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Originally published In Press as doi:10.1074/jbc.M203580200 on June 28, 2002

J. Biol. Chem., Vol. 277, Issue 37, 34618-34625, September 13, 2002
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The Long and Short Isoforms of Ret Function as Independent Signaling Complexes*

Brian A. Tsui-PierchalaDagger , Rebecca C. AhrensDagger , Robert J. Crowder§, Jeffrey Milbrandt§, and Eugene M. Johnson Jr.Dagger

From the Departments of Dagger  Molecular Biology and Pharmacology, Neurology, and § Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, April 15, 2002, and in revised form, June 21, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1, 2). The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) function via activation of the RTK, Ret (3, 4). 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 (5). 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 GFRalpha s. GFRalpha family members (GFRalpha 1-4) demonstrate preferential binding to a particular GFL, thus providing specificity to Ret activation depending upon which GFL and GFRalpha 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 (4, 6). These studies have identified multiple tyrosines that are autophosphorylated in Ret (7) 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 (8, 9). Phospholipase Cgamma and GRB2 bind to Tyr1015 and Tyr1096, respectively (10-12). 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 (8, 11, 13-19). 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 (20). These relatively minor differences have dramatic functional consequences; Ret9 is critically important for kidney morphogenesis and enteric nervous system development, whereas Ret51 is dispensable (21). Furthermore, transgenic overexpression of Ret51 only partially compensates for the loss of Ret9 in kidney and enteric nervous system development (21). In contrast, Ret51, but not Ret9, is required for the metabolism and growth of mature sympathetic neurons via a GFL-independent mechanism of activation (22). 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 (15, 16, 23), 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 (11, 12, 17, 24) but not FRS2 (17). Ret51 also has two additional tyrosine residues, Tyr1090 and Tyr1096, that may participate in signaling events. Consistent with this possibility, Tyr1096 appears to contribute to phosphatidylinositol 3-kinase and mitogen-activated protein kinase activation (16, 25). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sympathetic Neuron Cultures and Treatments-- Sympathetic neurons from the rat superior cervical ganglia were dissociated and maintained in vitro as described previously (26). 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.

Neuroblastoma Cell Line Maintenance and Transfection-- 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 mM L-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.

Detergent Extraction and Immunoprecipitation-- 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 (22).

Immunoblotting-- 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.

Antibody Production-- 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 mM glycine, 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).

Plasmids-- Ret expression plasmids used in this study have been described previously (27). 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 (7).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Antibodies to Specific Autophosphorylation Sites in Ret-- 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 (28, 29) 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.


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  		Fig. 1.   Specificity of PY905Ret, PY1015Ret, PY1062Ret, and PY1096Ret antibodies. A, schematic diagram of the autophosphorylation sites in Ret that phosphorylation state-specific antibodies were generated against. Also depicted in this diagram are signaling proteins reported to associate with these tyrosines upon their autophosphorylation and downstream signaling pathways that are subsequently activated. B, CHP126 neuroblastoma cells were transiently transfected with expression vectors encoding Ret9, Ret51, or Ret51 encoding a K758M, Y905F, Y1015F, Y1062F, or a Y1096F mutation. Two days after transfection, the cells were detergent-extracted, and Ret was immunoprecipitated with anti-Ret9 or anti-Ret51 agarose. SDS-PAGE was performed on the immunoprecipitates, and five identical blots were produced from the extracts. These blots were then evaluated by Western analysis by using anti-PY905Ret, anti-PY1015Ret, anti-PY1062Ret, and anti-PY1096Ret (antibodies designated on the right). To determine how much of each Ret mutant was expressed, the fifth blot was probed with anti-pan-Ret (bottom panel). CHP126 cells expressed differing amounts of the Ret mutants and, therefore, greater amounts of the K758MRet and Y905RRet immunoprecipitates were loaded on the gels because they frequently were expressed at lower levels than Ret9 or Ret51. This experiment was performed twice with similar results. PI-3-K, phosphatidylinositol 3-kinase; TM, transmembrane; PLCgamma , phospholipase Cgamma ; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; BMK, big MAP kinase.

Tyrosines 905, 1015, 1062, and 1096 in Ret Are Autophosphorylation Sites in Sympathetic Neurons-- 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).


