The Long and Short Isoforms of Ret Function as Independent Signaling Complexes
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m203580200
ISSN1083-351X
AutoresBrian A. Tsui-Pierchala, Rebecca Ahrens, Robert J. Crowder, Jeffrey Milbrandt, Eugene M. Johnson,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoRet, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr905 and Tyr1062 was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr1015 was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo. Ret, the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor family ligands (GFLs), is alternatively spliced to yield at least two isoforms, Ret9 and Ret51, which differ only in their C termini. To identify tyrosines in Ret that are autophosphorylation sites in neurons, we generated antibodies specific to phosphorylated Y905Ret, Y1015Ret, Y1062Ret, and Y1096Ret, all of which are autophosphorylated in cell lines. All four of these tyrosines in Ret became phosphorylated rapidly upon activation by GFLs in sympathetic neurons. These tyrosines remained phosphorylated in sympathetic neurons in the continued presence of GFLs, albeit at a lower level than immediately after GFL treatment. Comparison of GFL activation of Ret9 and Ret51 revealed that phosphorylation of Tyr905 and Tyr1062 was greater and more sustained in Ret9 as compared with Ret51. In contrast, Tyr1015 was more highly phosphorylated over time in Ret51 than in Ret9. Surprisingly, Ret9 and Ret51 did not associate with each other in sympathetic neurons after glial cell line-derived neurotrophic factor stimulation, even though they share identical extracellular domains. Furthermore, the signaling complex associated with Ret9 was markedly different from the Ret51-associated signaling complex. Taken together, these data provide a biochemical basis for the dramatic functional differences between Ret9 and Ret 51 in vivo. receptor tyrosine kinase glial cell line-derived neurotrophic factor GDNF family ligand neurturin days in vitro nerve growth factor phospho-Ret Trophic factors sculpt the nervous system during development by regulating neuronal number, size, and phenotype. Many neurotrophic factors function via activation of receptor tyrosine kinases (RTKs),1 which autophosphorylate in trans upon ligand-induced dimerization (1Hunter T. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1998; 353: 583-605Crossref PubMed Scopus (364) Google Scholar, 2Schlessinger J. Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3557) Google Scholar). The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) function via activation of the RTK, Ret (3Airaksinen M.S. Titievsky A. Saarma M. Mol. Cell. Neurosci. 1999; 13: 313-325Crossref PubMed Scopus (388) Google Scholar, 4Takahashi M. Cytokine Growth Factor Rev. 2001; 12: 361-373Crossref PubMed Scopus (367) Google Scholar). The GFL family consists of GDNF, neurturin (NRTN), persephin, and artemin, and they promote the survival and growth of central nervous system and peripheral nervous system neurons both in vitro and in vivo (5Baloh R.H. Enomoto H. Johnson E.M., Jr. Milbrandt J. Curr. Opin. Neurobiol. 2000; 10: 103-110Crossref PubMed Scopus (408) Google Scholar). Ret is not activated via direct binding of GFLs to its extracellular domain but rather is activated by complexes formed by GFLs associated with glycerophosphatidylinositol-anchored co-receptor proteins called GFRαs. GFRα family members (GFRα1–4) demonstrate preferential binding to a particular GFL, thus providing specificity to Ret activation depending upon which GFL and GFRα are present. Signal transduction pathways activated by Ret have been analyzed mostly in cell lines transiently expressing the receptor components, in cell lines that express MEN-2A and MEN-2B constitutively activated forms of Ret, or in neuroblastoma cell lines (4Takahashi M. Cytokine Growth Factor Rev. 2001; 12: 361-373Crossref PubMed Scopus (367) Google Scholar, 6Manie S. Santoro M. Fusco A. Billaud M. Trends Genet. 