40 PCT/1)S%/I81Q7 -37- the wells were inspected When hybrids cells covered 10 to 50% of the surface area of the well, the culture supernatants were assayed tor antibody by HLISA 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 5 buffer, pH 9 6 After an overnight incubation at 4 "C, the wells were washed with 0 05M phosphate buffered saline, pH 7 2, containing 0 05% Tween (PBS-T) Nonspecific binding was blocked with PBS-T containing 3% non-fatty milk and 1 % goat normal serum Supernatant samples were incubated 4 hours at room temperature Peroxidase goat anti-mouse antibody was used and the substrate was 10 o-phenylenediamme dihydrochloridc (OPD) Plates were read at 492 nm in an ELISA reader Negative controls included completed medium and normal mouse serum The hybrids were grown in HAT medium up to two weeks after fusion Cells were subsequently grown in HT medium 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 supernatants were done by ELISA Upon recloning, 5 positive hybridoma clones were choscn and the cells were maintained in complete DMEM for 30 days Isotyping of monoclonal antibodies 20 The class and subclass of the monoclonal antibodies were determined by ELISA using a DAKO panel for isotyping of mouse monoclonal antibodies All five 5 monoclone <tntibcidics were characterized as lgG, Purification of monoclonal antibodies Monoclonal antibodies from culture supernatants were purified by Protein G 25 Sepharose fast flow (Pharmacia, Biotech) according to manufacturer's instructions Culture supernatants were concentrated and filtered through a 0 45 jim membrane (Schleicheer and, Schull, Germany) and then pumped overnight through the column previously equilibrated with 20mM sodium phosphate, pH 7 0 lg was elutcd with 0 05M glycine buffer 30 Example 8 A motor neuron cell line showing biological and biochemical responses to Printed from Mimosa 07 11 15 WO 97/18240 PCT/l)S9(i/lH197 -38- GDNF MN-1 cell monolayers were exposed to increasing concentrations of GDNr in scrum-free medium and assayed 3 days later tor cell survival and growth by measurement of acid phosphatase activity (Clontech) GDNF was produced and 5 purified irom baculoviras mlected insect cells as previously described (Trupp et al ,supra) GDNr treatment ot serum deprived-MN-1 monolayers increased cell number in a dose-dependent manner (Fig 10a) The biological response of MN-1 cells correlated with biochemical and transcriptional responses to GDNF treatment MN-1 cell monolayers were exposed to 50 ng/ml GDNF for increasing 10 periods of tune, cell lysates were fractionated by SDS/PAGE and Western blots probed with an anti-phosphotvrosine antibody (UBi) Several proteins were seen to have increased tyrosine phosphorylation after GDNF treatment ot MN1 cells, including a protein with an electrophoretic mobility of 42K (Fig 10b) Based on comparison of us size with descriptions ot growth 15 tactor-mduced protein tyrosine phosphorylation elsewhere (Boulton, T G , et al Cell 65, 663-75 (1991), the 42K species would appear to be p42"kl, a serine-threonine kinase member of the extracellular signal-regulated kinase (ERK) family The identity of this protein as ERK2 was confirmed after immunoprecipitation with an antiERK2 polyclonal antiserum followed by analysis of tyrosine phosphorylation 20 (Fig 10c) Lysates of GDNF-stimulated MN-1 cells were immunoprecipitated with an anti-ERK2 antiserum (Santa Cruz) that also recognizes ERKI followed by antiphosphotyrosme Western blotting This analysis further revealed that another member of the ERK family, p44erkl, was also phosphorylated on tyrosine alter GDNF treatment of MN-1 cells (Fig 10c) Activation of the ERK pathway has 25 previously been shown to induce a rapid and transient increase in the transcription of immediate early genes, including the c-fos proto-oncogene (Gille et al . Nature 358, 414-7 (1992) Example 9 30 The product of the c-ret proto-oncogene as a signal transducing receptor for GDNF GDNF receptor complexes from MN-1 cells could be recovered by Printed from Mimosa 07 11 15 WO 97/1S240 pcr/usofi/isw -39- lmmunoprecipilatton with anti-GDNF antibodies or by binding to lectin-Sepharose beads (Fig 11a) Unexpectedly, the 180KD receptor complex (i e , e-RET, 180kD -23kD = 157kD, which is approximately equal to the 155kD receptor identified as c-RET - see Example 2, infra) could also be recovered by immunoprecipitation 5 with anti-phosphotyrosine antibodies (Fig 11a), indicating that the GDNF binding protein in this complex