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• W2046304984 abstract "The activation of Group 1 metabotropic glutamate receptors, mGluR5 andmGluR1α, triggers intracellular calcium release; however, mGluR5activation is unique in that it elicits Ca2+ oscillations. A shortregion of the mGluR5 C terminus is the critical determinant and differs fromthe analogous region of mGluR1α by a single amino acid residue, Thr-840,which is an aspartic acid (Asp-854) in mGluR1α. Previous studies showthat mGluR5-elicited Ca2+ oscillations require protein kinase C(PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylationsite. However, direct phosphorylation of mGluR5 has not been studied indetail. We have used biochemical analyses to directly investigate thephosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 isdirectly phosphorylated by PKC, whereas Thr-840 plays a permissive role.Although Ser-839 is conserved in mGluR1α (Ser-853), it is notphosphorylated, as the adjacent residue (Asp-854) is not permissive; however,mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylationof Ser-853 on mGluR1α. We investigated the physiological consequences ofmGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations thateliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependentCa2+ oscillations. However, mutation of Thr-840 to alanine, whichprevents potential Thr-840 phosphorylation but is still permissive for Ser-839phosphorylation, has no effect on Ca2+ oscillations. Thus, weshowed that it is phosphorylation of Ser-839, not Thr-840, that is absolutelyrequired for the unique Ca2+ oscillations produced by mGluR5activation. The Thr-840 residue is important only in that it is permissive forthe PKC-dependent phosphorylation of Ser-839. The activation of Group 1 metabotropic glutamate receptors, mGluR5 andmGluR1α, triggers intracellular calcium release; however, mGluR5activation is unique in that it elicits Ca2+ oscillations. A shortregion of the mGluR5 C terminus is the critical determinant and differs fromthe analogous region of mGluR1α by a single amino acid residue, Thr-840,which is an aspartic acid (Asp-854) in mGluR1α. Previous studies showthat mGluR5-elicited Ca2+ oscillations require protein kinase C(PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylationsite. However, direct phosphorylation of mGluR5 has not been studied indetail. We have used biochemical analyses to directly investigate thephosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 isdirectly phosphorylated by PKC, whereas Thr-840 plays a permissive role.Although Ser-839 is conserved in mGluR1α (Ser-853), it is notphosphorylated, as the adjacent residue (Asp-854) is not permissive; however,mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylationof Ser-853 on mGluR1α. We investigated the physiological consequences ofmGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations thateliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependentCa2+ oscillations. However, mutation of Thr-840 to alanine, whichprevents potential Thr-840 phosphorylation but is still permissive for Ser-839phosphorylation, has no effect on Ca2+ oscillations. Thus, weshowed that it is phosphorylation of Ser-839, not Thr-840, that is absolutelyrequired for the unique Ca2+ oscillations produced by mGluR5activation. The Thr-840 residue is important only in that it is permissive forthe PKC-dependent phosphorylation of Ser-839. Metabotropic glutamate receptors(mGluRs) 1The abbreviations used are: mGluR, metabotropic glutamate receptor;IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; GST,glutathione S-transferase; PMA, phorbol 12-myristate 13-acetate. playimportant roles throughout the nervous system, including the activation of ionchannels and the regulation of synaptic plasticity(1Hermans E. Challiss R.A. Biochem. J. 2001; 359: 465-484Crossref PubMed Scopus (340) Google Scholar). In addition, they havebeen implicated in a variety of neurological diseases(2Spooren W.P. Gasparini F. Salt T.E. Kuhn R. Trends Pharmacol. Sci. 2001; 22: 331-337Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 3Pellegrini-Giampietro D.E. TrendsPharmacol. Sci. 2003; 24: 461-470Scopus (80) Google Scholar, 4Conn P.J. Ann. N. Y. Acad.Sci. 2003; 1003: 12-21Crossref PubMed Scopus (136) Google Scholar, 5Kenny P.J. Markou A. TrendsPharmacol. Sci. 2004; 25: 265-272Scopus (210) Google Scholar).There are eight different mGluRs, and these are subdivided into three groupsbased on sequence identity and pharmacological properties. Group 1 mGluRs(mGluR1 and mGluR5) are linked to phospholipase C, whereas Group 2 mGluRs(mGluR2 and mGluR3) and Group 3 mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8)are negatively linked to adenylate cyclase. Activation of Group 1 mGluRstriggers phospholipase C, resulting in increases in IP3 anddiacylglycerol production and the concomitant release of intracellularCa2+ and activation of protein kinase C (PKC)(6Conn P.J. Pin J.P. Annu.Rev. Pharmacol. Toxicol. 