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  		Fig. 2.   Tyr905, Tyr1015, Tyr1062, and Tyr1096 are Ret autophosphorylation sites in sympathetic neurons. Dissociated sympathetic neurons from the superior cervical ganglia of ret+/+, ret-/-, or ret+/- mice were maintained in NGF for 12 DIV. The neurons were starved of NGF for 2 days and were then treated with medium alone, medium containing NGF (50 ng/ml), or medium containing GDNF (50 ng/ml) for 15 min. The neurons were detergent-extracted, and the extracts were evaluated by PY905Ret, PY1015Ret, PY1062Ret, and PY1096Ret immunoblotting (labeled on the right). The total amount of Ret in each sample was determined by pan-Ret immunoblotting of the same extracts (bottom panel). This experiment was performed twice with similar results.

To confirm that the 180-kDa protein identified by the P-Ret antibodies was Ret, dissociated 12 DIV superior cervical ganglia neurons from ret+/+, ret+/-, or ret-/- 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 in ret+/+ and ret+/- neurons but not ret-/- neurons (Fig. 2). A gene dosage effect occurred because less P-Ret was detected in ret+/- 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 (30). Coulpier et al. (30) found, as we did, that Tyr905, Tyr1015, Tyr1062, and Tyr1096 are autophosphorylated in a coordinated manner by GFL stimulation.

GDNF and NRTN Promote the Maintenance of Tyr905, Tyr1015, Tyr1062, and Tyr1096 Autophosphorylation in Sympathetic Neurons-- 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 Tyr1096 within 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).


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  		Fig. 3.   Kinetics of Tyr905, Tyr1015, Tyr1062, and Tyr1096 autophosphorylation stimulated by GDNF and NRTN in sympathetic neurons. A, dissociated sympathetic neurons were treated with medium alone (MA), GDNF, or NRTN (50 ng/ml each) for the length of time specified above the blots. The neurons were then detergent-extracted, and the extracts were analyzed by PY-Ret and pan-Ret immunoblotting as described in the legend to Fig. 2. This experiment was performed three times with similar results. B, densitometric analysis was conducted on two of the experiments in A, and the PY-Ret/pan-Ret ratio was calculated. These values were divided by the PY-Ret/Pan-Ret ratio for the control condition treated with medium alone, and these values, which represent the percentage change in the amount of phosphorylation with GFNF or NRTN, were graphed as a function of time. Each data point represents the mean ± range.

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 Tyr1062 showed 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.

Ret9 and Ret51 Have Distinct Kinetics of Autophosphorylation of Tyr905, Tyr1015, and Tyr1062 after GDNF Stimulation-- 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). Tyr1096 was 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).


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  		Fig. 4.   Ret9 and Ret51 differ in their kinetics of Tyr905, Tyr1015, and Tyr1062 phosphorylation 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 B because Ret9 and Ret51 display some basal autophosphorylation in the absence of GFLs at this neuronal age. The values graphed in both B and C represent the means ± range.

Because the levels of both Ret9 and Ret51 declined after GDNF treatment (Fig. 4A), the immunoblots were quantified to determine the percentage of Ret that was autophosphorylated on these residues. Densitometric analysis revealed that Ret 9 and Ret51 accounted for 20 and 80%, respectively, of Ret expressed in 12-DIV sympathetic neurons, and they declined to a similar extent after GDNF treatment. Comparison of the PY-Ret/Ret values between Ret9 (Fig. 4B, left) and Ret51 (Fig. 4B, right) for each tyrosine residue indicated that Tyr905 was more highly phosphorylated in Ret9 than in Ret51 and was maintained to a greater extent over time after GDNF stimulation (Fig. 4B). Tyr1062 was initially phosphorylated to the same extent in both Ret9 and Ret 51, but Tyr1062 phosphorylation in Ret9 was maintained at a higher level by 24 h after GDNF treatment than it was in Ret51 (Fig. 4B). In contrast, Tyr1015 was more highly phosphorylated at all times after GDNF treatment in Ret51 than in Ret9 (Fig. 4B). Because the basal level of phosphorylation of Tyr905, Tyr1015, and Tyr1062 was higher in Ret51 than in Ret9 at this age in vitro, the percentage of change in the phosphorylation state of these three residues upon GDNF stimulation (Fig. 4C) was greater than the increase in the amount of phosphorylation (Fig. 4B). Therefore, Ret9 and Ret51 have distinctly different kinetics of Tyr905, Tyr1015, and Tyr1062 autophosphorylation upon GDNF stimulation of sympathetic neurons.

