Calcineurin Inhibitor Protein (CAIN) Attenuates Group I Metabotropic Glutamate Receptor Endocytosis and Signaling
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.050872
ISSN1083-351X
AutoresLucimar T. Ferreira, Lianne B. Dale, Fabíola M. Ribeiro, Andy V. Babwah, Macarena Pampillo, Stephen S. G. Ferguson,
Tópico(s)Ion channel regulation and function
ResumoGroup I metabotropic glutamate receptors (mGluRs) are coupled via phospholipase Cβ to the hydrolysis of phosphoinositides and function to modulate neuronal excitability and synaptic transmission at glutamatergic synapses. The desensitization of Group I mGluR signaling is thought to be mediated primarily via second messenger-dependent protein kinases and G protein-coupled receptor kinases. We show here that both mGluR1 and mGluR5 interact with the calcineurin inhibitor protein (CAIN). CAIN is co-immunoprecipitated in a complex with Group I mGluRs from both HEK 293 cells and mouse cortical brain lysates. Purified CAIN and its C-terminal domain specifically interact with glutathione S-transferase fusion proteins corresponding to the second intracellular loop and the distal C-terminal tail domains of mGluR1. The interaction of CAIN with mGluR1 could also be blocked using a Tat-tagged peptide corresponding to the mGluR1 second intracellular loop domain. Overexpression of full-length CAIN attenuates the agonist-stimulated endocytosis of both mGluR1a and mGluR5a in HEK 293 cells, but expression of the CAIN C-terminal domain does not alter mGluR5a internalization. In contrast, overexpression of either full-length CAIN or the CAIN C-terminal domain impairs agonist-stimulated inositol phosphate formation in HEK 293 cells expressing mGluR1a. This CAIN-mediated antagonism of mGluR1a signaling appears to involve the disruption of receptor-Gαq/11 complexes. Taken together, these observations suggest that the association of CAIN with intracellular domains involved in mGluR/G protein coupling provides an additional mechanism by which Group I mGluR endocytosis and signaling are regulated. Group I metabotropic glutamate receptors (mGluRs) are coupled via phospholipase Cβ to the hydrolysis of phosphoinositides and function to modulate neuronal excitability and synaptic transmission at glutamatergic synapses. The desensitization of Group I mGluR signaling is thought to be mediated primarily via second messenger-dependent protein kinases and G protein-coupled receptor kinases. We show here that both mGluR1 and mGluR5 interact with the calcineurin inhibitor protein (CAIN). CAIN is co-immunoprecipitated in a complex with Group I mGluRs from both HEK 293 cells and mouse cortical brain lysates. Purified CAIN and its C-terminal domain specifically interact with glutathione S-transferase fusion proteins corresponding to the second intracellular loop and the distal C-terminal tail domains of mGluR1. The interaction of CAIN with mGluR1 could also be blocked using a Tat-tagged peptide corresponding to the mGluR1 second intracellular loop domain. Overexpression of full-length CAIN attenuates the agonist-stimulated endocytosis of both mGluR1a and mGluR5a in HEK 293 cells, but expression of the CAIN C-terminal domain does not alter mGluR5a internalization. In contrast, overexpression of either full-length CAIN or the CAIN C-terminal domain impairs agonist-stimulated inositol phosphate formation in HEK 293 cells expressing mGluR1a. This CAIN-mediated antagonism of mGluR1a signaling appears to involve the disruption of receptor-Gαq/11 complexes. Taken together, these observations suggest that the association of CAIN with intracellular domains involved in mGluR/G protein coupling provides an additional mechanism by which Group I mGluR endocytosis and signaling are regulated. Metabotropic glutamate receptors (mGluRs) 2The abbreviations used are: mGluRmetabotropic glutamate receptorPKCprotein kinase CHAhemagglutininGSTglutathione S-transferaseORFopen reading frameILintracellular loopPBSphosphate-buffered salineGFPgreen fluorescent proteinHBSSHEPES-buffered saline solutionIPinositol phosphate. 