1997; 37: 205-237Crossref PubMed Scopus (2736) Google Scholar). Group 1 mGluR-elicitedCa2+ release stimulates PKC translocation to the plasma membraneand oscillations of both PKC activation and IP3 production(7Dale L.B. Babwah A.V. Bhattacharya M. Kelvin D.J. Ferguson S.S.G. J. Biol. Chem. 2001; 276: 35900-35908Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Although both mGluR1 and mGluR5 stimulate intracellular Ca2+release, they differ in that mGluR5 activation results in Ca2+oscillations, whereas mGluR1 activation results in a single Ca2+transient with or without subsequent low frequency oscillations(9Kawabata S. Kohara A. Tsutsumi R. Itahana H. Hayashibe S. Yamaguchi T. Okada M. J. Biol.Chem. 1998; 273: 17381-17385Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). A previous study(10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar) demonstrated that a shortstretch of amino acids in the C terminus of mGluR5 was the criticaldeterminant regulating Ca2+ oscillations, specifically the identityof a single amino acid residue, Thr-840. In addition, it was reported that thedirect phosphorylation of Thr-840 by PKC was the mechanism regulating theCa2+ oscillations(10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). In the current study, wehave investigated the direct phosphorylation of mGluR5 by PKC usingphosphopeptide analysis. Surprisingly, we demonstrated that Thr-840 is not aPKC substrate but only plays a permissive role for PKC phosphorylation of theadjacent amino acid Ser-839. Consequently, it is the PKC phosphorylation ofSer-839 that regulates mGluR5-elicited Ca2+ oscillations.Furthermore, we showed that the analogous serine in mGluR1, Ser-853, is notphosphorylated due to an adjacent aspartic acid residue but can bephosphorylated when the adjacent amino acid is mutated to a permissiveresidue. Therefore, we have found that Thr-840 of mGluR5 and Asp-854 of mGluR1are the critical determinants of the distinct Ca2+ transientselicited by mGluR5 and mGluR1, respectively. However, our data clearlydemonstrate that this is because of the differential effect these residueshave on the PKC phosphorylation of a conserved adjacent serine, not because ofthe direct PKC phosphorylation of Thr-840 on mGluR5. DNA Constructs and Site-directed Mutagenesis—The mGluR5 cDNAwas a generous gift from Dr. S. Nakanishi (Kyoto University). The full-lengthC terminus (amino acids 828–1171) and first one-third of the C terminus(amino acids 828–944) of mGluR5 were amplified from rat mGluR5 cDNAusing PCR. The first one-third of the C terminus (amino acids 842–958)of rat mGluR1α was also amplified by PCR. The PCR products were thendigested with EcoRI and subcloned in-frame into pGEX-4T-1 GST fusion vector(Amersham Biosciences). mGluR5 (S839A, T840A, T840D, S834A, T837A, and T838A)and mGluR1α (S853A, D854A, D854T, and S853A/D854A) mutations on GSTfusion constructs or pRK5-mGluR5 were generated using the QuikChangesite-directed mutagenesis system (Stratagene, La Jolla, CA) following themanufacturer's instructions. Mutations were confirmed by sequenceanalysis. Fusion Protein Production and in VitroPhosphorylation—Fusion proteins for wild-type and mutant C terminiof mGluR5 or mGluR1α were purified for the most part in accordance withthe protocol provided by the manufacturer (Amersham Biosciences). In brief,DNA constructs were transformed into BL21 bacterial cells. 