could be a receptor tyrosine kinase The product of the c-ret proto-oncogene is highly expressed in primary motor neurons (Pachnis et al , supra, and Tsuzuki, T , et al Oncogene 10, 191-8 (1995) and is of similar molecular weight as the major GDNF receptor component 10 detected in MN-1 cells (Takahashi, M , et al Oncogene 3, 571-578 (1988) We tested whether this species represented a C-RET-GDNF cross-linked complex by immunoprecipitation with anti c-RET antibodies 115I-GDNF was cross-linked to MN-1 cells using EDAC and receptor complexes were precipitated with antibodies against GDNF (Trupp et al , supra), 15 lectin Sepharose beads (Formica), anti-phosphotyrosine antibodies (UBI), anti-c- RET antibodies (Santa Cruz) and control antibodies from non-immune rabbits An antipeptide c-RET rabbit antiserum readily immunoprecipitated the major 180kD ligand-receptor complex in MN-1 cells (Fig lid), while a number of unrelated monoclonal and polyclonal antibodies used as controls failed to immunoprecipitatc 20 this complex (Fig 11 a and data not shown) Because the product of the c-ret gene is a receptor tyrosine kinase, we investigated whether GDNF eould stimulate tyrosine phosphorylation of the c-RET protein in MN-1 cells MN-1 cell monolayers were exposed to GDNF at different concentrations or for different periods of tune and cell lysates were 25 immunoprecipitated with anti-c-RET antibodies and analyzed by SDS/PAGE and Western blotting with antiphosphotyrosine antibodies as disclosed above GDNF treatment stimulated rapid c-RET tyrosine phosphorylation in MN-1 cells (Fig lib) Maximal phosphorylation was reached 5 minutes after GDNF treatment and lasted for at least 60 minutes A dose-response analysis of GDNF induced c-RET 30 phosphorylation in MN-1 cells showed maximal phosphorylation at 30 ng/ml of GDNF (Fig lib), which is similar to the response of both serum deprived MN-l cells (Fig 10a) and embryonic sympathetic neurons (Trupp ct al supra) to Printed from Mimosa 07 11 15 WO <>7/18240 I'CI/US96/1819*7 -40- GDNF Taken together, these data indicate that the c-RET receptor may be an important component in the signal transduction mechanism of GDNF Example 10 5 c-ret transaction reconstitutes GDNF binding and biological activities to GDNF Experiments were conducted to determine whether expression ot 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 experiments were performed in naive 3T3 fibroblasts, and 3T1 cells stably transfected with either a 10 wild type c-ret or an oncogenic form of this gene found in MEN2a patients (Mulligan et al , supra) For c-ret expression in transfected cells, human wild type c-ret and MEN2a-ret cDNAs were subcloned in pcDNA3 (Invitrogen) Cold GDNF was used at 50x molar excess For survival/growth assays, cells were cultured tor 6 days in serum-free medium supplemented with the indicated concentrations of 15 GDNF, medium and GDNF were replaced every two days Cell number was quantified by measurementof acid phosphatase activity (Clontech) After immunoprecipitation with c-RET antibodies, GDNF-labeled receptor complexes of approximately 180K were dctcctcd in both MEN2a-ret and c-ret transfected 3T3 fibroblasts, but not in untransfected cells (Fig 12a) The labeling 20 could be displaced by excess cold GDNF, indicating that it represented specific GDNF binding (Fig 12a) Experiments were also conducted to determine whether c-ret could mediate a biological response to GDNF upon transfection in non-responsive cells Survival and growth responses to GDNF were investigated in untransfected and c-ret 25 transfected 3T3 fibroblasts cultured in serum-free medium GDNF elicited a dose-dependent increase in cell number in c-ret transfected, but not in untransfected, 3T3 cells (Fig 12b) which was comparable 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 transfection (see Fig 12a), it was concluded 30 that c-ret expression was sufficient for mediating a biological response to GDNF in these cells Printed from Mimosa 07 11 15 WO 97/18240 PCI/US96/181U7 -41- Examplc 11 c-ret expression in adult brain and dopaminergic neurons of the substantia nigra Experiments were conducted to determine whether the r-ret product may 5 mediate the neurotrophic effects of GDNF in the brain by examining the expression of c-ret in different regions of the rat central nervous system A rat c-ret riboprobe was generated using as template a cDNA fragment obtained by PCR with primers based on sequences U22513 and U22514 (Genbank accession