2The abbreviations used are: mGluRmetabotropic glutamate receptorPKCprotein kinase CHAhemagglutininGSTglutathione S-transferaseORFopen reading frameILintracellular loopPBSphosphate-buffered salineGFPgreen fluorescent proteinHBSSHEPES-buffered saline solutionIPinositol phosphate. play an essential role in regulating neuronal plasticity, development, and neurotoxicity and belong to the G protein-coupled receptor superfamily of integral membrane proteins (1Choi D.W. Rothman S.M. Annu. Rev. Neurosci. 1990; 13: 171-182Crossref PubMed Scopus (2018) Google Scholar, 2Bliss T.V. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9396) Google Scholar, 3Hermans E. 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Biochem. Mol. Biol. 1999; 34: 215-251Crossref PubMed Scopus (96) Google Scholar). At the level of the receptor, Group I mGluR activity is regulated by a process termed desensitization, which protects against both acute and chronic receptor overstimulation (9Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (853) Google Scholar, 10Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). The attenuation of Group I mGluR signaling can be mediated by both phosphorylation-dependent and phosphorylation-independent processes (11Ferguson S.S.G. Trends Pharmacol. Sci. 2007; 28: 173-179Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The phosphorylation-independent attenuation of Group I mGluR signaling is mediated by GRK2 (G protein-coupled receptor kinase 2), which is composed of three functional domains: an N-terminal RGS (regulator of G protein signaling) homology domain, a central catalytic domain, and a C-terminal Gβγ-binding pleckstrin homology domain (12Lodowski D.T. Pitcher J.A. Capel W.D. Lefkowitz R.J. Tesmer J.J.G. Science. 2003; 300: 1256-1262Crossref PubMed Scopus (309) Google Scholar). GRK2-mediated desensitization of Group I mGluRs does not require catalytic activity but rather requires the interaction of the GRK2 RGS homology domain with both the second intracellular loop domain of mGluR1 and the α-subunit of Gαq/11, thereby attenuating heterotrimeric G protein coupling (13Dhami G.K. Anborgh P.H. Dale L.B. Sterne-Marr R. Ferguson S.S.G. J. Biol. Chem. 2002; 277: 25266-25272Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Dhami G.K. Dale L.B. Anborgh P.H. O'Connor-Halligan K.E. Sterne-Marr R. Ferguson S.S.G. J. Biol. Chem. 2004; 279: 16614-16620Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 15Dhami G.K. Babwah A.V. Sterne-Marr R. Ferguson S.S.G. J. Biol. Chem. 2005; 280: 24420-24427Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Phosphorylation-independent desensitization of mGluR1 signaling is also mediated by optineurin, an effect that is enhanced by the expression of mutant huntingtin (16Anborgh P.H. Godin C. Pampillo M. Dhami G.K. Dale L.B. Cregan S.P. Truant R. Ferguson S.S. J. Biol. Chem. 2005; 280: 34840-34848Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Phosphorylation-dependent desensitization of Group I mGluR responsiveness involves the phosphorylation of PKC consensus sequence localized within the intracellular loop and C-terminal tail domains of mGluR1 and mGluR5 by PKC (17Gereau 4th, R.W. Heinemann S.F. Neuron. 1998; 20: 143-151Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 18Sato M. Tabata T. Hashimoto K. Nakamura K. Nakao K. Katsuki M. Kitano J. Moriyoshi K. Kano M. Nakanishi S. Eur. J. Neurosci. 2004; 20: 947-955Crossref PubMed Scopus (10) Google Scholar). It is proposed that calcineurin and mGluR5 may exist in a signaling complex in the brain and that calcineurin may function to modulate mGluR5 signaling by directly dephosphorylating the receptor at a PKC consensus site that contributes to mGluR5 desensitization (19Alagarsamy S. Saugstad J. Warren L. Mansuy I.M. Gereau 4th, R.W. Conn P.J. Neuropharmacology. 