50-ml cultures weregrown until the cultures reached the mid-log phase of growth(A550 = 0.5–1.0) after adding 1 ml of overnightcultures. Isopropyl 1-thio-β-d-galactopyranoside (1mm) was added to cultures to induce fusion protein expression.After 4 h of induction, the cells were lysed with 5 ml of B-PER bacterialprotein extraction reagent (Pierce, Rockford, IL). The bacterial lysates wereincubated with glutathione-Sepharose 4B (Amersham Biosciences) for 30 min, andthen the beads were washed with 10× bed volume of phosphate-bufferedsaline. The fusion proteins were eluted with 50 mm Tris-HCl (pH8.0) containing 10 mm reduced glutathione. The fusion proteins werephosphorylated in 20 mm HEPES, pH 7.4, 1.67 mmCaCl2, 1 mm dithiothreitol, 10 mmMgCl2, 200 mm cold ATP, 1 pmol of[γ-32P]ATP (3000 Ci/mmol) with 25 ng of purified PKC(Promega, Madison, WI) at 30 °C for 30 min. The reactions were stopped byadding SDS-PAGE sample buffer, and the samples were boiled for 5 min. Thephosphorylated proteins were resolved by SDS-PAGE and transferred tonitrocellulose membrane (Schleicher & Schuell, Keene, NH). The bands werevisualized by autoradiography and were excised for in vitrophosphopeptide mapping. Two-dimensional Phosphopeptide Mapping—Peptide mapping wasperformed as previously described(11Roche K.W. O'Brien R.J. Mammen A.L. Bernhardt J.P. Huganir R.L. Neuron. 1996; 16: 1179-1188Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar,12Roche K.W. Huganir R.L. Crawley J. Gerfen C. McKay R. Rogawski M. Sibley D. Skolnick P. Current Protocols in Neuroscience. John Wiley and Sons, Inc., Hoboken, New Jersey2000: 1-15Google Scholar). Briefly, phosphorylatedproteins were resolved by SDS-PAGE and transferred to nitrocellulose. Therelevant bands were excised and soaked for 1 h in tubes containing 1 ml of 1%polyvinylpyrrolidone-40 in 100 mm acetic acid. After washing with0.4% NH4HCO3, the proteins on the membrane were digestedwith trypsin overnight at 37 °C. Supernatants containing the trypticdigestion products were dried in a SpeedVac, washed twice with 900 μl ofH2O, and resuspended in 5 μl of H2O. One or two μlof the dissolved phosphopeptides were spotted onto a cellulose thin layerchromatography plate (Merck). The phosphopeptides were resolved in the firstdimension by electrophoresis in buffer containing 2.5% formic acid and 7.8%acetic acid. Separation by ascending chromatography in the second dimensionwas performed using buffer containing 62.5% isobutyric acid, 4.8% pyridine,1.9% butanol, and 2.9% acetic acid. The thin layer chromatography plate wasair-dried, and the peptide map was visualized by PhosphorImager analysis. Measurement of Ca2+Oscillations—HeLa cells (American Type Culture CollectionCCL-2) were grown in Dulbecco's modified Eagle's medium containing 10% fetalbovine serum on coverglasses. The cells were transfected with wild-type ormutant constructs of mGluR5 using Cal-Phos mammalian calcium phosphatetransfection kit (BD Biosciences). The cells were incubated with transfectants12 h before changing to fresh medium and analyzed 36–48 h followingtransfection. The cells were loaded with 5 μm Oregon Green 488BAPTA-1 AM (Molecular Probes, Eugene, OR) for 30 min at 37 °C and thenwashed three times with HEPES-buffered solution containing 135 mmNaCl, 5.4 mm KCl, 0.9 mm MgCl2, 1.8mm CaCl2, and 10 mm HEPES. After 1 h ofincubation in the same buffer, confocal microscopy was performed on a ZeissLSM-510 laser-scanning microscope after the treatment of a mGluR5 agonist (500μm glutamate). A time scan was performed for 300 s for eachsample. Cells exhibiting a Ca2+ response to agonist applicationwere classified as oscillating or non-oscillating, with oscillating cellsdefined as those having oscillation frequencies >1/min with constantamplitude and frequency and non-oscillating cells defined as those having anycalcium transients other than oscillations defined above, including a singlecalcium peak, a single sustained calcium plateau, a single calcium peak or aplateau with slow oscillations in a later phase, or a single peak with ashoulder(8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Kawabata S. Kohara A. Tsutsumi R. Itahana H. Hayashibe S. Yamaguchi T. Okada M. J. Biol.Chem. 1998; 273: 17381-17385Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). Generation of Phosphorylation State-specific Antibodies andImmunoblotting—Rabbit phosphospecific antibodies againstphosphorylated Ser-839 of mGluR5 were generated by BIOSOURCE International(Camarillo, CA). Rabbits were immunized with a synthetic peptideN-C(EE)FTT[pS]TVVR-C. The sera were collected and affinity-purified using theantigen peptide. Amino acids in parentheses represent