2001; 17: 580-589Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). These studies have identified multiple tyrosines that are autophosphorylated in Ret (7Liu X. Vega Q.C. Decker R.A. Pandey A. Worby C.A. Dixon J.E. J. Biol. Chem. 1996; 271: 5309-5312Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) including Tyr905, Tyr1015, Tyr1062, and Tyr1096 (see Fig. 1A). Tyr905 is an autocatalytic tyrosine that is conserved in many RTKs and is a binding site for GRB10 (8Durick K., Wu, R.-Y. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 9Pandey A. Duan H., Di Fiore P.P. Dixit V.M. J. Biol. Chem. 1995; 270: 21461-21463Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Phospholipase Cγ and GRB2 bind to Tyr1015 and Tyr1096, respectively (10Borrello M.G. Alberti L. Arighi E. Bongarzone I. Battistini C. Bardelli A. Pasini B. Piutti C. Rizzetti M.G. Mondellini P. Radice M.T. Pierotti M.A. Mol. Cell. Biol. 1996; 16: 2151-2163Crossref PubMed Google Scholar, 11Lorenzo M.J. Gish G.D. Houghton C. Stonehouse T.J. Pawson T. Ponder B.A.J. Smith D.P. Oncogene. 1997; 14: 763-771Crossref PubMed Scopus (114) Google Scholar, 12Alberti L. Borrello M.G. Ghizzoni S. Torriti F. Rizzetti M.G. Pierotti M.A. Oncogene. 1998; 17: 1079-1087Crossref PubMed Scopus (78) Google Scholar). Tyr1062 is a binding site for SHC, Dok4/5, IRS-1, and FRS-2 when phosphorylated and is a binding site for Enigma in a phosphorylation-independent manner (8Durick K., Wu, R.-Y. Gill G.N. Taylor S.S. J. Biol. Chem. 1996; 271: 12691-12694Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 11Lorenzo M.J. Gish G.D. Houghton C. Stonehouse T.J. Pawson T. Ponder B.A.J. Smith D.P. Oncogene. 1997; 14: 763-771Crossref PubMed Scopus (114) Google Scholar, 13Asai N. Murakami H. Iwashita T. Takahashi M. J. Biol. Chem. 1996; 271: 17644-17649Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14Arighi E. Alberti L. Torriti F. Ghizzoni S. Rizzetti M.G. Pelicci G. Pasini B. Bongarzone I. Piutti C. Pierotti M.A. Borrello M.G. Oncogene. 1997; 14: 773-7852Crossref PubMed Scopus (108) Google Scholar, 15Grimm J. Sachs M. Britsch S., Di Cesare S. Schwarz-Romond T. Alitalo K. Birchmeier W. J. Cell Biol. 2001; 154: 345-354Crossref PubMed Scopus (138) Google Scholar, 16Hayashi H. Ichihara M. Iwashita T. Murakami H. Shimono Y. Kawai K. Kurokawa K. Murakumo Y. Imai T. Funahashi H. Nakao A. Takahashi M. Oncogene. 2000; 19: 4469-4475Crossref PubMed Scopus (190) Google Scholar, 17Kurokawa K. Iwashita T. Murakami H. Hayashi H. Kawai K. Takahashi M. Oncogene. 2001; 20: 1929-1938Crossref PubMed Scopus (81) Google Scholar, 18Melillo R.M. Santoro M. Ong S.H. Billaud M., A., F. Hadari Y.R. Schlessinger J. Lax I. Mol. Cell. Biol. 2001; 21: 4177-4187Crossref PubMed Scopus (112) Google Scholar, 19Melillo R.M. Carlomagno F., De Vita G. Formisano P. Vecchio G. Fusco A. Billaud M. Santoro M. Oncogene. 2001; 20: 209-218Crossref PubMed Scopus (49) Google Scholar). Thus, Tyr905, Tyr1015, Tyr1062, and Tyr1096 contribute to various aspects of Ret signal transduction. However, the extent of autophosphorylation of these tyrosine residues and their functions in Ret signal transduction in neurons are unknown. Ret is alternatively spliced to produce at least two isoforms that differ only in the C-terminal residues; Ret9 has 9 amino acids that differ from the unique C-terminal 51 residues of Ret51 (20Tahira T. Ishizaka Y. Itoh F. Sugimura T. Nagao M. Oncogene. 1990; 5: 97-102PubMed Google Scholar). These relatively minor differences have dramatic functional consequences; Ret9 is critically important for kidney morphogenesis and enteric nervous system development, whereas Ret51 is dispensable (21de Graaff E. Srinivas S. Kilkenny C. D'Agati V. Mankoo B.S. Costantini F. Pachnis V. Genes Dev. 2001; 15: 2433-2444Crossref PubMed Scopus (202) Google Scholar). Furthermore, transgenic overexpression of Ret51 only partially compensates for the loss of Ret9 in kidney and enteric nervous system development (21de Graaff E. Srinivas S. Kilkenny C. D'Agati V. Mankoo B.S. Costantini F. Pachnis V. Genes Dev. 2001; 15: 2433-2444Crossref PubMed Scopus (202) Google Scholar). In contrast, Ret51, but not Ret9, is required for the metabolism and growth of mature sympathetic neurons via a GFL-independent mechanism of activation (22Tsui-Pierchala B.A. Milbrandt J. Johnson E.M., Jr. Neuron. 