Ret9 and Ret51 Do Not Associate with Each Other during GFL-dependent Activation in Sympathetic Neurons-- The observation that PY1096Ret was not detected in Ret9 immunoprecipitates (Fig. 4A) suggested that Ret9 and Ret51 did not associate upon GDNF stimulation in sympathetic neurons, which was unexpected because Ret9 and Ret51 share identical extracellular domains that participate in ligand-mediated dimerization and activation. To determine whether Ret9 and Ret51 associate upon GFL-dependent activation in sympathetic neurons, 10-12 DIV dissociated sympathetic neurons were treated with either medium alone or medium containing GDNF. As expected, both Ret9 and Ret51 were activated after GDNF treatment, as determined by phosphotyrosine immunoblotting of Ret9 or Ret51 immunoprecipitates from these neurons (Fig. 5A). Although Ret51 was abundant in Ret51 immunoprecipitates, Ret51 was not detected in Ret9 immunoprecipitates (Fig. 5A). Furthermore, Ret51 immunoprecipitation depleted Ret51 from the cellular extracts, whereas Ret9 immunoprecipitation did not alter the amount of Ret51 left in the detergent extracts (Fig. 5A), indicating that no detectable Ret51 was associated with Ret9 after stimulation with GDNF. Conversely, Ret9 was not detected in Ret51 immunoprecipitates from GDNF-stimulated sympathetic neurons (data not shown). Therefore, the association of Ret9 with Ret51 could not be detected in sympathetic neurons stimulated with GDNF.


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  		Fig. 5.   Ret9 and Ret51 do not associate with each other upon activation in sympathetic neurons. A, dissociated sympathetic neurons were treated with medium alone or medium containing GDNF (50 ng/ml) for 15 min. The neurons were then subjected to detergent extraction followed by Ret9 or Ret51 immunoprecipitation (IP). The immunoprecipitates were then evaluated by phosphotyrosine (P-Tyr) immunoblotting (top panel), and thereafter the blots were stripped and reprobed with Ret51 antibodies (middle panel). The supernatants from the immunoprecipitation were subjected to Ret51 Western analysis (W, bottom panel) to confirm that Ret51 immunoprecipitation depleted Ret51 from the extracts. This experiment was performed three times with similar results. B, CHP126 cells were transfected with Ret9, Ret51, or both Ret9 and Ret51 expression plasmids. Two days after transfection the cells were detergent-extracted, and the extracts were subjected to Ret51 (top panel) or Ret9 (bottom panel) immunoprecipitation (left three lanes). After the first immunoprecipitation, these same extracts were then immunoprecipitated with Ret9 or Ret51 antibodies, respectively (right three lanes), and the immunoprecipitates evaluated by Ret9 (top panel) or Ret51 (bottom panel) immunoblotting. This experiment was conducted three times with similar results.

To determine whether any association between Ret9 and Ret51 could be detected with our Ret immunoprecipitation conditions, we transiently expressed Ret9 alone, Ret51 alone, or Ret9 and Ret51 together in CHP126 cells. Ret9 could only be detected in Ret51 immunoprecipitates from cells that expressed both Ret9 and Ret51 (Fig. 5B), indicating that the Ret51 antibodies do not immunoprecipitate Ret9 directly. Conversely, Ret51 was only detected in Ret9 immunoprecipitates from cells that express both Ret9 and Ret51 (Fig. 5B). Therefore, Ret9 and Ret51 did associate with each other when transiently expressed in CHP126 cells. In fact, a significant amount of Ret9 and Ret51 associated with each other in CHP126 cells, as determined by comparing the amounts of co-immunoprecipitated Ret described above to the amount of Ret9 or Ret51 that was left remaining in the extracts by immunoprecipitation of this remaining Ret9 or Ret51 with the appropriate antibody (Fig. 5B). Quantitative analysis revealed that 44% of the transiently expressed Ret9 was associated with Ret51 in Ret51 immunoprecipitates, and 28% of Ret51 was immunoprecipitated with Ret9. Therefore, significant amounts of Ret9 and Ret51 associated with each other during the activation promoted by transient overexpression of Ret9 and Ret51 in neuroblastoma cell lines, in contrast to sympathetic neurons stimulated with GDNF. These data suggest that experiments on Ret signal transduction in cell lines must be interpreted cautiously, because correlation with how Ret functions in neurons and other cell types that normally express Ret is sometimes lacking.

