A Novel Site on the Gα-protein That Recognizes Heptahelical Receptors
2001; Elsevier BV; Volume: 276; Issue: 5 Linguagem: Inglês
10.1074/jbc.m004880200
ISSN1083-351X
AutoresJaroslav Blahoš, T Fischer, Isabelle Brabet, Daniela Stauffer, Giorgio Rovelli, Joël Bockaert, Jean‐Philippe Pin,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoSpecific domains of the G-protein α subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between α4 and α5 helices of the G-protein α subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Gα15 and its human homologue Gα16, whereas metabotropic glutamate receptor 8 activated Gα15 only. The extreme C-terminal 20 amino acid residues are identical between the Gα15 and Gα16 and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Gα16 that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of Gα16. One module comprises α4 helix, α4−β6 loop (L9 Loop), β6 sheet, and α5 helix. The other, not described previously, is located within the loop that links the N-terminal α helix to the β1 strand of the Ras-like domain of the α subunit. Coupling of Gα16 protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully. Specific domains of the G-protein α subunit have been shown to control coupling to heptahelical receptors. The extreme N and C termini and a region between α4 and α5 helices of the G-protein α subunit are known to determine selective interaction with the receptors. The metabotropic glutamate receptor 2 activated both mouse Gα15 and its human homologue Gα16, whereas metabotropic glutamate receptor 8 activated Gα15 only. The extreme C-terminal 20 amino acid residues are identical between the Gα15 and Gα16 and are therefore unlikely to be involved in coupling selectivity. Our data reveal two regions on Gα16 that inhibit its coupling to metabotropic glutamate receptor 8. On a three-dimensional model, both regions are found in a close proximity to the extreme C terminus of Gα16. One module comprises α4 helix, α4−β6 loop (L9 Loop), β6 sheet, and α5 helix. The other, not described previously, is located within the loop that links the N-terminal α helix to the β1 strand of the Ras-like domain of the α subunit. Coupling of Gα16 protein to the metabotropic glutamate receptor 8 is partially modulated by each module alone, whereas both modules are needed to eliminate the coupling fully. G-protein coupled receptor metabotropic glutamate receptors phospholipase C Dulbecco's modified Eagle's medium human embryonic kidney inositol phosphate hemagglutinin The specific signaling pathway of a given G-protein-coupled receptor (GPCR)1 depends on the subset of G-proteins it can activate. Transduction of signals by activated GPCR requires the interaction of the receptors with heterotrimeric G-proteins. Recent progress in defining the structure of G-proteins and site-directed mutagenesis studies of receptors and G-proteins bring increasingly more specific and precise descriptions of the contact sites between these proteins (1Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar, 2Bockaert J. Pin J.-P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1221) Google Scholar). From the G-protein α, β, and γ subunits, probably both Gα and βγ dimers contact the receptors (3Iiri T. Bell S.M. Baranski T.J. Fujita T. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 499-504Crossref PubMed Scopus (58) Google Scholar). The Gα subunit is likely to play a decisive role in discriminating between different receptor subtypes (4Savarese T.M. Fraser C.M. Biochem. J. 1992; 283: 1-19Crossref PubMed Scopus (441) Google Scholar, 5Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (526) Google Scholar, 6Wess J. FASEB J. 1997; 11: 346-354Crossref PubMed Scopus (509) Google Scholar) and also between different functional states of receptors (7Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1065) Google Scholar,8Spengler D. Waeber C. Pantaloni C. Holsboer F. Bockaert J. Seeburg P.H. Journot L. Nature. 1993; 365: 170-175Crossref PubMed Scopus (1116) Google Scholar). Several regions along the sequence of the Gα-protein are involved in the selectivity of its activation by GPCR (1Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar, 9Lichtarge O. Bourne H.R. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7507-7511Crossref PubMed Scopus (167) Google Scholar, 10Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H. Science. 1997; 275: 381-384Crossref PubMed Scopus (196) Google Scholar). The best characterized is the extreme C terminus, where residues at position −3 and −4 (the residue −1 being the last one) are decisive for coupling of Gα proteins with specific receptors (1Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar, 11Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (603) Google Scholar, 12Conklin B.R. Herzmark P. Ishida S. Voyno-Yasenetskaya T.A. Sun Y. Farfel Z. Bourne H. Mol. Pharmacol. 1996; 50: 885-890PubMed Google Scholar, 13Kostenis E. Gomeza J. Lerche C. Wess J. J. Biol. Chem. 1997; 272: 23675-23681Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 14Blahos J., II Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 15Bahia D.S. Wise A. Fanelli F. Lee M. Rees S. Milligan G. Biochemistry. 1998; 37: 11555-11562Crossref PubMed Scopus (80) Google Scholar). Residue −4 in Gαi, Gαo, and Gαt is the cysteine residue that is ADP-ribosylated by pertussis toxin, a covalent modification that prevents the interaction of the G-protein with the receptors. In the Gq family, the residue −4 is a tyrosine residue that has to be phosphorylated for an efficient coupling to the PLC-activating receptors (16Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar). Conformational changes in the C-terminal structures upon coupling to the GPCR probably cause activation of the Gα−protein. Another region determining coupling selectivity is the extreme N terminus. In the case of Gq/11-proteins, this N terminus has been shown to restrict coupling of these G-proteins to a subset of GPCRs, namely to those known to activate PLC (17Kostenis E. Zeng F.Y. Wess J. J. Biol. Chem. 1998; 273: 17886-17892Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Finally, the region between α4 and α5 helices that includes the L9 loop and β6 sheet (18Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (706) Google Scholar) is also involved in coupling selectivity probably by directly interacting with the receptors of the rhodopsin-like family (family 1 GPCRs) (10Onrust R. Herzmark P. Chi P. Garcia P.D. Lichtarge O. Kingsley C. Bourne H. Science. 1997; 275: 381-384Crossref PubMed Scopus (196) Google Scholar, 19Mazzoni M.R. Hamm H.E. J. Biol. Chem. 1996; 271: 30034-30040Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20Bae H. Anderson K. Flood L.A. Skiba N.P. Hamm H.E. Graber S.G. J. Biol. Chem. 1997; 272: 32071-32077Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Science. 1988; 241: 832-835Crossref PubMed Scopus (393) Google Scholar, 22Bockaert J. Pin J.-P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Google Scholar). The overall surface charge of a region that comprises the structures close to the C terminus is probably defining either selectivity or coupling properties in general (23Bae H. Cabrera-Vera T.M. Depree K.M. Graber S.G. Hamm H.E. J. Biol. Chem. 1999; 274: 14963-14971Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Among the various G-proteins identified so far, the Gα15subunit has unique properties. This G-protein that is found exclusively in the murine hematopoietic cell lineage shares the closest sequence similarity with the Gαq protein and activates PLCβs (24Wilkie T.M. Scherle P.A. Strathmann M.P. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10049-10053Crossref PubMed Scopus (256) Google Scholar, 25Amatruda III, T.T. Steele D.A. Slepak V.Z. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5587-5591Crossref PubMed Scopus (238) Google Scholar). In cells normally expressing this G-protein as well as in heterologous expression systems it was found that it can couple to many GPCRs, including those that naturally do not stimulate PLC. This was observed with the members of family 1 GPCRs (26Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 27Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 28Milligan G. Marshall F. Rees S. Trends Pharmacol. Sci. 1996; 17: 235-237Abstract Full Text PDF PubMed Scopus (107) Google Scholar) and the mGlu receptor family (family 3 GPCRs) (29Gomeza J. Mary S. Brabet I. Parmentier M.