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• W1974824979 abstract "The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFRα-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFRα-1 or GFRα-2, a novel receptor related to GFRα-1. Both GFRα-1 and GFRα-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFRα-1 or GFRα-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFRα-1 bind GDNF more efficiently than neurturin, while cells expressing GFRα-2 bind neurturin preferentially. Cross-linking and Ret activation data also suggest that while there is cross-talk, GFRα-1 is the primary receptor for GDNF and GFRα-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFRα receptor family. This putative receptor, designated GFRα-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFRα-1 and GFRα-2. Analysis of the tissue distribution of GFRα-1, GFRα-2, GFRα-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFRαs and Ret are expressed in many of the same tissues, suggesting that GFRαs mediate the action of GDNF family ligands in vivo. The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFRα-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFRα-1 or GFRα-2, a novel receptor related to GFRα-1. Both GFRα-1 and GFRα-2 mediate neurturin-induced Ret phosphorylation. GDNF can also bind to either GFRα-1 or GFRα-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFRα-1 bind GDNF more efficiently than neurturin, while cells expressing GFRα-2 bind neurturin preferentially. Cross-linking and Ret activation data also suggest that while there is cross-talk, GFRα-1 is the primary receptor for GDNF and GFRα-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFRα receptor family. This putative receptor, designated GFRα-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFRα-1 and GFRα-2. Analysis of the tissue distribution of GFRα-1, GFRα-2, GFRα-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFRαs and Ret are expressed in many of the same tissues, suggesting that GFRαs mediate the action of GDNF family ligands in vivo. Glial cell line-derived neurotrophic factor (GDNF) 1The abbreviations used are: GDNF, glial cell line-derived neurotropic factor; NTN, neurturin; PTK, protein tyrosine kinase; RT-PCR, reverse transcriptase-polymerase chain reaction; CM, conditioned medium; PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag; kb, kilobase pairs(s). 1The abbreviations used are: GDNF, glial cell line-derived neurotropic factor; NTN, neurturin; PTK, protein tyrosine kinase; RT-PCR, reverse transcriptase-polymerase chain reaction; CM, conditioned medium; PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag; kb, kilobase pairs(s). is a multipotent neurotrophic factor that has a variety of effects on cells of both the central and peripheral nervous systems (1Yan Q. Matheson C. Lopez O.T. Nature. 1995; 373: 341-344Crossref PubMed Scopus (537) Google Scholar, 2Beck K.D. Valverde J. Alexi T. Poulsen K. Moffat B. Vandlen R.A. Rosenthal A. Hefti F. Nature. 1995; 373: 339-341Crossref PubMed Scopus (615) Google Scholar, 3Tomac A. Lindqvist E. Lin L.F. Ogren S.O. Young D. Hoffer B.J. Olson L. Nature. 1995; 373: 335-339Crossref PubMed Scopus (986) Google Scholar, 4Kearns C.M. Gash D.M. Brain Res. 1995; 672: 104-111Crossref PubMed Scopus (330) Google Scholar, 5Zurn A.D. Baetge E.E. Hammang J.P. Tan S.A. Aebischer P. Neuroreport. 1994; 6: 113-118Crossref PubMed Scopus (157) Google Scholar, 6Henderson C.E. Phillips H.S. Pollock R.A. Davies A.M. Lemeulle C. Armanini M. Simpson L.C. Moffet B. Vandlen R.A. Koliatsos V.E. Rosenthal A. Science. 