numbers) High levels of c-ret mRNA were found in MN-1 cells and in rat spinal cord (data not shown) 10 High c-ret niRNA expression was also found in the adult pons, medulla, locus coeruleus and hypothalamus (Fig 13a), as well as in thalamus and cerebellum (data not shown) c-ret mRNA was expressed at barely detectable levels in striatum, hippocampus and cerebral cortex (Fig 13a) In the ventral mesencephalon, containing the cell bodies of GDNF-rcsponsive dopaminergic neurons, c-ret mRNA 15 levels increased progressively during post-natal development (Fig 13b) A peak of expression was detected between post natal day 6 (P6) and P8, at which time axons of dopaminergic neurons of the substantia nigra begin innervation of the striatum, and coincident with an increase in GDNF mRNA expression in this target region (Fig 13b) For mRNA quantification, a glyceraldehyde-3-P dehydrogenase 20 (GAPDH) riboprobe was included m the RPA, and values of relative mRNA expression, obtained atter densitometry scanning of gel autoradiograms, were normalised using the GAPDH signal of each RNA sample RPA for GDNF mRNA has been previously described (Trupp et al supra) In situ hybridisation and unmunohistochemistry were performed as 25 previously described (Arenas, E & Persson, H Nature 367, 368-371 (1994), Neveu, I & Arenas, E J Cell Biol in press (1996) c-RET protein was detected using a hamster monoclonal anti-mouse c-RET antibody which also recognises rat c-RET (Lo, supra) followed by fluorescein-conjugated rabbit anti-hamster secondary antibodies (Southern Biotechnologies) In situ hybridization on sections 30 through the adult substantia nigra revealed strong labelling over neurons throughout this structure (Figs 14 a-b) In addition, cells positive for c-RET-hke immunoreactivity (c-RET-LI) were found throughout the adult substantia nigra. Printed from Mimosa 07 11 15 WO 97/1X240 I'CT/USWIHW -42- with strong labelling over cell bodies (Fig 14c) in order to establish that c-ret expression in the adult substantia ninra was confined to dopaminergic neurons, these cells were selectively lesioned with a unilateral injection of 6-hydro\ydopamine (6-OHDA), the cells were then analy7ed 5 for c-ret mRNA expression by in utu hybridisation Lesions of dopaminergic neurons of the substantia nigra wereperformed by stereotaxic injections of 8 pig 6-OHDA in tlit. medial forebrain bundle at the following coordinates 1 6 mm caudal to bregma, 1 3 mm lateral to midhne, and 8 4 mm under the dural surface with the incisor bar 5 mm over the interaural line Animals were pretreated with 25 10 nig/kg desipramuie (i p ) 30 minutes prior to 6-OHDA injection 0 75 x 10*' GDNF-expressing fibroblast cells in 3 n\ of medium were injected supramgrally at the following coordinates 3,1 mm from interaural line, 2 mm lateral to midhne, and 7 mm under the dural surface, with the incisor bar at -3 3 mm Lesion and grafting in the locus coeruleus were as previously described (Arenas et al , 15 Neuron 15, 1465-1473 (1995) The generation and characterisation of GDNF expressing fibroblast*, have been described previously (Arenas et al , supra) Five hours after the lesion, no difference could be seen between ipsi and contralateral sides in c-ret mRNA expression (Fig 14d) However, a marked reduction in c-ret mRNA expression was seen in the lesioned substantia nigra 20 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 lesion was, however, not affected (Fig 14d) This result indicated that in the adult substantia nigra, c-ret mRNA expression was confined to dopaminergic neurons 25 Example 12 GDNF rescues c-RET-positive dopaminergic and noradrenergic neurons Experiments were conducted to determine whether c-RET expressing neurons of the adult substantia nigra and locus coeruleus responded to GDNF For this, nigral dopaminergic neurons lesioned with 6-OHDA, and were then examined 30 to determine whether grafts of GDNr expressing fibroblasts induced responses on c-RET immunoreactive neurons In lesioned animals that received a graft of control fibroblasts, no c-RET-LI could be detected, indicating a depletion of c-ret- Printed from Mimosa 07 11 15 WO 97/18240 PCT/US96/I8I97 -43- cxprcssmg cells by selective lesion of dopaminergic neurons in the adult substantia nigra (Fig 14e) However, c-RET-LI could be rescued by the GDNF-exprcssmg graft, where c-RET immunopositive fibers could be seen surrounding and penetrating the graft (Fig 14f) Similar results were obtained in the locus 5 coeruleus, where lesion with 6-OHDA