2005; 49: 135-145Crossref PubMed Scopus (75) Google Scholar). Calcineurin is also linked to the regulation of endocytosis via its interaction with dynamin-1 (20Lai M.M. Hong J.J. Ruggiero A.M. Burnett P.E. Slepnev V.I. De Camilli P. Snyder S.H. J. Biol. Chem. 1999; 274: 25963-25966Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). On the basis of the observation that calcineurin may form a complex with Group I mGluRs, we hypothesized that CAIN (calcineurin inhibitor protein) might also interact with Group I mGluRs and modulate their endocytosis and signaling. CAIN, also known as Cabin1 (calcineurin-binding protein), was first identified as a protein that binds to calcineurin and was shown to inhibit calcineurin catalytic activity (21Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 22Sun L. Youn H.D. Loh C. Stolow M. He W. Liu J.O. Immunity. 1998; 8: 703-711Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 23Jang H. Cho E.J. Youn H.D. Biochem. Biophys. Res. Commun. 2007; 359: 129-135Crossref PubMed Scopus (16) Google Scholar). Previous studies also demonstrated that CAIN may interact with amphiphysin-1, dynamin-1, and α-adaptin and led to the suggestion that CAIN functions as a component of synaptic endocytic complexes (24Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Consistent with this hypothesis, the overexpression of CAIN in human embryonic kidney (HEK 293) cells resulted in attenuated transferrin receptor endocytosis. We show here that CAIN interacts with the second intracellular loop and C-terminal tail domains of Group I mGluRs, inhibits Group I mGluR internalization, and attenuates mGluR1a signaling by disrupting receptor-Gαq/11 complexes. Taken together, these results describe an additional mechanism by which Group I mGluR activity may be regulated. HEK 293 cells were obtained from the American Type Culture Collection (Manassas, VA). Tissue culture reagents were purchased from Invitrogen. Quisqualate was purchased from Tocris Cookson Inc. (Ellisville, MO). myo-[3H]Inositol was acquired from PerkinElmer Life Sciences. The Dowex 1-X8 resin (formate form) with 200–400 mesh was purchased from Bio-Rad. EZ-Link sulfo-NHS-SS-biotin and immobilized NeutrAvidin were purchased from Pierce. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG secondary antibodies, protein G-Sepharose beads, enhanced chemiluminescence (ECL) Western blotting detection reagents, and mouse anti-hemagglutinin (HA) monoclonal antibody (12CA5) were purchased from GE Healthcare. Mouse anti-glutathione S-transferase (GST) monoclonal antibody and rabbit anti-Gαq/11 polyclonal antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit anti-mGluR5 and anti-mGluR1 polyclonal antibodies were obtained from Upstate (Lake Placid, NY). Rabbit anti-CAIN polyclonal antibody was obtained from Calbiochem. Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG were purchased from Invitrogen. All other biochemical reagents were purchased from Sigma, Fisher, and VWR Scientific (Mississauga, Ontario, Canada). All recombinant cDNA procedures were carried out in accordance with standard protocols. The HA-CAIN construct was generously provided by Dr. Solomon H. Snyder (Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore). This construct contained the entire rat CAIN open reading frame (ORF) (nucleotides 1–6549) and its 3′-untranslated region. The HA-CAIN and its 3′-untranslated region were subcloned into the SalI-XbaI sites of the Escherichia coli expression vector pPROEX-HT to create His6-CAIN. A His6-tagged C-terminal domain CAIN construct (nucleotides 5643–6519, amino acid residues 1519–2182) was generated by PCR and cloned into the Bg1II-XbaI sites of pPROEX-HT to create His6-CAIN-CTer. This construct encodes both the amphiphysin- and calcineurin-binding domains of CAIN (24Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The GST-tagged rat mGluR1a constructs were generated by PCR and cloned into the EcoRI-XhoI sites of the E. coli expression vector pGEX-4T (Amersham Biosciences). The PCR-generated sequences encode mGluR1a intracellular loop 1 (GST-IL1, nucleotides 1777–1950 of the ORF, amino acid residues 593–650), loop 2 (GST-IL2, nucleotides 1969–2181 of the ORF, amino acid residues 657–727), loop 3 (GST-IL3, nucleotides 2251–2421 of the ORF, amino acid residues 751–807), the membrane-proximal portion of the C-terminal tail (CT-N, nucleotides 2446–2580 of the ORF, amino acid residues 816–860), and the rest of the C-terminal tail (CT-C, nucleotides 2581–3597 of the ORF, amino acid residues 861–1199). pPROEX-HT- and pGEX-4T-based constructs were transformed into E. coli BL21. Full-length CAIN was cloned into the SalI and XbaI sites of pEGFP-C3, and the C-terminal portion of the CAIN ORF was cloned into the HindIII and SacII sites of pEGFP-C3. All constructs were subjected to automated DNA sequencing to confirm sequence integrity. HEK 293 cells were maintained in Eagle's minimal essential medium supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 50 μg/ml gentamycin. Cells were transfected using a modified calcium phosphate method as described previously (25Ferguson S.S. Ménard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Following transfection (18 h), the cells were incubated with fresh medium and allowed to recover for 24 h for co-immunoprecipitation studies. Otherwise, they were allowed to recover for 6–8 h and reseeded either into 12-well dishes coated with collagen or containing collagen-coated coverslips or into 24-well dishes and then grown for an additional 18 h prior to experimentation. Primary neuronal cultures were prepared from the cortexes of embryonic day 15 CD1 mouse embryos. Animal procedures were approved by the University of Western Ontario Animal Care Committee. Cells were plated on either 100-mm dishes or 15-mm glass coverslips coated with poly-l-ornithine (Invitrogen) in Neurobasal medium with B-27 and N-2 supplements, 2 mm GlutaMAX, 50 μg/ml penicillin, and 50 mg/ml streptomycin. Cells were incubated at 37 °C and 5% CO2 in a humidified incubator and cultured for up to 21 days in vitro with media replenishment every 3 days. GST-mGluR1a peptides were generated by growing recombinant E. coli BL21 at 37 °C to an A600 of 06–1.0. His6-CAIN and His6-CAIN-CTer (amino acid residues 1519–2182) cultures were induced for 3 h with 0.6 mm isopropyl β-d-thiogalactopyranoside, whereas GST-mGluR1a cultures were induced for 3 h with 1 mm isopropyl β-d-thiogalactopyranoside. After induction, cultures were pelleted, resuspended in phosphate-buffered saline (PBS) containing protease inhibitors, and lysed by mild sonication. The bacterial lysates were cleared of cellular debris by centrifugation, and the His6-CAIN and His6-CAIN-CTer lysates were then applied to Talon metal affinity resin. GST-mGluR1a lysates were applied to glutathione-Sepharose 4B and incubated on an orbital shaker overnight at 4 °C. Matrix-bound His6-CAIN, His6-CAIN-CTer, and GST-mGluR1a fusion proteins were washed extensively with PBS-containing 0.3% Triton X-100 (PBS-T) and then eluted off the resin in phosphate-buffered 500 mm imidazole. The imidazole was subsequently removed by chromatography using NAP-10 columns, and the proteins were recovered in phosphate buffer. One μg of His6-CAIN, His6-CAIN-CTer, and GST-mGluR1a peptides was used in the pulldown assays. Glutathione-Sepharose 4B-bound GST fusion proteins and either purified His6-CAIN or His6-CAIN-CTer were incubated together and mixed overnight on an orbital shaker at 4 °C. The Sepharose-bound protein complexes were washed extensively with PBS-T and then eluted off the Sepharose in SDS-PAGE loading buffer. Eluted samples were analyzed by immunoblotting with anti-CAIN antibody (1:1000 