2002; 33: 261-273Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The biochemical differences between Ret9 and Ret51 that account for these functional differences are unknown. Tyrosine 1062, which is required for the majority of mitogen-activated protein kinase activation, phosphatidylinositol 3-kinase activation, and Ret function by GFL stimulation in neuroblastoma cells (15Grimm J. Sachs M. Britsch S., Di Cesare S. Schwarz-Romond T. Alitalo K. Birchmeier W. J. Cell Biol. 2001; 154: 345-354Crossref PubMed Scopus (138) Google Scholar, 16Hayashi H. Ichihara M. Iwashita T. Murakami H. Shimono Y. Kawai K. Kurokawa K. Murakumo Y. Imai T. Funahashi H. Nakao A. Takahashi M. Oncogene. 2000; 19: 4469-4475Crossref PubMed Scopus (190) Google Scholar, 23De Vita G. Melillo R.M. Carlomagno F. Visconti R. Castellone M.D. Bellacosa A. Billaud M. Fusco A. Tsichlis P.N. Santoro M. Cancer Res. 2000; 60: 3727-3731PubMed Google Scholar), is only two residues N-terminal to the C-terminal Ret splice site, which alters the context of this residue between Ret9 and Ret51. Consistent with this, Tyr1062 in Ret9 and Ret51 does appear in some cases to have altered interactions with SHC and GRB2 (11Lorenzo M.J. Gish G.D. Houghton C. Stonehouse T.J. Pawson T. Ponder B.A.J. Smith D.P. Oncogene. 1997; 14: 763-771Crossref PubMed Scopus (114) Google Scholar, 12Alberti L. Borrello M.G. Ghizzoni S. Torriti F. Rizzetti M.G. Pierotti M.A. Oncogene. 1998; 17: 1079-1087Crossref PubMed Scopus (78) Google Scholar, 17Kurokawa K. Iwashita T. Murakami H. Hayashi H. Kawai K. Takahashi M. Oncogene. 2001; 20: 1929-1938Crossref PubMed Scopus (81) Google Scholar, 24Ishiguro Y. Iwashita T. Murakami H. Asai N. Iida K.-I. Goto H. Hayakawa T. Takahashi M. Endocrinology. 1999; 140: 3992-3998Crossref PubMed Google Scholar) but not FRS2 (17Kurokawa K. Iwashita T. Murakami H. Hayashi H. Kawai K. Takahashi M. Oncogene. 2001; 20: 1929-1938Crossref PubMed Scopus (81) Google Scholar). Ret51 also has two additional tyrosine residues, Tyr1090 and Tyr1096, that may participate in signaling events. Consistent with this possibility, Tyr1096appears to contribute to phosphatidylinositol 3-kinase and mitogen-activated protein kinase activation (16Hayashi H. Ichihara M. Iwashita T. Murakami H. Shimono Y. Kawai K. Kurokawa K. Murakumo Y. Imai T. Funahashi H. Nakao A. Takahashi M. Oncogene. 2000; 19: 4469-4475Crossref PubMed Scopus (190) Google Scholar, 25Besset V. Scott R.P. Ibanez C.F. J. Biol. Chem. 2000; 275: 39159-39166Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). To determine whether Ret9 and Ret51 autophosphorylation and downstream signaling differ in neurons, we generated antibodies that specifically recognize Ret when phosphorylated on Tyr905, Tyr1015, Tyr1062, or Tyr1096. Additionally, antibodies were generated that stoichiometrically immunoprecipitate Ret9 or Ret51. Using these antibodies we show that upon ligand binding significant differences in the kinetics of Tyr905, Tyr1015, and Tyr1062 autophosphorylation occur between Ret9 and Ret51 in neurons. Furthermore, despite co-expression, Ret9 and Ret51 did not associate with each other upon activation in neurons and formed distinctly different signaling complexes in sympathetic neurons. These data indicate that Ret9 and Ret51 functioned as independent receptors for GFLs even within the same cell, providing a biochemical basis for the functional uniqueness of these Ret isoforms in vivo. Sympathetic neurons from the rat superior cervical ganglia were dissociated and maintained in vitro as described previously (26Deckwerth T.L. Johnson E.M., Jr. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (515) Google Scholar). Sympathetic neurons were maintained for 9–12 days in vitro(DIV) in the presence of NGF (50 ng/ml) and then switched to medium containing a lower concentration of NGF (2 ng/ml) for 24 h prior to stimulation with either GDNF or NRTN (both at 50 ng/ml) for the length of time described in each experiment. CHP126 neuroblastoma cells were seeded into Primaria 6-well tissue culture dishes (Falcon, Becton Dickinson and Company, Franklin Lakes, NJ) and maintained in normal growth medium (10% fetal bovine serum, 1.4 mml-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Sigma)) to 50–70% confluence. The cells were then transfected with various expression vectors by using LipofectAMINE reagent (Invitrogen) according to the manufacturer's instructions. The transfected cells were maintained for 48 h in normal growth medium prior to lysis. After the described treatments, sympathetic neurons and transfected CHP126 cells were washed twice with ice-cold phosphate-buffered saline, pH 7.4, and then extracted with immunoprecipitation buffer (Tris-buffered saline, pH 7.4, 1% Nonidet P-40, 10% glycerol, protease inhibitors, and 1 mm sodium orthovanadate) with gentle rocking at 4 °C. The detergent extracts were cleared of insoluble debris and nuclei by centrifugation at 13,000 × g in a refrigerated microcentrifuge for 10 min. The cleared extracts were either immunoprecipitated with Ret antibodies or diluted 2-fold with 2× sample buffer and boiled for SDS-PAGE as described previously (22Tsui-Pierchala B.A. Milbrandt J. Johnson E.M., Jr. Neuron. 2002; 33: 261-273Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The cell extracts or immunoprecipitates were subjected to SDS-PAGE in 4–12% gradient mini-gels (Novex, San Diego, CA) or 7.5% slab gels, and the separated proteins were transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). The blots were then blocked with either 2% bovine serum albumin (PY-Ret antibodies, Pan-Ret antibodies) or 4% heat-inactivated horse serum (phosphotyrosine antibody) in TBST (0.1% Tween 20 in Tris-buffered saline) for 1 h. The blots were next incubated with the primary antibody in the appropriate blocking buffer for 2 h, washed three times with TBST, and then incubated with the appropriate secondary antibody (1:10,000 dilution; Cell Signaling Technology, Beverly, MA) in blocking buffer for 1 h. The immunoblots were again washed three times with TBST and developed with a chemiluminescent substrate (Supersignal; Pierce). The dilutions and sources of the antibodies used for immunoblot analysis were as follows: anti-PY905Ret, anti-PY1015Ret, anti-PY1062Ret, and anti-PY1096Ret were diluted 1:1000–1:3000; anti-pan-Ret was diluted 1:1000 (R & D Systems, Minneapolis, MN; or a rabbit polyclonal antibody to the extracellular domain of Ret produced previously); anti-Ret9 was diluted 1:1000 (C19, Santa Cruz, Inc., Santa Cruz, CA); anti-Ret51 was 1:1000 (C20, Santa Cruz); anti-phosphotyrosine was 1:2000 (Upstate Biotechnology Inc., Beverly, MA). The blots were quantified by using the UN-SCAN-IT software (Silk Scientific, Orem, UT) after confirmation that each antibody was in the linear range for the protein of interest by a dose-response analysis. Antibodies to specific phosphorylated tyrosines of mouse Ret were produced by first generating peptides containing the appropriate phosphotyrosine residues (Biomolecules Midwest Inc., Waterloo, IL). The Ret phospho-peptides were as follows: PY905Ret was CEEDSY(PO4)VKKS; PY1015Ret was CVKSRDY(PO4)LDLA; PY1062Ret was CIENKLY(PO4)GMSD; and PY1096Ret was CANDSVY(PO4)ANWM. The peptides were covalently bound to the large carrier protein keyhole limpet hemocyanin via their N-terminal cysteine residues by using maleimide-activated keyhole limpet hemocyanin (Pierce). The protein conjugates were then injected into rabbits (Covance Research Products Inc., Richmond, CA), and the sera were tested for specific antibody production by Western analysis. The highest titer antiserum was affinity purified by first binding the antibodies to affinity columns produced by covalently linking the corresponding phosphopeptide to an agarose resin (Pierce). The nonspecific antibodies were washed off the column, and the specific antibodies were eluted by using both acidic (100 mmglycine, pH 