depleted c-RET-tmmunoreaetive cell bodies (Fig 14g), which could be rescued by exogenous administration of GDNF (Fig 14h) In both brain regions, the rescue of c-RET-LI positive cells and sprouting in the animals grafted with GDNF-expressing fibroblasts paralleled that of tyrosine hydroxylase lminunoieactivity (data not shown), demoastrating that c-RET-10 expressing adult dopaminergic and noradrenergic neurons respond to GDNF Example 13 Identification of GDNF c-RET receptors PC 12 cells and NB2/a cells were washed three tunes with serum free 15 RPMI-1640 or DMEM, respectively, plated on noncoated (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 tells were harvested (100,000 cells, five parallels), incubated with 10 ng/ml human 125I-GDNF 20 (lodinated by Chloramine T method, 100 ^Ci/^g) in the presence or absence of 50-fold unlabeled GDNF for 120-150 min on ice, the unbound factor was removed by centrifugation through 30% sucrose cushion, and the cell-associated radioactivity counted on 1271 RIAGAMMA counter (LKB Wallac) Recombinant human GDNF promoted survival of about 20% of serum 25 deprived rat pheochromocytoma PC 12 cells at concentration of 50 ng/ml (Fig 15a) Serum-deprived PC12 cells are also maintained by nerve growth factor (NGF) Upon treatment with (NGF), PCI2 cells also stop dividing and differentiate into sympathetic 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 30 induce differentiation of PC12 cells at the concentrations studied, presumably because of the differences in signal transduction of NGFactivated trkA receptors and GDNF receptors Printed from Mimosa 07 11 15 WO 97/18240 ITT/IIS96/18197 -44- Human neuroblastoma NB2/a cells were plated in serum-free medium in the presence or absence of 50 ng/ml ot GDNF and the number of cells was countcd after 48 hr of culture GDNF significantly increased the number of NB2/a cells (Fig 15b) Monkey COS cells, human SY5Y cells and mouse NIH 3T3 cells 5 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, indicating that both cell lines express functional GDNF receptors To determine, whether GDNF binds to the responsive cells, PC12 cells and NB2/a cells were incubated with 125l-labeied human GDNF at 40°C as indicated in 10 the legend to Fig 15 As shown in Fig 15c, both PC 12 and NB2/a cell lines bind GDNF 30 efficiently More importantly, the binding of l25I-labeled GDNF could be competed with a 50-fold excess of unlabeled GDNF (Fig 15b) Thus, tin. binding of GDNF to the receptors on PCI2 and NB2/a cells appears to be specific 15 Example 14 Identification of GDNF c-RET binding components PC12 cells, SY5Y neuroblastoma cells and NB2/a cells where chemically cross-linktd to l2iI-GDNF with EDC 3-5 x 106 cells or mechanically dissociated cells from 2 E20 rat kidneys were incubated with 10 ng/ml of '"I-GDNF for 1 hour 20 on ice and cross linked with 30 mM EDAC (Pierce) for 30 minutes on ice Detergent lysates were immunoprecipitated, the precipitates collected by Protein A-Sepharose, separated on 7% SDS-PAGE, and visualized by Phosphnrimager SI (Molecular Dynamics) The resulting complexes were immunoprecipitated with rabbit antibodies to 25 GDNF, analyzed by SDS-PAGE and visualized by autoradiography Embiyonic 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) submitted) Cross linked complexes of 170 and 190 kD were obtained from the extracts of PC 12 cells, SY5Y cells and NB2/a cells and a 190 kD complex from 30 embryonic kidney extracts (Fig 16) The molecular weights of the crosslinked proteins minus GDNF of approximately 25-30 kDsubstantially, if not exactly, correspond to the molecular Printed from Mimosa 07 11 15 WO '>7/182.10 i'c r/us*)6/iaiQ7 -45- weights of c-RET protooncogene, an orphan receptor tyrosine kinase (Takahashi, M , Ritz, J iVt Cooper, G M Cell. 42, 581-588, 1985, Takahashi, M et al , Oncogene. 