1994; 266: 1062-1064Crossref PubMed Scopus (1126) Google Scholar, 7Lin L.H. Doherty D.H. Lile J.D. Bektesh S. Collins F. Science. 1993; 260: 1130-1132Crossref PubMed Scopus (2815) Google Scholar, 8Arenas E. Trupp M. Akerud P. Ibanez C.F. Neuron. 1995; 15: 1465-1473Abstract Full Text PDF PubMed Scopus (301) Google Scholar, 9Buj-Bello A. Buchman V.L. Horton A. Rosenthal A. Davies A.M. Neuron. 1995; 15: 821-828Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 10Ebendal T. Tomac A. Hoffer B.J. Olson L. J. Neurosci. Res. 1995; 40: 276-284Crossref PubMed Scopus (122) Google Scholar, 11Li L. Wu W. Lin L.F. Lei M. Oppenheim R.W. Houenou L.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9771-9775Crossref PubMed Scopus (333) Google Scholar, 12Mount H.T. Dean D.O. Alberch J. Dreyfus C.F. Black I.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9092-9096Crossref PubMed Scopus (145) Google Scholar, 13Oppenheim R.W. Houenou L.J. Johnson J.E. Lin L.F. Li L. Lo A.C. Newsome A.L. Prevette D.M. Wang S. Nature. 1995; 373: 344-346Crossref PubMed Scopus (608) Google Scholar, 14Sauer H. Rosenblad C. Bjoerklund A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8935-8939Crossref PubMed Scopus (324) Google Scholar, 15Trupp M. Ryden M. Joernvall H. Funakoshi H. Timmusk T. Arenas E. Ibanez C.F. J. Cell Biol. 1995; 130: 137-148Crossref PubMed Scopus (517) Google Scholar). Recently, a novel neurotrophic factor called neurturin that is 42% identical in amino acid sequence to GDNF was reported (16Kotzbauer P.T. Lampe P.A. Heuckeroth R.O. Golden J.P. Creedon D.J. Johnson Jr., E.M. Milbrandt J. Nature. 1996; 384: 467-470Crossref PubMed Scopus (649) Google Scholar). Both GDNF and neurturin are synthesized in pre-pro forms and their precursor molecules are proteolytically processed to yield mature proteins of about 100 amino acids that assemble into disulfide-linked homodimers (7Lin L.H. Doherty D.H. Lile J.D. Bektesh S. Collins F. Science. 1993; 260: 1130-1132Crossref PubMed Scopus (2815) Google Scholar, 16Kotzbauer P.T. Lampe P.A. Heuckeroth R.O. Golden J.P. Creedon D.J. Johnson Jr., E.M. Milbrandt J. Nature. 1996; 384: 467-470Crossref PubMed Scopus (649) Google Scholar). Seven cysteine residues are present in both GDNF and neurturin and their spacing is identical (16Kotzbauer P.T. Lampe P.A. Heuckeroth R.O. Golden J.P. Creedon D.J. Johnson Jr., E.M. Milbrandt J. Nature. 1996; 384: 467-470Crossref PubMed Scopus (649) Google Scholar). Although the biological activities of neurturin have not yet been thoroughly investigated, initial results indicate that they are very similar to those of GDNF. Both neurturin and GDNF have been shown to promote the survival of sympathetic neurons derived from the superior cervical ganglia and of sensory neurons of both the nodose and dorsal root ganglia (1Yan Q. Matheson C. Lopez O.T. Nature. 1995; 373: 341-344Crossref PubMed Scopus (537) Google Scholar, 6Henderson C.E. Phillips H.S. Pollock R.A. Davies A.M. Lemeulle C. Armanini M. Simpson L.C. Moffet B. Vandlen R.A. Koliatsos V.E. Rosenthal A. Science. 1994; 266: 1062-1064Crossref PubMed Scopus (1126) Google Scholar, 9Buj-Bello A. Buchman V.L. Horton A. Rosenthal A. Davies A.M. Neuron. 1995; 15: 821-828Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 10Ebendal T. Tomac A. Hoffer B.J. Olson L. J. Neurosci. Res. 1995; 40: 276-284Crossref PubMed Scopus (122) Google Scholar, 13Oppenheim R.W. Houenou L.J. Johnson J.E. Lin L.F. Li L. Lo A.C. Newsome A.L. Prevette D.M. Wang S. Nature. 