dilution). Co-immunoprecipitation experiments were performed using 500–1000 μg of total cell lysate protein solubilized from HEK 293 cells transiently transfected with the cDNA constructs as described in the figure legends. Cells were solubilized in lysis buffer (25 mm HEPES, pH 7.5, 300 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, and 0.1% Triton X-100) containing protease inhibitors. FLAG-mGluR1a and FLAG-mGluR5a were immunoprecipitated with anti-FLAG monoclonal antibody M2 affinity Sepharose beads by 2 h of rotation at 4 °C. In experiments utilizing a peptide corresponding to the second intracellular loop domain of mGluR1a (IARILAGSKKKICTRKPRFMS) fused to the human immunodeficiency virus Tat protein membrane transducing domain (YGRKKRRQRRR), HEK 293 cells were treated with peptide for 2 h prior to co-immunoprecipitation with FLAG-mGluR1a. For the co-immunoprecipitation of endogenous proteins from cortical extracts, adult mouse brains were employed. Tissue was dissected and homogenized on ice in lysis buffer containing protease inhibitors. The particulate fraction was removed by centrifugation, and 2 mg of supernatant protein was incubated with rabbit anti-CAIN polyclonal antibody and protein G-Sepharose beads by 2 h of rotation at 4 °C. Afterward, the beads were washed twice with lysis buffer and once with PBS, and proteins were eluted in SDS-PAGE loading buffer by warming the samples at 55 °C for 5 min. Eluted samples were subjected to SDS-PAGE, followed by electroblotting onto nitrocellulose membranes for immunoblotting with antibodies described in the figure legends. Immunoblots were visualized by chemiluminescence using an ECL kit. Intensities of immunoblot signals were determined with the 4.0.2 version of the data acquisition and analysis software from Scion Corp. (Frederick, MD). Confocal microscopy was performed using a Zeiss LSM-510 META laser scanning confocal microscope equipped with a Zeiss 63×, 1.4 numerical aperture, oil immersion lens. HEK 293 cells expressing either green fluorescent protein (GFP)-CAIN alone or coexpressing GFP-CAIN and FLAG-mGluR1a or neuronal primary culture cells were seeded on 15-mm glass coverslips. Cells were washed with HEPES-buffered saline solution (HBSS; 116 mm NaCl, 20 mm HEPES, 11 mm glucose, 5 mm NaHCO3, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, and 1.2 mm KH2PO4, pH 7.4; Molecular Probes, Eugene, OR) and fixed in 4% paraformaldehyde. Afterward, HEK 293 cells were permeabilized with 0.05% Triton X-100 and incubated with rabbit anti-FLAG and mouse anti-HA primary antibodies for 1 h at room temperature. Cells were washed and labeled with Alexa Fluor 568-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse secondary antibodies. Primary neuronal cultures were permeabilized with 0.05% Triton X-100, stained for endogenous mGluR5a with rabbit anti-mGluR5a antibody (1:2000) conjugated to Alexa Fluor 568 following the manufacturer's specifications using a Zenon IgG labeling kit, and washed three times with HBSS. Cells were washed again with HBSS and labeled for 20 min with rabbit anti-CAIN antibody (1:500) conjugated to Alexa Fluor 488. Cells were washed three times with HBSS, fixed in 4% paraformaldehyde, washed again with HBSS, mounted in Immu-Mount (Thermo Shandon, Pittsburgh, PA) onto glass slides, and allowed to air dry before viewing. Co-localization studies were performed using dual excitation (488 and 543 nm) and emission (505–530 nm band pass and 560 nm long pass for Alexa Fluor 488 and 568, respectively) filter sets. The specificity of labeling and the absence of signal crossover were established by examination of single-labeled samples. HEK 293 cells transiently transfected with 3 μg of FLAG-mGluR1a construct and 3 μg of either empty plasmid vector or plasmid vector containing GFP-CAIN or GFP-CAIN-CTer were seeded into 24-well dishes. Inositol lipids were radiolabeled by incubating the