3, 571-578 (1988)) (140 kD and 160 kD, representing differently glycosylated forms ot c-RET , Tsuzuki, T , Takahashi, M , Asai, N , Iwashita, T , 5 Matsuyama, M & Asai, J Oncogene, 10 191-198 (1995) Example 15 Affinity Cross Linking of GDNF to c-RET The cross linked complexes were immunoprecipitated from the NB2/a cells 10 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 precipitated by anti-c-RET antibodies, which thus correspond to cross linked GDNFc-RET complexes Binding of I1SI-GDNF to c-RET proteins was completely abolished by 500-fold excess of unlabeled GDNF (lane 2) No proteins 15 were preeipitated by monoclonal anti-neurofilament antibodies (lane 3) or by Protein A-Sepharose only (lane 4) No cross linked complexes were obtained trom COS cells (not shown) Since c-ret proto-oncogene is a glycoprotein, ,2,I-labelcd NB2/a cell extracts were also immunoprecipitated with wheat germ agglutinin Again, proteins of 170 and 190 kD were obtained (lane 5) 20 To establish further that GDNF specifically binds c-RET, the mouse c ret cDNA was cloned into the mammalian expression vector PBK-CMV and transiently expressed in monkey COS cells Mouse c-ret cDNA (Pachnis, V , Mankoo, B Costantini, F Development, 119, 1005-1017 (1993)) in pbluesenpt SK' (Stratagene) was cleaved with Sacll and EcoRV and cloned into Sacll and Smal site 25 of pBK-CNV vector (Strategene) COS cells were transiently transfected with c-ret cDNA or with empty plasmid by electroporation (Bio Rad) with - 30 % efficiency by fluorescence of cotransfected Red Shift Green Fluorescent Protein in PEF-BOS vector 48 hours later, 10 x 10' transfected COS cells or 3-5 x 106 parental COS cells or NB2/a cells were treated With '"I-GDNF, cross linked and analysed as 30 specified in legends of Fig IS and Fig 16 First the expression of c RET protein by was examined Western blotting c-ref-transfccted COS ccIls (Fig 18a) and NB2/a cells (not shown) expressed Printed from Mimosa 07 11 15 WO 97/182*0 PCT/IJS96/I8197 -46- detectable amounts of tlic c-RET protein, whercus no c-RE'I protein was detected in mock-translectcd (with PHK-CMV plasmid) COS cells (Fig 18a) PCI 2 cells also express c-RET protein, ulbelt at considerably lower level than NB2/a cells or r-/rMranstected COS cells (not shown) COS cells, transiently expressing mouse t-5 ret proto-oncogene were incubated with 12SI-GDNF As shown in Fig 17b, those cells bound GDNF, and binding of psI GDNF can be competed with excess of unlabeled GDNF In contrast, no significant binding-of GDNF was observed m moek-transfectcd COS cells 10 Example 16 Phosphorylation of tyrosine residues 10 X 10h transfected COS cells (48 hr after transfection) were treated with 50 ng/ml of GDNF (Preprotech EC Ldt) for 5 minutes in serum-frcc DMEM, or not treated, and then quickly washed with the same medium NB2/a cells were 15 similarly treated (results not shown) c RET proteins were immunoprecipitated from detergent extracts by cocktail ot monoclonal (Lo, L & Anderson, D J Neuron 15, 527-539 (1995) and polyclonal (Santa Cruz) anti-c-ret antibodies, separated by 1% SDS-PAGE, transferred to nitrocellulose, probed by anti-c-ret antibodies (Santa Cruz), stripped and reprobed by anti-phosphotyrosine antibodies 20 (Sigma) This treatment resulted in significant increase in tyrosine phosphorylation ot 190 kD cRET proto-oncogene, the 170 kD form being less prominently phosphorylated (Fig 18b) In both cell lines, relatively high c-RET phosphorylation was detected also in the absence of GDNF (Fig 18), most 25 probably via endogeneous GDNF secreted by these cells and/or ligand independent receptor dimenzation Example 17 30 I2Sl-GDNF binds to c-ret-positive enteric neurons l:uI-GDNF was bound to developing rat tissue explants in situ In situ binding of human '"I-GDNF (PeproTech EC Ltd ), lodinated by Chloramine 1 Printed from Mimosa 07 11 15 WO 07/18240 l,Cr/HS')6/18l')7 -47- Mcthod, was carried out essentially ns described (Partancn and Fhcsleft, 1987) Briefly, explants of E15 rat gut were incubated with 10 ng/ml of '"I-GDNF in Eagle's minimal essential medium on the Nuclepore filter (Ccistar) tor 90 mm <U room temperature 250-fold excess of unlabeled GDNF was applied as a 5 competitor to control explants After careful washing, the explants were fixed with 3 5% paraformaldehyde in PBS, sectioned and exposed to NTn-2 emulsion (Kodak) The gastrointestinal tract was chosen as it strongly expresses GDNF mRNA (Suvanto et al , 1996), Figure 19 a and b) and c-RET-positive neurons arc absent 10 in the gastrointestinal tract in r-/^-deficient mice (Schuchart et al , 1994, Durbee et al , 1996) '"I-GDNF binds to a group of cells within the muscle layer ot embryonic day (E)15 rat gut (Figure 19 c and d) This binding was specific as it was totally competed with 250-fold excess of unlabeled GDNF (Figure 19h) The cells that bind GDNF were the enteric neurons of the myenteric plexus, as revealed 15 by penpherin lrrununoreactivity (Figure 190 Moreover, tlie^e neurons also expressed c-ret mRNA, as demonstrated by irt situ hybridization (Figure 19e) Cloning of the GDNF cDNA and in situ hybridization with GDNF probe was performed 