additional glutamic acidresidues that were added to the N terminus of peptides to promote solubility.Fusion proteins (wild-type GST-mGluR5-Cprox, GST-mGluR5-Cprox (S839A), and GSTalone) were phosphorylated by purified PKC in vitro as describedunder “Fusion Protein Production and in VitroPhosphorylation.” Proteins were resolved by SDS-PAGE and transferred toa polyvinylidene difluoride membrane. Phosphorylation of Ser-839 on mGluR5C-terminal fusion proteins was analyzed by immunoblotting with the pSer839phosphospecific antibody. PKC is an important modulator of mGluR function(10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar,13Ciruela F. Giacometti A. McIlhinney R.A. FEBS Lett. 1999; 462: 278-282Crossref PubMed Scopus (17) Google Scholar, 14Macek T.A. Schaffhauser H. Conn P.J. J. Neurosci. 1998; 18: 6138-6146Crossref PubMed Google Scholar, 15Francesconi A. Duvoisin R.M. Proc. Natl. Acad. Sci. 2000; 97: 6185-6190Crossref PubMed Scopus (102) Google Scholar, 16Rush A.M. Wu J. Rowan M.J. Anwyl R. J. Neurosci. 2002; 22: 6121-6128Crossref PubMed Google Scholar, 17Mundell S.J. Pula G. Carswell K. Roberts P.J. Kelly E. J. Neurochem. 2003; 84: 294-304Crossref PubMed Scopus (37) Google Scholar);however, the biochemical analyses characterizing the direct phosphorylation ofmGluRs by PKC have been lacking. Therefore, we began to characterize themGluR5 C terminus as the region most likely to contain PKC phosphorylationsites. To evaluate precisely the direct phosphorylation of the mGluR5 Cterminus by PKC, we generated a GST fusion protein containing the entiremGluR5 C terminus (GST-mGluR5-C; Fig.1A). Previous studies have reported the phosphorylationof specific residues, including Thr-840, on mGluR5 within the proximal Cterminus near the seventh transmembrane domain(10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar,18Gereau IV, R.W. Heinemann S.F. Neuron. 1998; 20: 143-151Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar); therefore, we alsogenerated a GST fusion protein containing the first one-third of the mGluR5 Cterminus, GST-mGluR5-Cprox (Fig.1A). We performed an in vitro phosphorylationassay to evaluate the direct phosphorylation of mGluR5 by PKC. GST-mGluR5fusion proteins were incubated with [γ-32P]ATP and PKC for 30min at 30 °C as described under “Experimental Procedures.” Thefusion proteins were then resolved by SDS-PAGE, the phosphorylated proteinswere proteolyzed with trypsin, and the peptide fragments were resolved in twodimensions, resulting in a distinct phosphopeptide map. The peptide map of themGluR5 C terminus revealed multiple phosphopeptides that varied in theirintensity (Fig. 1B).Interestingly, the peptide maps of GST-mGluR5-C and GST-mGluR5-Cprox wereindistinguishable (Fig.1B). This was compelling evidence that the major PKCphosphorylation sites on the mGluR5 C terminus were contained within theproximal one-third of this region. Therefore we used the GST-mGluR5-Cproxfusion protein for further analysis. We next wanted to evaluate the phosphorylation of Thr-840 within mGluR5,which had been reported to be the PKC substrate responsible for regulating theCa2+ oscillations(10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). We began by evaluatingPKC phosphorylation of the wild-type mGluR5 C terminus and comparing it to themGluR5 C terminus containing an alanine mutation of the critical threonine(GST-mGluR5-Cprox T840A). We performed in vitro phosphorylation ofthe wild-type GST-mGluR5-Cprox along with the GST-mGluR5-Cprox (T840A).However, we found no change in the peptide maps(Fig. 1C), which wassurprising, as Thr-840 had been reported to be a PKC substrate(10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). We also investigated thethreonine to aspartic acid mutation (GST-mGluR5-Cprox T840D) described in thatstudy (10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). In contrast toT840A, we found that T840D did result in the disappearance of a distinctphosphopeptide on the map compared with wild-type mGluR5(Fig. 1C). These dataare therefore inconsistent with the direct phosphorylation of Thr-840 by PKC,as both the alanine and aspartic acid mutation would abolish directphosphorylation of Thr-840. Instead, these experiments suggest that the T840Dmutant specifically disrupted the phosphorylation of some other site onmGluR5. We therefore tested the alternative