1995; 373: 344-346Crossref PubMed Scopus (608) Google Scholar, 15Trupp M. Ryden M. Joernvall H. Funakoshi H. Timmusk T. Arenas E. Ibanez C.F. J. Cell Biol. 1995; 130: 137-148Crossref PubMed Scopus (517) Google Scholar, 16Kotzbauer P.T. Lampe P.A. Heuckeroth R.O. Golden J.P. Creedon D.J. Johnson Jr., E.M. Milbrandt J. Nature. 1996; 384: 467-470Crossref PubMed Scopus (649) Google Scholar). Neurturin and GDNF mRNAs are widely distributed in a variety of both neuronal and non-neuronal tissues of embryos and adults (6Henderson C.E. Phillips H.S. Pollock R.A. Davies A.M. Lemeulle C. Armanini M. Simpson L.C. Moffet B. Vandlen R.A. Koliatsos V.E. Rosenthal A. Science. 1994; 266: 1062-1064Crossref PubMed Scopus (1126) Google Scholar,15Trupp M. Ryden M. Joernvall H. Funakoshi H. Timmusk T. Arenas E. Ibanez C.F. J. Cell Biol. 1995; 130: 137-148Crossref PubMed Scopus (517) Google Scholar, 16Kotzbauer P.T. Lampe P.A. Heuckeroth R.O. Golden J.P. Creedon D.J. Johnson Jr., E.M. Milbrandt J. Nature. 1996; 384: 467-470Crossref PubMed Scopus (649) Google Scholar, 17Springer J.E. Mu X. Bergmann L.W. Trojanowski J.Q. Exp. Neurol. 1994; 127: 167-170Crossref PubMed Scopus (184) Google Scholar, 18Schaar D.G. Sieber B.A. Dreyfus C.F. Black I.B. Exp. Neurol. 1993; 124: 368-371Crossref PubMed Scopus (263) Google Scholar). Both are found in brain, kidney, and lung, whereas neurturin mRNA is also expressed at high levels in neonatal blood (15Trupp M. Ryden M. Joernvall H. Funakoshi H. Timmusk T. Arenas E. Ibanez C.F. J. Cell Biol. 1995; 130: 137-148Crossref PubMed Scopus (517) Google Scholar, 16Kotzbauer P.T. Lampe P.A. Heuckeroth R.O. Golden J.P. Creedon D.J. Johnson Jr., E.M. Milbrandt J. Nature. 1996; 384: 467-470Crossref PubMed Scopus (649) Google Scholar, 17Springer J.E. Mu X. Bergmann L.W. Trojanowski J.Q. Exp. Neurol. 1994; 127: 167-170Crossref PubMed Scopus (184) Google Scholar, 18Schaar D.G. Sieber B.A. Dreyfus C.F. Black I.B. Exp. Neurol. 1993; 124: 368-371Crossref PubMed Scopus (263) Google Scholar, 19Arenas E. Trupp M. Akerud P. Ibanez C.F. Neuron. 1995; 15: 1465-1473Abstract Full Text PDF PubMed Scopus (295) Google Scholar, 20Poulsen K.T. Armanini M.P. Klein R.D. Hynes M.A. Phillips H.S. Rosenthal A. Neuron. 1994; 13: 1245-1252Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 21Stroemberg I. Bjoerklund L. Johansson M. Tomac A. Collins F. Olson L. Hoffer B. Humpel C. Exp. Neurol. 1993; 124: 401-412Crossref PubMed Scopus (318) Google Scholar). The striking structural and biological similarities between GDNF and neurturin suggest that their action may be mediated by the same or related receptors. The receptor for GDNF consists of a complex ofGDNF Family Receptorα-1 (GFRα-1, previously abbreviated as GDNFR-α) and the Ret protein tyrosine kinase (PTK) (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar, 23Treanor J.J. Goodman L. de-Sauvage F. Stone D.M. Poulsen K.T. Beck C.D. Gray C. Armanini M.P. Pollock R.A. Hefti F. Phillips H.S. Goddard A. Moore M.W. Buj-Bello A. Davies A.M. Asai N. Takahashi M. Vandlen R. Henderson C.E. Rosenthal A. Nature. 