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 20 shorter form (Takahashi et al , 1988) of c-ret was cloned into Notl-XhoJ site of pBSK + vector (Stratagene) cRNAs in antisense and sense onentation were labeled with digoxigenm-UTP (Boehnnger-Mannheim), hybridized to cryosections through El5 rat gut and visualized with alkaline phosphatase-coniugatcd anli-digoxigenin antibodies according to manufacturers instructions In both cases, only background 25 labeling was obtained with hybridization of corresponding probes in sense orientation (Figure 19g) Polyclonal anti-periphcnn antibodies (Bio-Rad) were applied to cryosections of E15 rat gut at a dilution of 1 100 for 1 hr and visualized by FITC-conjugated secondary antibodies (Jackson) Thus, GDNF specifically binds to c-RET-expressing enteric neurons of developing rat 30 Example 18 Affinity-crosslmking of GDNF to c-RET Printed from Mimosa 07 11 15 WO 97/18240 r(T/l)S<Hi/|8l')7 -48- PC12 cells and NB2/a cells were washed tlircu times wuh serum-fiee RPMI-1640 or DMEM, respectively, plated on uncoated (NB2/,i cells) or collagen coated (PC 12 cells) dishes (5000 6000 cells per dish in triplicate) in tlie presence or absence of 50 ng/ml of GDNF (PeproTech EC Ltd ), and the number of cells S microscopically counted alter 48 h For c-RET expression in transacted cells, the shorter form (Takahashi el al , 1988) of human wild-type c-ret cDNA was subcloned in pcDNA3 (Invitrogen) 3T3 fibroblasts were stably transfected with c-ret expression plasmid or with empty vector (mock-transfected cells) and positive cells lines selected with G418 10 Transient transfection of trkC 3T3 fibroblasts (Ip et al (1993) Neuron, 10,137-149) with human c-ret cDNA in pcDNA3 vector or with empty vector was performed by the lipofectin method (Gibco-BRI ) c-ret and mock-transtected cells (10 000 - 15 000 cells per well) in live parallels were treated with rat GDNF (Trupp ct al (1995) J Cell Biol 130, 137-148) at indicated 15 concentrations for five days NT-3 was used as positive control at 30 ng/ml Cell number was quantified by measurement of acid phosphatase activity using Abacus'" Cell Proliferation Kit (Clontcch) 3-5 x I0h PC 12 cells, NB2/a cells, COS cells or c-ret-3T3 as well as mock-3T3 cells or mechanically dissociated cells trom two F20 or from 17 El5 rat 20 kidneys were incubated with 10 ng/ml of l2<iI-GDNF (human GDNF from PeproTech EC Ltd or rat GDNF from C F lbanez) (Trupp et al , 1995), lodinated by Chloramine T method, for 1 hour on ice 250-fold cxcess ot unlabeled GDNF (PeproTech EC) or TGF-01 (kindly provided by Dr M Laiho) was applied to control sample l25I-GDNF was then crosslinked to the cells with 30 mM ot 25 ethyl-dimethylaminopropyl carbodnmide (EDAC) (Pierce) for 30 minutes on ice Detergent lysates of the cells were immunoprecipitated 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 neurofilament proteins (a gift of Dr 1 Virtanen) were used as control 30 antibodies The precipitates were collectcd by Protein A-Sepharose (Pharmacia) or by WGA-agarose (a gift from Dr O Renkoncn), separated on 7% SDS-PACE, and visualized with a Phosphonmagcr SI (Molecular DynanLics) Printed from Mimosa 07 11 15 WO 97/1824(1 l»rm]S96/IR»«)7 -49- First, '"I-GDNF was crosslinked to PCI2 cells. NB2/j cells and COS cells with ethyl-dnnethyiaminopropyl carbodnmide (EDAC), and the complexes were precipitated with anti-GDNF antibodies As shown on Figure 20a, complexes with molecular weight ot 190 kD and 170 kD were obtained trom PC 12 and NB2/A 5 cells, but not from COS cells The molecular weights of the cross linked proteins (minus GDNF monomer of -25K) correspond to those of c-RET, (140 kD and 160 kD, representing partially and fully glycosylated isoforms of c-RET, respectively) (Takahashi et al, 1988) Next '"I-GDNF was crosslinked to PC12 and NB2/a cells by EDAC and 10 immunoprecipitated formed complexes with anti-c-RET antibodies The bands with molecular weight of 190 kD were obtained from both cell lines (Figure 20 a and b) Formation of the complexes was abolished by 500-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 isoform by anti-c-RET 15 antibodies, is unclear The same complexes although much weaker, were also obtained when dithiobis(suecinimidylpropionate) was used as a crosslmkcr (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 20 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 GDNT Thus, only the fully glycosylated form of c-RET is expressed in embryonic kidney cells Crosslinked U5l-GDNF-c-RET