hypothesis that the identity of the 840amino acid affects the substrate specificity of PKC for a nearby residue. Thefirst one-third of the mGluR5 C terminus contains many potential PKCphosphorylation sites, with several serines and threonines included within thepredicted tryptic peptide fragment containing Thr-840(834SAFTTSTVVR843). One candidate residue for PKCphosphorylation was the adjacent Ser-839(Fig. 2A). To testthis hypothesis, we mutated Ser-839 to alanine (GST-mGluR5-Cprox S839A) andevaluated it using the in vitro phosphorylation assay. We found thatPKC phosphorylation of GST-mGluR5-Cprox (S839A) resulted in a phosphopeptidemap with a phosphopeptide missing (Fig.2B), yielding a map with the same pattern as the peptidemap of the T840D mutant (Fig.1C). This was consistent with the T840D mutationdisrupting the PKC phosphorylation of the adjacent Ser-839. Furthermore, pointmutations of the other serine and threonines within the predicted peptidefragment (S834A, T837A, T838A) did not affect phosphorylation and resulted inpeptide maps that did not differ from wild type(Fig. 2C). In additionto the peptide map analysis, we also generated a phosphorylationstate-specific antibody recognizing mGluR5 phosphorylated on Ser-839 to probewhether or not Ser-839 is directly phosphorylated. We performed an invitro PKC phosphorylation assay of GST, wild-type GST-mGluR5-Cprox, andGST-mGluR5-Cprox (S839A), resolved the proteins by SDS-PAGE, and immunoblottedwith our Ser-839 phosphoantibody. We found that the antibody specificallyrecognized PKC-phosphorylated wild-type GST-mGluR5-Cprox and did not recognizePKC-phosphorylated GST-mGluR5-Cprox (S839A), consistent with the directphosphorylation of Ser-839 in vitro(Fig. 2D). Based on our mutagenesis studies of mGluR5, it followed that mGluR1αmight be phosphorylated by PKC on serine 853, which is analogous to Ser-839 ofmGluR5 (see alignment in Fig.2A), when we replace the adjacent aspartic acid (Asp-854in wild-type mGluR1α) with a permissive amino acid. To characterize thedirect phosphorylation of mGluR1α, we made a GST fusion protein of theproximal one-third of the mGluR1α C terminus (GST-mGluR1α-Cprox).We performed an in vitro phosphorylation assay comparing the PKCphosphorylation of wild-type GST-mGluR1α-Cprox andGST-mGluR1α-Cprox mutants. Interestingly, the phosphopeptide map formGluR1α-Cprox D854A and mGluR1α-Cprox D854T revealed theappearance of a new phosphopeptide (Fig.2E). To confirm that the new phosphopeptide was theresult of the phosphorylation of Ser-853, we made a double mutation, includingboth S853A and D854A (GST-mGluR1α-Cprox S853A/D854A). PKCphosphorylation of this fusion protein resulted in a peptide map lacking thecritical phosphopeptide and resembling the peptide map of wild-typemGluR1α (Fig.2E). These results strongly suggest that the endogenousaspartic acid (Asp-854 of mGluR1α) affects the phosphorylation of theadjacent residue on mGluR1α (Ser-853), just as the adjacent threonine oraspartic acid affects the phosphorylation of Ser-839 on mGluR5. Our data, thus far, was entirely consistent with the direct phosphorylationof Ser-839 on mGluR5 by PKC and an absence of PKC phosphorylation of Thr-840.However, we wanted to explore the possibility that certain isoforms of PKCmight preferentially phosphorylate Thr-840 or other nearby residues inaddition to Ser-839. All of our phosphorylation assays had used a PKC mixturethat consisted predominantly of α, β, and γ PKC. We thereforeextended our analyses using a battery of PKC isoforms to phosphorylatewild-type GST-mGluR5-Cprox or GST-mGluR5-Cprox (S839A)(Fig. 3). We found that thephosphopeptide maps derived from the phosphorylation assays of different PKCisoforms were quite similar, indicating that most of the PKC phosphorylationsites could be phosphorylated by several different isoforms of PKC.Interestingly, many of the PKC isoforms phosphorylated the peptide containingSer-839 and Thr-840 (Fig.3A), with PKCα, PKCγ, and PKCθ beingmost efficient. In contrast, PKCδ, -ϵ, and -ζ did not appearto phosphorylate Ser-839 at all. It should be noted that our experiments onlyaddressed the differential specificity of the PKC isoforms in vitroand therefore do not necessarily reflect isoform specificity in vivo,where other factors, such as compartmentalization, may also play a criticalrole. However, importantly, the