2.5) and basic (100 mm triethylamine, pH 11) buffers. To purify the antibodies further, these eluates were counter-purified over a column made with the corresponding peptide that did not contain the phosphorylated tyrosine. After this, phosphotyrosine antibodies not specific to the particular tyrosine in Ret were removed from the eluate by counter-purifying the eluate from the prior two columns over a third column produced by using the three other unrelated PYRet peptides. These eluates were then dialyzed and concentrated with a Centriprep centrifugal device (Millipore). This triple purification produced antibodies highly specific to the phosphorylated tyrosine in Ret that was originally targeted. Antibodies specific to the short isoform of Ret (Ret9) and the longer isoform of Ret (Ret51) were generated in a manner similar to that of the PYRet antibodies. The peptides were: Ret9, CGRISHAFTRF; Ret51, CMVSPSAAKLMDTFDS, both of which are only contained in that particular Ret isoform. For immunoprecipitation using these Ret9 and Ret51 antibodies, anti-Ret9 and anti-Ret51 were covalently bound to an agarose support to avoid contamination of the immunoprecipitate with IgG released from the protein A beads after detergent solubilization. To generate anti-Ret9 and anti-Ret51 agarose, purified and unpurified antibodies were covalently bound to the agarose support by using AminoLink resin according to the manufacturer's instructions (Pierce). Ret expression plasmids used in this study have been described previously (27Tansey M.G. Baloh R.H. Milbrandt J. Johnson E.M., Jr. Neuron. 2000; 25: 611-623Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The Y905F, Y1015F, Y1062F, and K758M Ret mutants were generated by standard PCR-based point mutagenesis cloning techniques. The Y1096F Ret mutant was generously provided by Jack Dixon and Carolyn Worby (7Liu X. Vega Q.C. Decker R.A. Pandey A. Worby C.A. Dixon J.E. J. Biol. Chem. 1996; 271: 5309-5312Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). To determine whether specific tyrosine residues in Ret are bona fide autophosphorylation sites in neurons, we generated antibodies that recognize Ret only when phosphorylated on individual tyrosine residues. Antibodies to PY905Ret, PY1015Ret, PY1062Ret, and PY1096Ret (Fig.1A) were made by producing and purifying rabbit antisera against phosphorylated Ret antigens (see "Experimental Procedures"). Similar antibodies to PY1015Ret and PY1062Ret have been described previously (28Salvatore D. Barone M.V. Salvatore G. Melillo R.M. Chiappetta G. Mineo A. Fenzi G. Vecchio G. Fusco A. Santoro M. J. Clin. Endocrinol. Metab. 2000; 85: 3898-3907Crossref PubMed Scopus (57) Google Scholar, 29Yamamoto M., Li, M. Mitsuma N. Ito S. Kato M. Takahashi M. Sobue G. Brain Res. 2001; 912: 89-94Crossref PubMed Scopus (16) Google Scholar) and were used to demonstrate that these tyrosines are phosphorylated in Ret in transfected cell lines and transforming mutants of Ret. To confirm that these antibodies were specific for only the targeted tyrosine residue in Ret, we expressed Ret9, Ret51, or various tyrosine-to-phenylalanine mutants of Ret51 in the neuroblastoma cell line CHP126. CHP126 cells did not express Ret and were transfected with high efficiency (data not shown). Like other cell lines, transient overexpression of Ret in CHP126 cells resulted in high levels of Ret autophosphorylation in the absence of ligand stimulation (Fig. 1B and data not shown). Consistent with this observation, all four phospho-Ret antibodies recognized Ret51 when overexpressed in CHP126 cells (Fig.1B). In contrast, none of the P-Ret antibodies recognized Ret containing a K758M mutation that renders Ret kinase inactive (Fig. 1B). Of importance, none of the P-Ret antibodies recognized Ret that contained a tyrosine-to-phenylalanine mutation of the tyrosine that each antibody was directed against (Fig.1B). Anti-PY1096Ret also did not detect activated Ret9 because Ret9 does not contain Tyr1096 (Fig. 1B). Because Tyr905 is required for the catalytic activity of Ret, mutation