Protein Kinase C Phosphorylation of the Metabotropic Glutamate ReceptormGluR5 on Serine 839 Regulates Ca2+Oscillations (2005)

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 A was performed for for a Ca2+ to agonist as or with as with and frequency and cells as oscillations including a a single calcium a single calcium or with oscillations in a or a single with 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, 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, S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). of Ser-839 of mGluR5 were generated by were with a The were and using acids in that were added to the terminus of to proteins and were phosphorylated by purified PKC in as Protein Production and in were resolved by SDS-PAGE and transferred of Ser-839 on fusion proteins was analyzed by with the PKC is an important of S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google F. A. R.A. PubMed Scopus Google Scholar, H. P.J. J. 1998; PubMed Google Scholar, A. Sci. PubMed Scopus Google Scholar, J. M.J. R. J. 2002; 22: PubMed Google Scholar, P.J. E. J. 2003; PubMed Scopus Google the biochemical analyses the direct phosphorylation by PKC have been Therefore, we to C terminus as the region most to PKC the direct phosphorylation of the mGluR5 by PKC, we generated a GST fusion protein containing the C terminus Previous studies have reported the phosphorylationof including Thr-840, on mGluR5 the the S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google we a GST fusion protein containing the first one-third of the mGluR5 We performed an in to the direct phosphorylation of mGluR5 by proteins were incubated with and PKC for at 30 °C as proteins were resolved by the phosphorylated with and the peptide were resolved in resulting in a distinct The peptide map of C terminus phosphopeptides that in the peptide of and was that the on the mGluR5 C terminus were one-third of this we used the protein for analysis. We to the phosphorylation of Thr-840 been reported to the PKC substrate 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 by phosphorylation of the wild-type mGluR5 C terminus and it to C terminus containing an alanine mutation of the critical We performed in phosphorylation ofthe wild-type with the we found no in the peptide as Thr-840 been reported to a PKC S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). We also investigated to aspartic acid mutation in S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Scholar). In we found that in the of a on the map with wild-type with the direct phosphorylation of Thr-840 by both the alanine and aspartic acid mutation of Thr-840. these that the specifically the phosphorylation of We the that the identity of the acid the substrate of PKC for a one-third of the mGluR5 C terminus potential with and peptide containing One residue for was the adjacent we mutated Ser-839 to alanine it using the in phosphorylation We found phosphorylation of in a with a a map with the same as the of the mutant was with the the PKC phosphorylation of the adjacent Ser-839. of the serine and the T837A, not phosphorylation and that not differ from In the peptide map we also generated a mGluR5 phosphorylated on Ser-839 to or not Ser-839 is directly We performed an PKC phosphorylation of wild-type resolved the proteins by and our Ser-839 We found that the wild-type and not with the of Ser-839 in on our mutagenesis studies of it that phosphorylated by PKC on serine is analogous to Ser-839 ofmGluR5 in when we the adjacent aspartic acid wild-type with a permissive amino acid. thedirect phosphorylation of we a GST fusion protein of one-third of the mGluR1α C terminus performed an in phosphorylation the of wild-type the map and of a that the was of the phosphorylation of Ser-853, we a and of this fusion protein in a peptide map and the peptide map of results that the acid of the phosphorylation of theadjacent residue on mGluR1α (Ser-853), as the adjacent acid the phosphorylation of Ser-839 on mGluR5. was with the direct phosphorylationof Ser-839 on mGluR5 by PKC and an of PKC phosphorylation of we to the that of Thr-840 or to Ser-839. of our phosphorylation used a PKC of and We our analyses using a of PKC to or We found that from the phosphorylation of different were that most of the PKC phosphorylated by different of of the PKC phosphorylated the peptide and Thr-840 with and In and not Ser-839 at that our the differential of the PKC in not in as also a However, the same peptide was PKC when Ser-839 was mutated to that Ser-839 was the only residue the peptide that by the variety of PKC no or residue, that phosphorylated by these same PKC data of the PKC phosphorylation of serine PKC phosphorylation of Ser-839 is critical for oscillations, the mutation by mGluR5 investigate mGluR5 or in in intracellular Ca2+ in to μm were analyzed using The intracellular Ca2+ as 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, 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, S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google with a critical role for Ser-839 phosphorylation in the Ca2+ the mutation Ca2+ with mGluR5 studies 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 with activation of mGluR5 also in a of Ca2+ with our that it is Ser-839 and not Thr-840 is important