same peptide fragment was never phosphorylatedby any PKC isoform when Ser-839 was mutated to alanine(Fig. 3B). These datademonstrated that Ser-839 was the only residue within the peptide that wasphosphorylated by the variety of PKC isoforms tested. Under no conditions wasThr-840, or any other nearby residue, contained within that particular trypticpeptide, phosphorylated by these same PKC isoforms. These data further confirmthe specificity of the PKC phosphorylation of serine 839. If PKC phosphorylation of Ser-839 is critical for mGluR5-elicitedCa2+ oscillations, then the S839A mutation should alterCa2+ transients elicited by mGluR5 activation. To investigate this,we expressed full-length mGluR5 wild type or mutants in HeLa cells, andchanges in intracellular Ca2+ concentration in response to agonistapplication (500 μm glutamate, 250 s) were analyzed using aconfocal microscope. The intracellular Ca2+ transients wereclassified as oscillating (representative response,Fig. 4A), ornon-oscillating (see “Experimental Procedures”)(8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Kawabata S. Kohara A. Tsutsumi R. Itahana H. Hayashibe S. Yamaguchi T. Okada M. J. Biol.Chem. 1998; 273: 17381-17385Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar).Consistent with a critical role for Ser-839 phosphorylation in the regulationof Ca2+ signaling, the S839A mutation significantly prevented theoscillating Ca2+ response compared with mGluR5 wild type(Fig. 4B). Similar toprevious studies (9Kawabata S. Kohara A. Tsutsumi R. Itahana H. Hayashibe S. Yamaguchi T. Okada M. J. Biol.Chem. 1998; 273: 17381-17385Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar) and in agreement with ourphosphorylation data, activation of mGluR5 T840D also resulted in a pronouncedreduction of oscillating Ca2+ responses(Fig. 4B). However,consistent with our finding that it is Ser-839 and not Thr-840 phosphorylationthat is important for regulating Ca2+ signaling, T840A (which ispermissive for Ser-839 phosphorylation) exhibited Ca2+ oscillationsthat were indistinguishable from mGluR5 wild-type Ca2+ responses(Fig. 4B). Thusphosphorylation of Ser-839 is the critical molecular determinant for theCa2+ oscillations induced by mGluR5 activation. Previous studies (8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar) have characterized themGluR5-elicited Ca2+ oscillations pharmacologically. For example,using several PKC inhibitors, Kawabata et al.(9Kawabata S. Kohara A. Tsutsumi R. Itahana H. Hayashibe S. Yamaguchi T. Okada M. J. Biol.Chem. 1998; 273: 17381-17385Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar,10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar) determined that theoscillations were dependent on PKC but not on other kinases, such ascAMP-dependent protein kinase, protein kinase G, orcalcium/calmodulin-dependent protein kinase II. Therefore, we alsoinvestigated the PKC dependence of mGluR5-elicited Ca2+oscillations, specifically comparing wild-type mGluR5 to mGluR5 T840A. Cellsexpressing mGluR5 T840A were treated with PMA for 12 h to down-regulate PKCactivity (8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Under theseconditions, both mGluR5 wild-type and mGluR5 T840A no longer consistentlyproduced Ca2+ oscillations (Fig.5), further confirming that PKC phosphorylation of Thr-840 is notthe essential PKC event regulating Ca2+ oscillations. Many studies have implicated PKC as an important regulator of mGluR5function (10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar,14Macek T.A. Schaffhauser H. Conn P.J. J. Neurosci. 1998; 18: 6138-6146Crossref PubMed Google Scholar, 15Francesconi A. Duvoisin R.M. Proc. Natl. Acad. Sci. 2000; 97: 6185-6190Crossref PubMed Scopus (102) Google Scholar, 16Rush A.M. Wu J. Rowan M.J. Anwyl R. J. Neurosci. 2002; 22: 6121-6128Crossref PubMed Google Scholar).One of the most convincing examples of PKC regulating mGluR signaling is thatof mGluR5-mediated Ca2+ oscillations. The activation of the Group 1metabotropic glutamate receptors, mGluR1α and mGluR5, results inintracellular Ca2+ release, but mGluR5 activation is unique in thatit triggers Ca2+ oscillations. It is known that the proximal regionof the mGluR5 C terminus is the critical determinant regulating theCa2+ oscillations, and until now, it has been widely accepted thatPKC phosphorylation of Thr-840 within that region of mGluR5 was the essentialmechanism(8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Kawabata S. Kohara A. Tsutsumi R. Itahana H. Hayashibe S. Yamaguchi T. Okada M. J. Biol.Chem. 1998; 273: 17381-17385Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar,19Nakahara K. Okada M. Nakanishi S. J. Neurochem. 