GDNF Promotes the Formation of Distinct Ret9 and Ret51 Signaling Complexes in Sympathetic Neurons-- Because Ret9 and Ret51 do not associate with each other upon activation in sympathetic neurons, it is likely that Ret9 and Ret51 form distinct signaling complexes in GFL-stimulated neurons. To test this hypothesis, either Ret9 or Ret51 was transiently expressed in CHP126 cells, and the Ret signaling complexes were evaluated by Ret9 or Ret51 immunoprecipitation followed by phosphotyrosine immunoblotting of the immunoprecipitates. The complex of tyrosine-phosphorylated proteins associated with Ret9 was distinctly different from the complex associated with Ret51 (Fig. 6A). At least 5-7 distinctly different proteins were associated with only Ret9 (asterisks) or Ret51 (arrowheads) under these conditions (Fig. 6A). Identical complexes associated with Ret9 and Ret51 were immunoprecipitated by two different antibodies to both Ret9 and Ret51, indicating that these complexes were not dependent upon the immunoprecipitating antibody (Fig. 6A). In addition, the tyrosine-phosphorylated proteins were not immunoprecipitated nonspecifically because they were not immunoprecipitated from cells transfected with the empty vector (data not shown). Therefore, Ret9 and Ret51 form distinct signaling complexes when activated by transient expression in neuroblastoma cells.


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  		Fig. 6.   Activated Ret9 and Ret51 assemble unique signaling complexes in both neuroblastoma cells and sympathetic neurons. A, CHP126 cells were transfected with Ret9, Ret51, or both Ret9 and Ret51 expression plasmids. After 2 days the cells were detergent-extracted, and Ret9 or Ret51 was immunoprecipitated from the extracts. The immune complexes were then subjected to SDS-PAGE on minigels (not shown) or slab gels followed by phosphotyrosine (P-Tyr) immunoblotting (top panel). Prior to the immunoprecipitation (IP), levels of Ret were determined, and the volumes of the immunoprecipitates were adjusted such that similar amounts of Ret were evaluated (bottom panel). One third of the amount of Ret9 and Ret51 immune complexes from a separate experiment was loaded and evaluated by phosphotyrosine immunoblotting (shown on the right) to visualize the more abundant tyrosine-phosphorylated proteins. B, dissociated sympathetic neurons were treated with medium alone or medium containing GDNF (50 ng/ml) for 15 min and detergent-extracted. The extracts were analyzed by Ret9 or Ret51 immunoprecipitation followed by phosphotyrosine immunoblotting (top panels). Equal loading of Ret was confirmed as in (A). The experiments in A and B were performed twice with minigels and twice with slab gels, which yielded similar results. W, Western blotting.

To determine whether Ret9 and Ret51 create distinct signaling complexes in cells that express both receptors, Ret9 and Ret51 were co-expressed in CHP126 cells. Evaluation of the Ret9 and Ret51 complexes from transfected cells revealed that these signaling complexes appeared considerably more similar to each other (Fig. 6A). For example, the three lowest molecular mass proteins associated with Ret9 co-immunoprecipitated with both Ret9 and Ret51 when they were co-expressed (Fig. 6A). When expressed together, the Ret9 and Ret51 complexes still displayed clear differences (Fig. 6A), suggesting that even when transiently expressed in neuroblastoma cell lines, Ret9 and Ret51 preferred to homodimerize, consistent with the previous results (Fig. 5A). When Ret9 and Ret51 complexes were compared with each other from sympathetic neurons treated with GDNF, we found that these complexes displayed more striking differences than immune complexes purified from CHP126 cells that express both Ret9 and Ret51 (Fig. 6B). Ret51 immunoprecipitates were more highly enriched in at least three proteins than were Ret9 immune complexes (Fig. 6B, arrowheads). Likewise, several tyrosine-phosphorylated proteins displayed a preference for Ret9 as compared with Ret51 (Fig. 6B, asterisks). Therefore, Ret9 and Ret51 form unique signaling complexes upon activation in sympathetic neurons and neuroblastoma cells even when co-expressed in the same cell.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Ret isoforms Ret9 and Ret51 are highly conserved between species, suggesting that these C-terminal regions have important functions that are evolutionarily conserved. Support for this hypothesis was provided recently when it was shown that Ret9 is required for kidney and enteric nervous system development, in contrast to Ret51, which is dispensable for these two functions (21). Transgenic expression of Ret51 in the kidneys only partially compensates for the loss of Ret9 function (21), which is surprising given the modest differences between Ret9 and Ret51. The biochemical differences between Ret9 and Ret51 that account for their dramatic functional specificity, however, are unknown. To address this question we generated four affinity-purified antibodies that specifically recognize Ret when phosphorylated on Tyr905, Tyr1015, Tyr1062, or Tyr1096, as well as immunoprecipitating antibodies specific for Ret9 and Ret51. These specific P-Ret antibodies allowed us and others (30) to confirm that Tyr905, Tyr1015, Tyr1062, and Tyr1096 were bona fide Ret autophosphorylation sites in sympathetic neurons. The phosphorylation status of Tyr905, Tyr1015, and Tyr1062 was maintained for at least 24 h after GFL treatment in sympathetic neurons, whereas Tyr1096 phosphorylation decreased with time, returning to nearly basal levels within 24 h. These data suggest that signaling through Tyr1096 may be less important for long term signaling events. The total amount of phosphorylation of these tyrosines, however, diminished over time because of an apparent loss of Ret protein, perhaps caused by degradation upon GFL-dependent activation.