1996; 382: 80-83Crossref PubMed Scopus (960) Google Scholar). GFRα-1 is a glycosyl phosphatidylinositol-anchored cell surface molecule. GFRα-1 binds to GDNF but cannot signal independently since it lacks a cytoplasmic domain. GDNF signaling is accomplished via association of the complex of GDNF and GFRα-1 with Ret, resulting in activation of the Ret kinase. GFRα-1 mRNA is widely distributed in neuronal and non-neuronal tissues and is expressed throughout embryonic development to adulthood, implying a broad spectrum of biological functions (23Treanor J.J. Goodman L. de-Sauvage F. Stone D.M. Poulsen K.T. Beck C.D. Gray C. Armanini M.P. Pollock R.A. Hefti F. Phillips H.S. Goddard A. Moore M.W. Buj-Bello A. Davies A.M. Asai N. Takahashi M. Vandlen R. Henderson C.E. Rosenthal A. Nature. 1996; 382: 80-83Crossref PubMed Scopus (960) Google Scholar, 24Trupp M. Belluardo N. Funakoshi H. Ibanez C. J. Neurosci. 1997; 17: 3554-3567Crossref PubMed Google Scholar). The other component of the GDNF receptor complex, Ret, is a receptor type PTK encoded by the ret proto-oncogene (25Takahashi M. Cooper G.M. Mol. Cell. Biol. 1987; 7: 1378-1385Crossref PubMed Scopus (284) Google Scholar). Ret mRNA and protein are highly expressed in the central and peripheral nervous systems, as well as in the kidney (26Pachnis V. Mankoo B. Costantini F. Development. 1993; 119: 1005-1017Crossref PubMed Google Scholar, 27Tsuzuki T. Takahashi M. Asai N. Iwashita T. Matsuyama M. Asai J. Oncogene. 1995; 10: 191-198PubMed Google Scholar). Various mutations in the ret gene are associated with human inherited diseases, including familial medullary thyroid carcinoma (28Donis-Keller H. Dou S. Chi D. Carlson K. Toshima K. Lairmore T. Howe J. Moley J. Goodfellow P. Wells S. Hum. Mol. Genet. 1997; 2: 851-856Crossref Scopus (1151) Google Scholar, 29Hofstra R.M. Landsvater R.M. Ceccherini I. Stulp R.P. Stelwagen T. Yuo Y. Pasini B. Hoppener J.W.M. van Amstel H.K.P. Romeo G. Lips C.J.M. Buys C.H.C.M. Nature. 1994; 367: 375-376Crossref PubMed Scopus (1007) Google Scholar), multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B) (28Donis-Keller H. Dou S. Chi D. Carlson K. Toshima K. Lairmore T. Howe J. Moley J. Goodfellow P. Wells S. Hum. Mol. Genet. 1997; 2: 851-856Crossref Scopus (1151) Google Scholar, 29Hofstra R.M. Landsvater R.M. Ceccherini I. Stulp R.P. Stelwagen T. Yuo Y. Pasini B. Hoppener J.W.M. van Amstel H.K.P. Romeo G. Lips C.J.M. Buys C.H.C.M. Nature. 1994; 367: 375-376Crossref PubMed Scopus (1007) Google Scholar, 30Borrello M.G. Smith D.P. Pasini B. Bongarzone I. Greco A. Lorenzo M.J. Arighi E. Miranda C. Eng C. Alberti L. Bocciardi R. Mondellini P. Scopsi L. Romeo G. Pinder B.A.J. Pierotti M.A. Oncogene. 1995; 11: 2419-2427PubMed Google Scholar, 31Mulligan L. Kwok J. Healey C. Elsdon M. Eng C. Gardner E. Love D. Mole S. Moore J. Papi L. Ponder M. Telenius H. Tunnacliffe A. Ponder A. Nature. 1993; 363: 458-460Crossref PubMed Scopus (1707) Google Scholar, 32Santoro M. Carlomagno F. Romeo A. Bottaro D. Dathan N. Grieco M. Fusco A. Vecchio G. Matoskova B. Kraus M. Di Fiore P. Science. 1995; 267: 381-383Crossref PubMed Scopus (792) Google Scholar), and Hirschsprung's disease (33Romeo G. Ronchetto P. Yuo Y. Barone V. Seri M. Ceccherini I. Pasini B. Bocciardi R. Lerone M. Kaariainen H. Martucciello G. Nature. 1994; 367: 377-378Crossref PubMed Scopus (642) Google Scholar, 34Edery P. Lyonnet S. Mulligan L. Pelet A. Dow E. Abel L. Holder S. Nihoul-Fekete C. Ponder B. Munnich A. Nature. 1994; 367: 378-380Crossref PubMed Scopus (660) Google Scholar). Targeted disruption of the ret gene in knockout mice results in severe phenotypic defects, including renal agenesis or severe dysgenesis and lack of the entire enteric nervous system (35Schuchardt A. D'Agati V. Larsson-Blomberg L. Costantini F. Pachnis V. Nature. 