1997; 69: 1467-1475Crossref PubMed Scopus (123) Google Scholar, 20Sorensen S.D. Conn P.J. Neuropharmacology. 2003; 44: 699-706Crossref PubMed Scopus (53) Google Scholar, 21Uchino M. Sakai N. Kashiwagi K. Shirai Y. Shinohara Y. Hirose K. Iino M. Yamamura T. Saito N. J. Biol. Chem. 2004; 279: 2254-2261Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22Sato 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).This is based on mutagenesis data in which altering Thr-840 to aspartic acidperturbs mGluR5 Ca2+ oscillations. We have used biochemicalphosphorylation assays to specifically characterize the PKC phosphorylation ofthe portion of the mGluR5 C terminus surrounding Thr-840. Strikingly, we foundthat Thr-840 is not a PKC substrate, and its phosphorylation does not play anyrole in the PKC regulation of mGluR5-elicited Ca2+ oscillations.Instead, an adjacent residue, Ser-839, is directly phosphorylated by PKC, andphosphorylation of this residue regulates mGluR5-dependent Ca2+oscillations. Although Thr-840 is not directly phosphorylated, both our study and that ofKawabata et al. (10Kawabata S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar)show that a threonine residue at the 840 position, not an aspartic acid, isessential for mGluR5-elicited Ca2+ oscillations. We thendemonstrated that this could be attributed to a permissive or structural rolefor Thr-840 in allowing the phosphorylation of Ser-839 on mGluR5. Although theoriginal study by Kawabata et al. used peptide map analysis toanalyze the phosphorylation of the critical region of mGluR5, they neverevaluated the T840A mutation. This experiment would have revealed that Thr-840was not the residue directly phosphorylated by PKC. We also showed that the analogous residue Asp-854 in mGluR1α inhibitsthe PKC phosphorylation of the conserved serine in mGluR1α, Ser-853.When mGluR1α Asp-854 is mutated to a permissive residue, D854A, weobserved PKC phosphorylation of Ser-853. These findings are entirelyconsistent with the original findings in which activation of mGluR5 containingthe T840D mutation resulted in mGluR1α-like intracellular calciumrelease, and activation of mGluR1α containing the D854T mutationresulted in mGluR5-like Ca2+ oscillations. Although our experiments are the first to demonstrate that Thr-840 is notdirectly phosphorylated by PKC, other studies suggest that Thr-840 may not bea substrate for PKC. For example, the T840A and T840D mutations in mGluR5 havebeen reported to differentially regulate PKC oscillation patterns upon mGluR5activation (7Dale L.B. Babwah A.V. Bhattacharya M. Kelvin D.J. Ferguson S.S.G. J. Biol. Chem. 2001; 276: 35900-35908Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,21Uchino M. Sakai N. Kashiwagi K. Shirai Y. Shinohara Y. Hirose K. Iino M. Yamamura T. Saito N. J. Biol. Chem. 2004; 279: 2254-2261Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). These results cannot beexplained by the direct phosphorylation of Thr-840 on mGluR5 by PKC (or lackof phosphorylation of serine 854 of mGluR1α), because both of thesemutations would abolish phosphorylation of this particular residue and wouldbe predicted to have similar phenotypes. However, in neither study was analternative mechanism for PKC regulation of mGluR5 signaling identified. Ourfindings that PKC directly phosphorylates Ser-839 and that Thr-840 ispermissive for Ser-839 phosphorylation are consistent with these reports andreconcile much of the data found in the literature. Taken together, these dataare consistent with a role for PKC phosphorylation of Ser-839 specificallyregulating mGluR5-elicited Ca2+ (and perhaps PKC andIP3) oscillations. We used an in vitro phosphorylation assay followed byphosphopeptide map analyses to determine that Ser-839 is phosphorylated by PKCbut that Thr-840 is not. Initially we used a PKC mixture that consistedpredominantly of α, β, and γ isoforms in all of ourphosphorylation assays. However, we wanted to explore the possibility thatparticular PKC isoforms might preferentially phosphorylate Ser-839 