The experiments described here provide compelling evidence that Ret9 and Ret51 are not only functionally distinct but are biochemically distinct GFL receptors as well. First, Ret9 and Ret51 had differing kinetics of autophosphorylation after GDNF stimulation. The phosphorylation of Tyr905 and Tyr1062 in Ret9 were maintained at higher levels over time than the level in Ret51, in contrast to Tyr1015 phosphorylation, which was maintained at a higher level in Ret51 than in Ret9. This implies that signaling pathways activated by phosphorylation of these residues will be sustained to differing extents by Ret9 and Ret51. Ret51, for example, may promote a greater and more sustained activation of protein kinase C because of the higher level of Tyr1015 phosphorylation in Ret51 (10). Second, Ret9 and Ret51 did not associate with each other upon activation in dissociated sympathetic neurons, even though they share identical ligand-binding extracellular domains. Ret9 and Ret51, however, were capable of associating with each other when transiently expressed in CHP126 cells, consistent with data from other neuroblastoma cells (31). The mechanism that accounts for the inability of Ret9 and Ret51 to associate in sympathetic neurons is unclear. Cholesterol depletion did not enable Ret9 and Ret51 to associate with each other after GDNF stimulation in sympathetic neurons (data not shown), indicating that preferential partitioning of one isoform into lipid rafts does not account for this phenomenon. A differential localization of Ret9 and Ret51 within neurons or mechanisms regulated by either the 9- or 51-amino acid regions unique to these isoforms may account for their lack of association. Third, the signaling molecules associated with activated Ret9 were considerably different compared with those associated with activated Ret51, consistent with their altered autophosphorylation kinetics and lack of association in neurons. These data suggest that Ret9 and Ret51 activate a distinct assortment of signaling pathways in neurons, which provides a biochemical foundation for the differential functions of Ret9 and Ret51. Importantly, these data further imply that Ret9 and Ret51 serve different signaling functions even within the same cell.

Many RTKs, such as the fibroblast growth factor and vascular endothelial growth factor receptors, are alternatively spliced in their extracellular domains, often yielding soluble versions of their ligand-binding domains. The intracellular domains of some RTKs are also alternatively spliced to produce forms with inserts in the juxtamembrane and kinase domains. The fibroblast growth factor receptor, for example, is alternatively spliced to produce two intracellular isoforms that differ only in whether a valine and a threonine are present in the juxtamembrane region (32). The VT+ insert of the fibroblast growth factor receptor, among other functions, alters fibroblast growth factor receptor activity by creating a protein kinase C phosphorylation site that regulates kinase activity (33). In the nervous system, some neurotrophic factor receptors, such as the NT-3 receptor TrkC, contain inserts in the intracellular domain that alter receptor function. Kinase domain insertions in TrkC inhibit signal transduction pathways as well as neurite outgrowth promoted by TrkC activation (34-38). In contrast to TrkC, the alternative splicing of Ret alters, rather than dampens, the signaling capacities of these isoforms. The alternative splicing of ret in effect creates two distinct signaling receptors for the GFLs, Ret9 and Ret51. This situation may be indicative of an evolutionarily conserved mechanism among some RTKs to expand the number of activities regulated by a particular growth factor. Because other RTKs expressed in the nervous system, such as the ephrin receptor EPHB2, contain alternatively spliced forms that generate novel C termini (39), this type of RTK regulation likely contributes to many aspects of neural development and plasticity.

    ACKNOWLEDGEMENTS

We thank Patricia A. Osborne, Mario Encinas, and Cynthia C. Tsui-Pierchala for critical comments on the manuscript as well as members of the Johnson and Milbrandt laboratories for helpful scientific discussions.

    FOOTNOTES

* This work is supported by National Institutes of Health Grants AG13729 (to E. M. J.), AG13730 (to J. M.), and F32-MH65111 (to B. A. T.-P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3926; Fax: 314-747-1772; E-mail: ejohnson@pcg.wustl.edu.

Published, JBC Papers in Press, June 28, 2002, DOI 10.1074/jbc.M203580200

    ABBREVIATIONS

The abbreviations used are: RTK, receptor tyrosine kinase; GDNF, glial cell line-derived neurotrophic factor; GFL, GDNF family ligand; NRTN, neurturin; DIV, days in vitro; NGF, nerve growth factor; P-Ret, phospho-Ret.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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