1994; 367: 380-383Crossref PubMed Scopus (1404) Google Scholar). These defects are very similar to those caused by GDNF null mutations (36Moore M.W. Klein R.D. Farinas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. Rosenthal A. Nature. 1996; 382: 76-79Crossref PubMed Scopus (1076) Google Scholar, 37Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Nature. 1996; 382: 73-76Crossref PubMed Scopus (995) Google Scholar, 38Sanchez M.P. Silos-Santiago I. Frisen J. He B. Lira S.A. Barbacid M. Nature. 1996; 382: 70-73Crossref PubMed Scopus (1033) Google Scholar), implying that GDNF-mediated signaling through Ret is required for the development of these tissues. Much less severe defects, however, were detected in a number of neuronal structures in which both GFRα-1 and Ret were expressed, such as the trigeminal and vestibular ganglia, the facial motor nucleus, the substantia nigra, and the locus coeruleus (35Schuchardt A. D'Agati V. Larsson-Blomberg L. Costantini F. Pachnis V. Nature. 1994; 367: 380-383Crossref PubMed Scopus (1404) Google Scholar, 36Moore M.W. Klein R.D. Farinas I. Sauer H. Armanini M. Phillips H. Reichardt L.F. Ryan A.M. Carver-Moore K. Rosenthal A. Nature. 1996; 382: 76-79Crossref PubMed Scopus (1076) Google Scholar, 37Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Nature. 1996; 382: 73-76Crossref PubMed Scopus (995) Google Scholar, 38Sanchez M.P. Silos-Santiago I. Frisen J. He B. Lira S.A. Barbacid M. Nature. 1996; 382: 70-73Crossref PubMed Scopus (1033) Google Scholar). This suggests that either GDNF signaling is not required for the embryonic development of these structures, or that some unknown signaling molecules similar to GDNF or Ret may exist that can substitute for them. Alternatively, the embryonic development of these tissues may rely completely on other as yet unknown signaling systems. In this paper we report the cloning of GFRα-2 and GFRα-3, two novel receptors related to GFRα-1, and provide evidence that GFRα-2 is a receptor for both GDNF and neurturin. Our data also indicate that GFRα-1 is a receptor for neurturin as well as for GDNF. We describe a related cDNA that codes for a protein, GFRα-3, that shares significant amino acid homology with both GFRα-1 and GFRα-2 and is likely to be a third member to the family of receptors for GDNF-related ligands. A search of the GenBank data base for sequences related to GFRα-1 resulted in the identification of a single related EST, H12981.Gb_Est1. Primers corresponding to nucleotides 47–65 (5′-CTGCAAGAAGCTGCGCTCC-3′) and 244–265 (5′-CTTGTCCTCATAGGAGCAGC-3′) of H12981.Gb_Est1 were synthesized and used for RT-PCR with human fetal brain mRNA (CLONTECH, catalog number 64019-1) as the template. A 218-nucleotide fragment was amplified, subcloned into pBlueScript (Stratagene, La Jolla, CA), and sequenced. The fragment was then radiolabeled with [32P]dCTP using a Random Prime DNA Labeling Kit (Stratagene) according to the manufacturer's instructions. The oligonucleotide primers described above were also used as primers for PCRs to screen DNAs isolated from 27 pools (1500 clones each) of a rat photoreceptor cDNA library (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). A single positive pool was identified, leading to the identification of an individual rat cDNA clone from this pool by hybridization to the radiolabeled human PCR fragment described above. Filters were prehybridized at 55 °C for 3.5 h in 200 ml of 6 × SSC, 1 × Denhardt's, 0.5% SDS, and 50 μg/ml salmon sperm DNA. Following the