cells overnight with 1 μCi/ml myo-[3H]inositol in inositol-free Dulbecco's modified Eagle's medium. Unincorporated myo-[3H]inositol was removed by washing the cells with HBSS. Cells were preincubated for 1 h in HBSS at 37 °C and then preincubated in 500 μl of the same buffer containing 10 mm LiCl for an additional 10 min at 37 °C. Next, cells were incubated in the absence or presence of quisqualate for 30 min at 37 °C. The reaction was stopped on ice by the addition of 500 μ1 of 0.8 m perchloric acid and then neutralized with 400 μ1 of 0.72 m KOH and 0.6 m KHCO3. The total myo-[3H]inositol incorporated into the cells was determined by counting the radioactivity present in 50 μ1 of the cell lysate. Total inositol phosphate was purified from the cell extracts by anion exchange chromatography using Dowex 1-X8 200–400 mesh anion exchange resin (formate form). [3H]Inositol phosphate formation was determined by liquid scintillation using a Beckman LS 6500 scintillation system. HEK 293 cells transiently coexpressing either FLAG-mGluR1 and HA-CAIN or FLAG-mGluR5 and either GFP-CAIN or GFP-CAIN-CTer were washed and incubated on ice in HBSS. Plasma membrane proteins were then biotinylated with EZ-Link sulfo-NHS-SS-biotin for 1 h on ice. To quench the biotinylation reaction, cells were washed and incubated for 30 min with cold 100 mm glycine in HBSS, followed by three washes with cold HBSS. Subsequently, cells were incubated for varying times with 30 μm quisqualate in HBSS at 37 °C to allow receptor internalization. Cells were then transferred to ice to stop internalization and washed once with ice-cold HBSS. Biotin remaining at the cell surface was stripped by incubating the cells with freshly prepared 150 mm mercaptoethanesulfonic acid sodium salt in HBSS at 4 °C for 45 min. To assess the amount of receptor initially in the plasma membrane, a control sample was kept on ice without mercaptoethanesulfonic acid sodium salt stripping. Cells were then lysed in the same lysis buffer described above. Biotinylated proteins were pulled down with NeutrAvidin-agarose resin, eluted from the beads with 3× SDS loading buffer, subjected to SDS-PAGE, and immunoblotted for the receptors. Internalization of the receptors at the various time points was expressed as a percentage of biotinylated receptor on the surface of the non-stimulated and non-stripped control. The means ± S.E. are shown for values obtained for the number of independent experiments indicated in the figure legends. GraphPad Prism software was used to analyze data for statistical significance as well as to analyze and fit dose-response and time-course data. The statistical significance was determined by t test. Previous studies demonstrated that calcineurin interacts with Group I mGluRs and that CAIN negatively regulates both calcineurin function and transferrin receptor endocytosis (19Alagarsamy S. Saugstad J. Warren L. Mansuy I.M. Gereau 4th, R.W. Conn P.J. Neuropharmacology. 2005; 49: 135-145Crossref PubMed Scopus (75) Google Scholar, 21Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 24Lai M.M. Luo H.R. Burnett P.E. Hong J.J. Snyder S.H. J. Biol. Chem. 2000; 275: 34017-34020Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Therefore, in initial experiments, we tested whether CAIN associates with either mGluR1a or mGluR5a by co-immunoprecipitation. We found that HA epitope-tagged CAIN was co-immunoprecipitated from HEK 293 cells with both FLAG-tagged mGluR1a and mGluR5a (Fig. 1, A and B). We also found that endogenous mGluR5 could be co-immunoprecipitated with endogenous CAIN from mouse cortical brain lysates using a rabbit CAIN-specific polyclonal antibody (Fig. 1C). Taken together, these data suggested that CAIN was associated in a protein complex with Group I mGluRs. Previously, Snyder and co-workers (21Lai M.M. Burnett P.E. Wolosker H. Blackshaw S. Snyder S.H. J. Biol. Chem. 1998; 273: 18325-18331Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) reported that CAIN is predominantly a cytosolic protein, whereas in lymphocytes, CAIN is localized to the nucleus, where it negatively regulates MEF2 transcription factor activity (22Sun L. Youn H.D. Loh C. Stolow M. He W. Liu J.O. Immunity. 