of this residue diminishes, but does not eliminate, Ret autophosphorylation (data not shown). Although anti-PY1015Ret could not detect Y905F Ret when expressed in CHP126 cells, anti-PY1062Ret, and anti-PY1096Ret did recognize Y905F Ret at lower levels than Ret51, demonstrating that some residual autophosphorylation remained. This indicates that anti-PY905Ret is specific for Tyr905 because anti-PY905Ret was unable to detect the residual autophosphorylation of Y905F Ret (Fig. 1B) even after long exposure times (data not shown). Therefore, purified antibodies directed against PY905, PY1015, PY1062, and PY1096 Ret specifically recognized Ret only when the tyrosine of interest was phosphorylated. To determine whether these tyrosine residues were autophosphorylated upon ligand-mediated Ret activation, sympathetic neurons from the superior cervical ganglion of mice were dissociated and maintained in vitro with NGF, which is required for their survival. After 8–12 DIV, the neurons were starved of NGF for 2 days and were treated with GDNF (50 ng/ml) or medium alone for 15 min. The sympathetic neurons were then detergent-extracted, and P-Ret immunoblotting was performed on the extracts. Tyr905, Tyr1015, Tyr1062, and Tyr1096 were phosphorylated upon GDNF stimulation, demonstrating that they are autophosphorylation sites in Ret (ret+/+neurons; Fig. 2). Proteins with a molecular mass smaller than 180 kDa were also detected with several of the P-Ret antibodies after GDNF treatment of sympathetic neurons and probably represent proteolytic degradation of Ret that may occur during detergent extraction (data not shown). To confirm that the 180-kDa protein identified by the P-Ret antibodies was Ret, dissociated 12 DIV superior cervical ganglia neurons fromret+/+, ret+/−, orret−/− mice were stimulated with GDNF, NGF, or medium alone and subjected to P-Ret antibody analysis. GDNF induced the appearance of a 180-kDa protein by all four P-Ret antibodies inret+/+ and ret+/−neurons but not ret−/− neurons (Fig. 2). A gene dosage effect occurred because less P-Ret was detected inret+/− than in ret+/+neurons after GDNF stimulation (Fig. 2). NGF treatment did not stimulate the appearance of the 180-kDa phosphoprotein, indicating that this protein was not a downstream tyrosine-phosphorylated substrate of neurotrophic factors in sympathetic neurons. Therefore, Tyr905, Tyr1015, Tyr1062, and Tyr1096 are Ret autophosphorylation sites in response to GDNF in neurons. While this manuscript was in preparation, a study was published that described phospho-Ret antibodies directed against the same four tyrosines (30Coulpier M. Anders J. Ibanez C.F. J. Biol. Chem. 2002; 277: 1991-1999Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Coulpier et al. (30Coulpier M. Anders J. Ibanez C.F. J. Biol. Chem. 2002; 277: 1991-1999Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) found, as we did, that Tyr905, Tyr1015, Tyr1062, and Tyr1096 are autophosphorylated in a coordinated manner by GFL stimulation. The kinetics of autophosphorylation of these four tyrosines stimulated by GDNF and NRTN was examined in dissociated sympathetic neurons. GDNF induced the rapid and coordinated phosphorylation of Tyr905, Tyr1015, Tyr1062, and Tyr1096within minutes of treatment, and this phosphorylation persisted for hours (Fig. 3A). NRTN also promoted a rapid and robust autophosphorylation of these tyrosine residues in sympathetic neurons, and no significant differences between GDNF and NRTN were observed (Fig. 3A). Within 4 h the levels of autophosphorylation of Tyr905, Tyr1015, Tyr1062, and Tyr1096 began to decrease and reached a minimum by 8–24 h of GDNF or NRTN treatment (Fig. 3A). The levels of Ret also decreased during this same time, suggesting either that Ret was degraded or that new Ret synthesis was inhibited after GFL treatment in sympathetic neurons (Fig.3A). Because the amount of Ret decreases over time, changes in the levels of phosphorylation of a particular tyrosine residue may not reflect the actual changes in the percentage of Ret that is