addition of 2 × 108 cpm of the radiolabeled probe, hybridization was continued for 18 h. Filters were then washed twice for 30 min each at 55 °C in 0.2 × SSC, 0.1% SDS and exposed to x-ray film overnight with an intensifying screen. The GenBank data base was searched for sequences related to GFRα-1 and GFRα-2 using the Wisconsin sequence analysis package (Wisconsin Package version 9.0, Genetics Computer Group, Madison, WI). Oligonucleotide primers corresponding to regions near the ends of an EST AA238748.Gb_New2 were synthesized. Primers corresponding to AA238748.Gb_New2 were used for PCR screening of 83 pools of 1000 clones each from a rat E15 embryonic cDNA library. 2S. Jing, Y. Yu, M. Fang, Z. Hu, P. L. Holst, and G. M. Fox, unpublished data. A single positive pool was identified by this method, and the DNA fragment amplified from this pool was subcloned into a plasmid vector and labeled with [32P]dCTP using a Random Primed DNA Labeling Kit (Stratagene) according to the manufacturer's instructions. Clones from the cDNA library pool that had been identified as positive by PCR were plated on 15-cm agarose plates and replicated on duplicate nitrocellulose filters for screening by hybridization to the radiolabeled insert. Hybridization conditions were the same as those described in the preceding section. Positive clones were sequenced as described below. DNA sequencing was performed using an automated Applied Biosystems 373A DNA sequencer and Taq DyeDeoxy Terminator cycle sequencing kits (Applied Biosystems, Foster City CA). Comparison of the GFRα-1 and GFRα-2 sequences with public data bases was carried out using the FASTA computer algorithm (39Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9328) Google Scholar). The peptide sequences of GFRα-1, GFRα-2, and GFRα-3 were aligned using the Lineup program. All sequence analysis programs used were included in the Wisconsin sequence analysis package (Wisconsin Package Version 9.0, Genetics Computer Group, Madison, WI). Recombinant human neurturin was expressed in Escherichia coli as insoluble protein sequestered into inclusion bodies. The inclusion bodies were isolated, solubilized, and the neurturin protein was re-folded and purified by ion exchange and hydrophobic interaction chromatography as described previously (7Lin L.H. Doherty D.H. Lile J.D. Bektesh S. Collins F. Science. 1993; 260: 1130-1132Crossref PubMed Scopus (2815) Google Scholar). [125I]NTN (∼1800 Ci/mmol) was prepared using the purified E. coli-expressed protein by Amersham (Arlington Heights, IL; custom iodination, catalog number IMQ1057). Recombinant human GDNF was also radioiodinated by Amersham to a similar specific activity (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). The isolated human GFRα-2 cDNA was subcloned into an eukaryotic expression plasmid, pBK RSV (Stratagene) to generate the GFRα-2 expression vector pHGLRSV.2 NNR-9, a cell line expressing GFRα-2, was derived from Neuro-2a cells (ATCC catalog number CCL 131) transfected with pHGLRSV. The transfection was accomplished by using the calcium phosphate transfection system (Life Technologies, Inc.) according to the manufacturer's directions. Transfected cells were selected for expression of the plasmid by growth in 400 μg/ml G418 antibiotic (Sigma). G418-resistant clones were expanded and assayed for GFRα-2 expression by Northern blot using the GFRα-2 cDNA as probe. Expression of GFRα-2 in individual clones was confirmed by binding to [125I]NTN. Binding of [125I]NTN