complexes from the cells ectopically 25 expressing c-ret were also demonstrated 3T3 cells were transfccted with c-ret cDNA or with empty plasmid, and established stable transfected cell lines (c-ret-3T3 cells or mock-3T3 cells) Cross linking of 125I-GDNF to these cclls followed by ant t-RET-precipitation revealed a 190 kD band that was abolished with 250-fold excess of unlabeled GDNF (Figure 20b) As GDNF is a distant member of 1GF-/3 30 family, wc also used a 250-fold cxcess of TGF-/31 as a competitor No competition was observed with TGF-/01 (Figure 20b) Taken together, these data show that GDNF directly and specifically binds to c-RET Printed from Mimosa 07 11 15 WO 97/18140 rrr/llS9<i/l8197 -so Example 19 GDNF specifically increases tyrosine phosphorylation of c-RET r-rer-3T3 cells and mock 3T3 cells were ticatcd with GDNF and the protein1! from these cells were immunoprecipitated with anti-c-RET antibodies 5 The precipitated proteins wt.ro then analyzed by Western blotting with anti- phosphotyrosine antibodies 10 \ 10" c-ret-3T3 cells were treated with different doses of GDNF (PeproTech LC Ltd or from C F Ibane/) (Trupp et al , 199S) for S min, or with 50 ng/ml of GDNF for indicated times in serum-free Dulbecco's modified Eagle's medium containing 1 mM Na,V04 and then quickly 10 washed with the same medium c-RET proteins were immunoprecipitated from detergent extracts, containing 1 mM Na3V04 by cocktail of monoclonal (Lo, L and Anderson, D J (1995) Neuron, 15 527-539) and polyclonal (Santa Cruz) anti-c-RET antibodies, separated by 1% SDS-PAGE and transacted to nitrocellulose which were probed by anti-phosphot>rosine antibodies to nitrocellulose with was 15 probed by anti-phosphotyrosine antibodies PY20 (Transduction Laboratories), then stripped and reprobed by anti-c-RET antibodies (Santa Cruz) As shown on Figure 21a, a short treatment of c-ret-3T3 cells with GDNF dose-dependently (beginning at 25 ng/ml) increased tyrosine phosphorylation ot the 160 kD c-RET isoform whereas the phosphorylation of the 140 kD isoform 20 remained unchanged An increase in c-RCT phosphorylation was evident at 25 ng/ml of GDNF and above it No c-RET proteins were detected in mock-3T3 cells c-ret-313 cells were also treated with GDNT (SO ng/ml) tor different times An increase in c-RET tyrosine phosphorylation was < vident after 5 minutes of treatment and continued at least tor one hour (Figure 21b) With prolonged 25 exposition, the increase in phosphorylation of lower c-RET isoform also became evident A basal level of c-RET phosphorylation was detected in the absence of GDNF, possibly via a ligand-independent dimerization of that receptor To reveal the amounts 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 30 was not changed by GDNF treatment in c-ret-3T3 cells (Figure 21a and b, lower panels) The finding that GDNF specifically activates c-RET indicates that c-RET is a signaling receptor for GDNF Printed from Mimosa 07 11 15 WO 97/18240 PC IVUS96/18197 -51- Evample 20 c-RET expression confers GDNF-responsiveness to 3T3 cells Mouse 3T3 fibroblast cell line expressing trkC (trkC-3T3) (lp et al, 1993) were transiently transformed with c-ret expression plasmid trkC-3T3 cells die 5 within 2-3 days in scruin-fret medium m the absence ot trkC ligand neurotrophin-3 (NT-3) (Ip et a!, 1993) and do not express detectable amounts of c-ret GDNF dose-dependently increased the number of c-ret-transfected but not of mock-transfected frA.C-3T3 cells (Figure 22), which was comparable to the response elicited by NT 3 Whether this is a proliferative or survival-promoting response 10 eould not be distinguished based upon the data Thus, introduction of c-RFT to GDNF-nonresponsive cells is sufficient to bring about the biological response to GDNF 15 Example 21 Isolation of GDNF rcceptor L6 myoblast cells were lysed with 1 % NP40 and cell lysates were fractionated by anionic exchange on a Q-Sepharose column Fractions eluted at 20 different ionic strength were dialyzed and assayed for binding to GDNF lmmoliblized on a chip in a Biacorc device (Pharmacia) A distinct binding component was detected in a fraction of L6 cell lystaes (Figure 23a) In the Figure, solid bars indicate total protein (as absorbance at 280 run), hatched bars indicate GDNF finding (in resonance units) This fraction was not particularly rich 25 in protein, indicating a substantial purification over the total protein mixture The equivalent fraction of a COS cell lysate did not show binding under the