phosphorylated on that residue at any given time. Therefore, the immunoblots were quantified, and the PY-Ret/Ret ratio was calculated to determine whether the percentage of autophosphorylation of these tyrosine residues changed with time after GFL treatment. Quantitative measurements demonstrated that GDNF and NRTN treatment caused a marked reduction of Ret protein, decreasing to 35.1% and 42.1% of unstimulated levels, respectively, after 24 h. Analysis of the P-Ret/Ret ratio revealed that the percentage of Ret that was phosphorylated on Tyr905, Tyr1015, and Tyr1062 reached a maximum within 1 h, declined somewhat thereafter, and, by 24 h, either reached a plateau or even increased (Fig. 3B). In contrast, the percentage of Ret with phosphorylated Tyr1096 declined with time and reached a minimum value after 24 h of GDNF or NRTN treatment (Fig. 3B). Although the percentage of phosphorylated Ret increased on some tyrosines at 24 h, this may reflect a differential localization or stability of the phosphorylated receptor as compared with the nonphosphorylated receptor as opposed to an enhanced activation of Ret occurring at 24 h. Densitometric analysis also revealed that Tyr905 and Tyr1062showed the greatest increases in phosphorylation with GFL stimulation, changing by 9- and 14-fold, respectively (Fig. 3B). GFL stimulation also markedly induced the phosphorylation of Tyr1015 and Tyr1096 with GFL stimulation, increasing by roughly 4-fold (Fig. 3B). Therefore, GDNF and NRTN promote the sustained autophosphorylation of Tyr905, Tyr1015, Tyr1062, and, to a lesser extent, Tyr1096. Furthermore, these data suggest that a significant portion of the decrease in Ret phosphorylation after GFL stimulation was because of the loss of Ret protein rather than the dephosphorylation of Ret. Sympathetic neurons, like other cell types, express both Ret9 and Ret51. To determine whether any differences in the kinetics of autophosphorylation occur between these Ret isoforms, dissociated sympathetic neurons were stimulated with GDNF for various lengths of time and were detergent-extracted. Ret9 or Ret51 were immunoprecipitated with immobilized antibodies specific for either isoform (see "Experimental Procedures" and Fig. 5), and the phosphorylation of Tyr905, Tyr1015, Tyr1062, and Tyr1096 was evaluated. Tyr905, Tyr1015, and Tyr1062 were rapidly phosphorylated in a synchronous manner in both Ret9 and Ret51 (Fig. 4A). Tyr1096was also rapidly phosphorylated in Ret51 and was not detected in Ret9 immunoprecipitates (Fig. 4A). The levels of phosphorylation of all four tyrosine residues began declining within 1–4 h of GDNF treatment (Fig. 4A), similar to the previous experiments (Fig. 3).Figure 4Ret9 and Ret51 differ in their kinetics of Tyr905, Tyr1015, and Tyr1062phosphorylation in sympathetic neurons.A, dissociated sympathetic neurons were treated with medium alone (MA) or medium containing GDNF (50 ng/ml) for the length of time indicated. The neurons were then detergent-extracted, and either Ret9 or Ret51 was immunoprecipitated from the extracts. Equal amounts of each immunoprecipitate was subjected to PY-Ret and pan-Ret Western analysis as in Fig. 2. This experiment was performed twice with similar results.B, the immunoblots generated in A were quantified and the PY-Ret/pan-Ret ratio calculated for each condition. These values were graphed as a function of time. C, the PY-Ret/pan-Ret values displayed in B were divided by the PY-Ret/pan-Ret value for the control (MA) conditions to determine the percentage of change of phosphorylation each tyrosine underwent after GDNF stimulation. The values graphed here were generally greater than the values in Bbecause Ret9 and Ret51 display some basal autophosphorylation in the absence of GFLs at this neuronal age. The values graphed in bothB and C represent the means ± range.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the levels of both Ret9 and Ret51 declined after GDNF treatment (Fig. 4A), the immunoblots were quantified to determine the perc
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