and [125I]GDNF to NNR-9 and NGR-38 cells were carried out as described previously (40Jing S.Q. Spencer T. Miller K. Hopkins C. Trowbridge I.S. J. Cell Biol. 1990; 110: 283-294Crossref PubMed Scopus (218) Google Scholar). Briefly, cells were seeded 1 day before the assay in 24-well Costar tissue culture plates precoated with polyornithine at a density of 3 × 104 cells/cm2. Cells were placed on ice for 5–10 min, washed once with ice-cold buffer (Dulbecco's modified Eagle's medium containing 25 mm HEPES, pH 7.0), and incubated at 4 °C in 0.2 ml of binding buffer (washing buffer containing 2 mg/ml bovine serum albumin) containing 50 pm[125I]NTN or [125I]GDNF in the absence or presence of 500 nm unlabeled ligand for 4 h. Cells were washed 4 times with 0.5 ml of ice-cold washing buffer and lysed with 0.5 ml of 1 m NaOH. The lysates were counted in a 1470 Wizard Automatic Gamma Counter (Wallac Inc., Gaithersburg, MD). The coding regions of the first 455 amino acids of the human GFRα-1 and the first 451 residues of human GFRα-2 cDNAs were fused in-frame with a DNA fragment encoding the Fc region of human IgG1 tagged with 6 histidine residues at the carboxyl terminus (41Culouscou J.-M. Carlton G.W. Aruffo A. J. Biol. Chem. 1995; 270: 12857-12863Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These constructs were then inserted into the expression vector pBK RSV as described previously (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). The GFRα-1/Fc and GFRα-2/Fc fusion constructs were transfected into 293T cells and conditioned media (CM, Dulbecco's modified Eagle's medium supplied with 0.5% fetal calf serum) containing the fusion proteins were collected 4 days after transfection. Aliquots of 1 ml of CM plus 50 μl of 1 m HEPES, pH 7.5, were incubated at 4 °C with 2 nm [125I]NTN or [125I]GDNF in the presence or absence of 1 μm unlabeled ligand for 4 h. A 40 mmbis-suberate (BS3 Pierce, Rockford, IL) stock solution in washing buffer was added to each binding mixture to a final concentration of 1 mm, mixed, and incubated at room temperature for 30 min. The reaction was quenched by adding 50 μl of 1 m glycine and incubating at room temperature for 15 min. Triton X-100 was added to a final concentration of 1% and the cross-linked product was precipitated directly with 200 μl of Protein-A Sepharose CL-4B (Pharmacia). The cross-linked products were analyzed by 7.5% SDS-PAGE (1:200 bis:acrylamide ratio) under reducing conditions. Ret autophosphorylation was examined by immunoblot analysis as described previously (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). Briefly, cells were seeded 24 h prior to the assay in 6-well tissue culture dishes at a density of 1.5 × 106 cells/well. Cells were washed once with binding buffer and treated with various concentrations of neurturin or GDNF (0.5 pm to 50 nm) in binding buffer at 37 °C for 10 min. Treated cells and untreated controls were lysed in Triton X-100 lysis buffer (50 mm HEPES, pH 7.5, 1% Triton X-100, 50 mm NaCl, 50 mm sodium fluoride, 10 mm sodium pyrophosphate, 1% aprotinin (Sigma, catalog number A-6279), 1 mm phenylmethylsulfonyl fluoride (Sigma, catalog number P-7626), 0.5 mm Na3VO4 (Fisher catalog number S454-50) and immunoprecipitated with an anti-Ret antibody (Santa Cruz Biotechnology) and protein-A Sepharose as described (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar). Immunoprecipitates were fractionated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes as described by