same conditions (data not shown) Further purification of the GDNF binding activity in the first 1M salt fraction was obtained after hydrophobic interaction chromatography (Figure 23b) 30 The data represents 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 Printed from Mimosa 07 11 15 WO 07/18240 ITT/US')fi/181«J7 -52- Alternatively, purification may be effected by crosshnking GDNF to cells in the presence of tracer amounts of radiolabeled ligand, and ligand/receplor complexes can by fractionated through ion exchange chromatography followed by hydrophobic interaction chromatography and SDS/PAGE Bands corresponding to 5 the molecular weights of GDNF-receptor complexes can by excised, dissociated, and then sequenced by mass spectrometry or Edman degradation, depending upon the yield of recovery Example 22 10 A novel GDNF-binding protein in brain By ligand blotting, we have identified another GDNF-bmding protein from total brain cxtract We bound l2M-GDNF to the filters carrying protein from the total extracts of brain and liver (a ligand blot assay) A major band with MW of about 50 KD was obtained from brain cxtract, but not from liver (Figure 24) This 15 binding is specific as ,J,1-GDNF did not bind to other proteins from total lysates, it is not found in liver lysates ( nor in some other tissues), and it can be competed with excess of unlabeled GDNF Binding of )251-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 Alternatively, by analogy with other receptors for 20 GDNF, c-RET might not bind GDNF directly, but might first bind to another nonsignalhng receptor that thereafter presents the ligand to c-RET, a signaling receptor A 50 kD GDNF-binding protein is a good candidate for the putative presenting receptor 25 Example 23 Protocol for isolating novel signaling receptors for GDNF In the absence of serum, 3T3 fibroblasts can be made dependent on a given exogenous growth factor provided appropriate receptors are expressed on the cell surface An expression library can be made using RN33B cDNA, which can then 30 be transfected into 3T3 fibroblasts by procedures well known in the art (Maniatis et al , supra) Stable transfectants can be selected in senim-free media supplemented with GDNF Fibroblast clones that express signaling GDNF receptors will Printed from Mimosa 07 11 15 WO 97/18240 PCT/lJSflfi/ISl1)? -53- selectivcly grow in the presence of GDNF in serum-free media The selection step may allow detection of even very reare clones due to their differential growth advantage Further analysis of the recovered clones in media with or without GDNF would help to distinguish GDNl'-dependent from GDNF-independent 5 survival of clones All references cited herein are hereby incorporated by reference in their entireties 10 Printed from Mimosa 0711 15 </div>

Claims

<div class="application article clearfix printTableText" id="claims">



WO 97/18240 

PCT/US96/18197 

-54- , t , 

WHAT IS CLAIMED IS: 

1 An isolated receptor which binds glial cell line-denved neurotrophic factor (GDNF), said receptor comprising at least one polypeptide having a 

5 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 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
3 The method of claim 2 wherein the receptors are polypeptides which bind GDNF selected from the group consisting of polypeptides about 55kD, 70kD, 135kD, 155kD, and 300kD molecular weight 

^
4 The method of claim 3 wherein the polypeptide is about !55kD molecular weight
5 The method of claim 2 wherein the isolated receptor is c-RET
6 The method of claim 2 wherein the labeled GDNF is 12fI-GDNF 

5
7 An isolated receptor which binds glial cell line-derived neurotrophic factor (GDNF), said receptor compnsmg a polypeptide having a molecular weight of about 155 kD, as determined by SDS-PAGE on 4-20% gradieat gels and at least one polypeptide having a molecular weight selected from the group consisting 

0 of polypeptides of about 55kD, 70kD, 135kD, and 300kD molecular weight, as determined by SDS-PAGE on 4-20% gradient gels 

9 

-55-
8 An isolated receptor as claimed in claim 1 substantially as herein descnbed with reference to any example thereof and to the accompanying drawings.
9 A method as claimed in claim 2 substantially as herein descnbed with reference to any example thereof and to the accompanying drawings
10 An isolated receptor as claimed m claim 7 substantially as herein descnbed with reference to any example thereof and to the accompanying drawings h
11 jl:J t;:9 



</div>