for regulating Ca2+ for Ser-839 Ca2+ were from mGluR5 wild-type Ca2+ of Ser-839 is the critical determinant for theCa2+ oscillations by mGluR5 Previous studies 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 Ca2+ oscillations PKC 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) that were on PKC but not on protein protein kinase protein kinase Therefore, we the PKC of mGluR5-elicited Ca2+oscillations, specifically wild-type mGluR5 to mGluR5 mGluR5 were with for h to 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). both mGluR5 wild-type and mGluR5 no Ca2+ oscillations that PKC phosphorylation of Thr-840 is PKC regulating Ca2+ oscillations. studies have implicated PKC as an important of S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google H. P.J. J. 1998; PubMed Google Scholar, A. Sci. PubMed Scopus Google Scholar, J. M.J. R. J. 2002; 22: PubMed Google of the most of PKC regulating is Ca2+ oscillations. The activation of the Group glutamate receptors, mGluR1α and results Ca2+ but mGluR5 activation is unique in triggers Ca2+ oscillations. is that the the mGluR5 C terminus is the critical determinant regulating theCa2+ oscillations, and until it has been phosphorylation of Thr-840 that region of mGluR5 was the 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, 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, S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google Okada M. Nakanishi S. J. 1997; PubMed Scopus Google Scholar, P.J. 2003; PubMed Scopus Google Scholar, M. N. Y. Y. M. T. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. M. J. M. Nakanishi S. J. 2004; PubMed Scopus Google is on mutagenesis data in Thr-840 to aspartic mGluR5 Ca2+ oscillations. We have used to specifically the PKC phosphorylation ofthe of the mGluR5 C terminus Thr-840. we Thr-840 is not a PKC and phosphorylation not in the PKC regulation of mGluR5-elicited Ca2+ an adjacent residue, Ser-839, is directly phosphorylated by PKC, of this residue regulates Although Thr-840 is not directly both our and that S. Tsutusmi R. Kohara A. Yamaguchi T. Nakanishi S. Okada M. Nature. 1996; 383: 89-92Crossref PubMed Scopus (248) Google that a residue at the not an aspartic acid, for mGluR5-elicited Ca2+ oscillations. We that this to a permissive or Thr-840 in the phosphorylation of Ser-839 on mGluR5. Although by used peptide map the phosphorylation of the critical region of they the have that not the residue directly phosphorylated by We also showed that the analogous residue Asp-854 in mGluR1α PKC phosphorylation of the conserved serine in mGluR1α Asp-854 is mutated to a permissive residue, D854A, PKC phosphorylation of are with the in activation of mGluR5 mutation in intracellular and activation of mGluR1α containing the in Ca2+ oscillations. Although our are the first to that Thr-840 is phosphorylated by PKC, studies that Thr-840 not substrate for the and mutations in mGluR5 havebeen reported to PKC mGluR5activation 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 M. N. Y. Y. M. T. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). results by the direct phosphorylation of Thr-840 on mGluR5 by PKC phosphorylation of serine of because both of phosphorylation of this residue and to have However, in was mechanism for PKC regulation of mGluR5 that PKC directly Ser-839 and that Thr-840 for Ser-839 phosphorylation are with these of the data found in the these with a role for PKC phosphorylation of Ser-839 mGluR5-elicited Ca2+ PKC oscillations. We used an in phosphorylation map analyses to that Ser-839 is phosphorylated by that Thr-840 is we used a PKC that of and in of However, we to the PKC Ser-839 or reported that specifically no biochemical of phosphorylation were M. N. Y. Y. M. T. N. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). that used a of PKC to wild-type We found that Thr-840 was not phosphorylated by of the used in our including these a permissive or role for Thr-840 in to a substrate for PKC to mGluR5 Ca2+ oscillations were we mGluR5 with for a of We found that PKC Thus, mGluR5 Ca2+ oscillations with PKC directly Ser-839 Thr-840. results demonstrated a role for the PKC phosphorylationof Ser-839 in regulating mGluR5 Ca2+ oscillations in our studies a unique that the regulation of the Group intracellular Ca2+ a in intracellular mGluR5 is and our studies the substrate mGluR5 that is phosphorylated However, it is also that the regulation Ca2+ oscillations phosphorylation of a single The that cells to the that are at is also in the Group 1 Ca2+ 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 Sci. S. A. PubMed Scopus Google Scholar, A. R. J. D.J. PubMed Scopus Google Scholar, R. J. 2002; PubMed Scopus Google Scholar). The role of Ca2+ oscillations in is not it is that the and are critical for the regulation of in P. 1998; PubMed Scopus Google Scholar, Nature. 1998; PubMed Scopus Google Scholar, J. M. Nature. 1998; PubMed Scopus Google reported that Asp-854 on mGluR1α to mGluR5) not oscillations in M. T. M. J. M. Nakanishi S. J. 2004; PubMed Scopus Google it oscillations in and cells in in the regulation of Ca2+ previously and the in determinants PKC phosphorylation of our and analyses of PKC, and IP3 oscillations in We the in Dr. for and in the of the confocal We the for DNA sequence analysis.