or Thr-840.A recent study reported that PKCδ specifically phosphorylates Thr-840,although no biochemical assays of phosphorylation were conducted(21Uchino M. Sakai N. Kashiwagi K. Shirai Y. Shinohara Y. Hirose K. Iino M. Yamamura T. Saito N. J. Biol. Chem. 2004; 279: 2254-2261Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). To address thepossibility that specific isoforms might preferentially phosphorylate Thr-840,we used a battery of PKC isoforms to phosphorylate wild-type GST-mGluR5-Cproxor GST-mGluR5-Cprox (S839A) (Fig.3). We found that Thr-840 was not phosphorylated by any of the PKCisoforms used in our assay, including PKCδ. Once again these datasupported a purely permissive or structural role for Thr-840 in allowingSer-839 to be a substrate for PKC phosphorylation. Finally, to explore whetherthe mGluR5 T840A Ca2+ oscillations were PKC-dependent, we treatedcells expressing mGluR5 T840A with PMA for a prolonged period of time todown-regulate PKC. We found that PKC down-regulation inhibited theoscillations. Thus, mGluR5 T840A-elicited Ca2+ oscillations wereindeed PKC-dependent, consistent with PKC directly phosphorylating Ser-839 andnot Thr-840. Our results clearly demonstrated a central role for the PKC phosphorylationof Ser-839 in regulating mGluR5 Ca2+ oscillations in heterologouscells. Furthermore, our studies revealed a unique substrate-specificphosphorylation event that differentiates the regulation of the Group 1mGluR-mediated intracellular Ca2+ release. Such a preciselyregulated difference in intracellular signaling between mGluR5 andmGluR1α is extremely provocative, and our studies defining the precisePKC substrate within mGluR5 that is phosphorylated will facilitate morerigorous studies. However, it is also clear that the regulation ofmGluR5-elicited Ca2+ oscillations must be more complicated than thePKC phosphorylation of a single residue. The fact that all responding cells donot oscillate (Fig. 4) lendssupport to the notion that other factors are at play. This is also apparent bythe many complexities in the Group 1 mGluR Ca2+ signalingliterature (8Nash M.S. Schell M.J. Atkinson P.J. Johnston N.R. Nahorski S.R. J. Biol. Chem. 2002; 277: 35947-35960Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,23Flint A.C. Dammerman R.S. Kriegstein A.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12144-12149Crossref PubMed Scopus (85) Google Scholar, 24Crawford J.H. Wainwright A. Heavens R. Pollock J. Martin D.J. Scott R.H. Seabrook G.R. Neuropharmacology. 2000; 39: 621-630Crossref PubMed Scopus (54) Google Scholar, 25Sosa R. Hoffpauir B. Rankin M.L. Bruch R.C. Gleason E.L. J. Neurochem. 2002; 81: 973-983Crossref PubMed Scopus (29) Google Scholar). The role of Ca2+ oscillations in neurons is not understood,although it is clear that the frequency, duration, and spatial location ofcalcium signals are critical for the precise regulation of signal transductionand gene expression in other cellular systems(26De Koninck P. Schulman H Science. 1998; 279: 227-230Crossref PubMed Scopus (1088) Google Scholar, 27Dolmetsch R.E. Xu K. Lewis R.S. Nature. 1998; 392: 933-936Crossref PubMed Scopus (1683) Google Scholar, 28Li W. Llopis J. Whitney M. Zlokarnik G. Tsien R.Y. Nature. 1998; 392: 936-941Crossref PubMed Scopus (777) Google Scholar).A recent study reported that mutating Asp-854 on mGluR1α to threonine(to mimic mGluR5) does not yield oscillations in Purkinje neurons(22Sato 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), making it unclear howCa2+ oscillations in glia and heterologous cells relate toCa2+ transients in neurons. These recent findings suggest that, atleast in Purkinje neurons, the regulation of Ca2+ transients may bemore complex than previously believed and illustrate the importance ofevaluating mGluR signaling in neurons. By precisely characterizing themolecular determinants underlying PKC phosphorylation of mGluR5, our studywill facilitate more vigorous and accurate analyses of mGluR-regulatedCa2+, PKC, and IP3 oscillations in vivo. We thank the NINDS Light Microscopy Imaging Facility, in particular thefacility manager Dr. Carolyn Smith, for advice and expertise in the collectionand analysis of the confocal images. We acknowledge the NINDS SequencingFacility for automated DNA sequence analysis." @default.
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