Harlow and Lane (42Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1988Google Scholar). The membranes were blocked with 5% bovine serum albumin (Sigma) and tyrosine phosphorylation of the Ret receptor was detected by probing with an anti-phosphotyrosine monoclonal antibody 4G10 (UBI, catalog number 05-321) at room temperature for 2 h. The amount of Ret protein in each lane was determined by stripping and re-probing the same membrane with the anti-Ret antibody. Detection was accomplished using sheep anti-mouse secondary antibody or protein-A conjugated to horseradish peroxidase (Amersham, catalog number NA931) in conjunction with chemiluminescence reagents (ECL, Amersham) following the manufacturer's instructions. For blot hybridization analysis, the cloned rat GFRα-1, GFRα-2, and GFRα-3 cDNA was labeled using the Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's instructions. Rat and mouse RNA blots purchased from CLONTECH were hybridized with the probe and washed at high stringency using the reagents of the ExpressHyb Kit (CLONTECH) according to the manufacturer's instructions. Following exposure on x-ray film, the filters were stripped of probe by boiling in 0.5% SDS for 10 min and rehybridized with a β-actin probe (CLONTECH) as a control for total RNA loading. A human expressed sequence tag (EST) with significant homology to GFRα-1 was found by a search of the publicly available nucleic acid sequence data bases. 3L. Hillier, WashU-Merck EST Project, unpublished data. Oligonucleotides corresponding to the ends of this EST were synthesized and used in a reverse transcription-polymerase chain reaction (RT-PCR) with human fetal brain mRNA as the template. A fragment of the expected length was isolated, labeled, and used as a hybridization probe to screen a human fetal brain cDNA library. The longest clone isolated in this manner was sequenced and found to contain an open reading frame coding for a 464-amino acid protein related in sequence to GFRα-1. We have named this protein GDNFFamily Receptor α-2 (GFRα-2). The oligonucleotides described above were also used to screen pools from a rat photoreceptor cDNA library (22Jing S. Wen D. Yu Y. Holst P.L. Luo L. Fang M. Tamir R. Antonio L. Hu Z. Cupples R. Louis J.C. Hu S. Altrock B.W. Fox G.M. Cell. 1996; 85: 1113-1124Abstract Full Text Full Text PDF PubMed Scopus (1039) Google Scholar) by PCR and a product of the expected length was obtained from a single pool. An individual cDNA clone from this pool was identified by hybridization to the radiolabeled human GFRα-1 PCR product and sequenced. This clone contained an open reading frame coding for a 460-amino acid peptide that is nearly identical to human GFRα-2 and almost certainly represents its rat ortholog. Publicly available sequence data bases were searched using GFRα-1 and GFRα-2 as query sequences and a short EST with homology to both GFRα-1 and GFRα-2 was found. 4M. Marra, WashU-HHMI Mouse EST Project, unpublished data. Oligonucleotides corresponding to the ends of this EST were used as primers in RT-PCR with total rat embryo RNA as the template. A 225-nucleotide fragment was amplified, cloned into a plasmid vector, and sequenced to verify that it corresponded to the original GFRα-1/GFRα-2-related EST. Plasmid DNAs isolated f" @default.
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• W1974824979 title "GFRα-2 and GFRα-3 Are Two New Receptors for Ligands of the GDNF Family" @default.
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