1998; 8: 703-711Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 23Jang H. Cho E.J. Youn H.D. Biochem. Biophys. Res. Commun. 2007; 359: 129-135Crossref PubMed Scopus (16) Google Scholar, 26Youn H.D. Sun L. Prywes R. Liu J.O. Science. 1999; 286: 790-793Crossref PubMed Scopus (232) Google Scholar). Therefore, because the current information in the literature regarding the subcellular distribution of CAIN is contradictory, we examined the subcellular localization of GFP-CAIN in HEK 293 cells in the absence and presence of mGluR1a expression. In HEK 293 cells lacking FLAG-mGluR1a expression, GFP-CAIN localization was restricted to the nucleus (Fig. 2A), consistent with the report indicating that CAIN is a nuclear protein. However, in cells expressing both FLAG-mGluR1a and GFP-CAIN, the subcellular distribution of GFP-CAIN was altered such that CAIN was localized to the cytoplasm and, to a lesser extent, the nucleus (Fig. 2B). In addition, primary mouse cortical neurons that stained positive for endogenous mGluR5 were positive for the expression of CAIN in both the nucleus and the cytoplasm (Fig. 3C). Taken together, the data indicated that CAIN was appropriately localized to the cytoplasm in both HEK 293 cells and neurons, which was consistent with the observation that CAIN forms a complex with Group I mGluRs.FIGURE 3Purified His6-CAIN binds to GST fusion proteins corresponding to the mGluR1a second intracellular loop and distal C-terminal tail domains. A, shown are the full-length CAIN and CAIN C-terminal domains purified from E. coli as His6 fusion proteins and the localization of the coil-coiled (CC) domain, the proline-rich domain (PRD) that binds amphiphysin, and the calcineurin-binding domain (CBD). B, the schematic representation of mGluR1a illustrates the regions of mGluR1a that were prepared as GST fusion proteins. The GST fusion proteins correspond to the first (GST-IL1), second (GST-IL2), and third (GST-IL3) intracellular loops as well as the proximal (GST-CT-N) and distal (GST-CT-C) C-terminal tails. C, 1 μg of purified GST-IL1, GST-IL2, GST-IL3, GST-CT-N, and GST-CT-C was incubated with 1 μg of either full-length His6-tagged CAIN (HIS6CAIN) or the His6-tagged CAIN C-terminal domain (HIS6CAIN-CTer). CAIN eluted with the GST fusion proteins was determined by immunoblotting (IB) with rabbit anti-CAIN polyclonal antibody. Data are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether CAIN interacts directly with Group I mGluRs and to ascertain which intracellular domains bind to CAIN, both full-length CAIN and the C terminus of CAIN were expressed in and purified from E. coli as His6 fusion proteins (Fig. 3A). The purified CAIN proteins were subsequently tested for their ability to bind in vitro to GST fusion proteins corresponding to the mGluR1a intracellular loops (IL1, IL2, and IL3) as well as the proximal (GST-CT-C) and distal (GST-CT-N) portions of the mGluR1a C-terminal tail (Fig. 3B). We found that CAIN specifically bound to GST fusion proteins corresponding to the IL2 and CT-N domains of mGluR1a (Fig. 3C). In addition, the CAIN-CTer construct, which encodes the CAIN calcineurin- and amphiphysin-binding domains, also bound to the IL2 and CT-N domains of mGluR1a (Fig. 3C). Treatment of the cells with increasing concentrations of Tat-tagged peptide corresponding to the second intracellular loop domain of mGluR1a resulted in a dose-dependent attenuation of HA-CAIN co-immunoprecipitation with FLAG-mGluR1a (Fig. 4A). HA-CAIN co-immunoprecipitated with FLAG-mGluR1a was reduced by 78 ± 9% of
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