-L. Restituito S. Bockaert J. Pin J.-P. Mol. Pharmacol. 1996; 50: 923-930PubMed Google Scholar, 30Parmentier M.L. Joly C. Restituito S. Bockaert J. Grau Y. Pin J.P. Mol. Pharmacol. 1998; 53: 778-786Crossref PubMed Scopus (70) Google Scholar). Gα15 is therefore a G-protein that is not able to discriminate between a variety of receptors (26Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16010Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Functional characterization of the human homologue Gα16 revealed that this protein also couples to many GPCRs (27Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar) but is not as promiscuous as Gα15. It is of interest to understand what determines the lack of selectivity of Gα15 and which sequences restrict promiscuity of the Gα16 subunit. There are several examples of receptors that cannot activate Gα16 (31Arai H. Charo I.F. J. Biol. Chem. 1996; 271: 21814-21819Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 32Kuang Y. Wu Y. Jiang H. Wu D. J. Biol. Chem. 1996; 271: 3975-3978Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Among the family 3 receptors we reported that the group II metabotropic glutamate receptor mGlu2 activated both Gα15 and Gα16, whereas the group III receptors (mGlu4, -7, and -8 receptors) activated Gα15only (29Gomeza J. Mary S. Brabet I. Parmentier M.-L. Restituito S. Bockaert J. Pin J.-P. Mol. Pharmacol. 1996; 50: 923-930PubMed Google Scholar, 30Parmentier M.L. Joly C. Restituito S. Bockaert J. Grau Y. Pin J.P. Mol. Pharmacol. 1998; 53: 778-786Crossref PubMed Scopus (70) Google Scholar). Gα15 and Gα16 proteins share 85% sequence identity. Interestingly, they share identical C termini, indicating this sequence element is not responsible for their differential coupling. Such a situation appears ideal to identify other regions in these G-proteins involved in their specific interaction with GPCRs. In the present study, the coupling of both mGlu2 and mGlu8 receptors to a series of Gα15/Gα16 chimeric G-proteins was analyzed. This study revealed that amino acids within α4 helix, β6 strand, L9 loop, and α5 helix constitute one of the determinants that allows G-protein α subunit to discriminate between members of family 3 receptors, a situation corresponding to data reported for family 1 GPCRs. In addition we identified a new region within the G-protein α subunit involved in the selective recognition of group II versus group III mGlu receptors. This new region is located in the loop that connects αN helix with β1 sheet. Chemicals including glutamate were obtained from Sigma (L'Isle d'Abeau, France) unless otherwise indicated. Serum, culture media, and other solutions used for cell culture were from Life Technologies, Inc. (Cergy Pontoise, France). The plasmids expressing mGlu receptors were as described previously (14Blahos J., II Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) or modified (see below). The hemagglutinin epitope-tagged Gαq was kindly provided by Dr. Bruce Conklin (The Gladstone Institute, San Francisco, CA). The plasmids pCIS-Gα15 and pCIS-Gα16were kindly provided by Dr. M. Simon (Caltech, Los Angeles, CA). HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.) supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin, 100 units/ml final). Electroporation was performed in a total volume of 300 μl with 10 μg of carrier DNA, mGlu2, or mGlu8 receptor plasmid DNA (2 and 4 μg, respectively), wild-type or mutated Gα subunit plasmid DNA (1 μg), and 10 million cells in electroporation buffer (K2HPO4, 50 mm; CH3COOK, 20 mm; KOH, 20 mm). After electroporation (260 V, 960 microfarads, Bio-Rad gene pulser electroporator), cells were resuspended in DMEM supplemented with 10% fetal calf serum and antibiotics and split in 12-well clusters (Falcon, Paris, France) (10 million cells per cluster) previously coated with poly-l-ornithine (15 μg/ml; M r40,000), (Sigma, Paris, France) to favor adhesion of the cells. The procedure used for the determination of IP accumulation in transfected cells was adapted from previously published methods (33Berridge M. Dawson R. Downes C. Heslop J. Irvine R. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1540) Google Scholar, 34Bone E. Fretten P. Palmer S. Kirk C. Michell R. Biochem. J. 1984; 221: 803-811Crossref PubMed Scopus (181) Google Scholar). Cells were washed 2–3 h after electroporation and incubated for 14 h in DMEM-glutamax-I (Life Technologies, Inc., Paris, France) containing 1 μCi/ml myo-[3H]inositol (23.4 Ci/mol), (PerkinElmer Life Sciences, Paris, France). Cells were then washed three times and incubated for 1–2 h at 37 °C in 1 ml of Hepes buffer saline (NaCl, 146 mm; KCl, 4.2 mm;MgCl2, 0.5 mm; glucose, 0.1%; Hepes, 20 mm, pH 7.4) supplemented with 1 unit/ml glutamate pyruvate transaminase (Roche Molecular Biochemicals, Meylan, France) and 2 mm pyruvate (Sigma, Lisle d'Abeau, France). Cells were then washed again twice with the same buffer, and LiCl was added to a final concentration of 10 mm. The agonist was applied 5 min later and left for 30 min. The reaction was stopped by replacing the incubation medium with 0.5 ml of perchloric acid (5%), on ice. Supernatants were recovered, and the IPs were purified on Dowex columns. Total radioactivity remaining in the membrane fraction was counted after treatment with 10% Triton X-100, 0.1 n NaOH for 30 min and used as standard. Results are expressed as the amount of IP produced over the radioactivity present in the membranes. The dose-response curves were fitted according to the equationy = ((y max −y min)/1 + (x/EC50)n) + y minusing the kaleidagraph program (Abelbeck software). The anti-Gα15/16 antibodies were raised in rabbits against a synthetic peptide corresponding to the last 10 amino acid residues that are common for both the Gα15 and Gα16. The antiserum was tested in several dilutions (data not shown). The 1:2000 dilution gave optimal results. ECL chemiluminescence system was used to stain the secondary antibodies (Amersham Pharmacia Biotech, Paris, France). The chimeric GαqX5 and GαqX6 were constructed using a unique Xba I site of the cDNA encoding the Gαq-HA-tagged protein. The Xba I sites in the C-terminal part of Gα15 and Gα16 were introduced by silent mutagenesis into the consensus sequence Met-Asp-Leu. This resulted in the expression of chimeric proteins that were detectable by both the HA antibodies and our anti-Gα15/16 C-terminal antibody. Cells were transfected and treated as described above. Cells cultured in one well were harvested for immunodetection, and the rest of the cells were used for IP assay. Protein samples (10 μg per lane) were separated using SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane. Prior to the immunoblotting, total protein on the membranes was visualized with Ponceau S to confirm that the same amounts of protein were loaded on the gel. The G-proteins were detected using primary monoclonal antibodies against hemagglutinin-epitope (generous gift of Dr. B. Mouillac, Montpellier, France) or the newly described polyclonal anti-Gα15/16 C-terminal antibodies. The His6-tagged receptors were detected using specific antibodies recognizing the MRGS-His epitope (Qiagen, Paris, France). We have noticed considerable lower expression levels of the human Gα16 protein than those of Gα15 in transfected HEK 293 cells. To get higher expression levels of Gα16, we removed most of the 5′-untranslated region and introduced a Kozak sequence corresponding to that of Gα15 by polymerase chain reaction using the unique Eco RI-Pst I sites. These modifications were sufficient to raise the Gα16 protein expression obtained with this new Gα16PLUS construct to levels comparable with those reached by Gα15. This construct was used through out the study and is referred as to Gα16 in this text. Silent mutagenesis approach using Pfu-based QuickChangeTMtechnique (Stratagene) was used to introduce a Bam HI, aPst I, and a Bam HI restriction site at the positions indicated in Fig. 2 as sites 1, 2, and 4, respectively. Together with the existing Xba I site (position 3 in Figs.Figure 2, Figure 3, Figure 4) and the Hin dIII site in the 3′-untranslated region, these sites were used to construct the chimeric proteins described in Figs. 3 and 4 using conventional subcloning techniques. The same approach was used for making the point mutations.Figure 3a, chimeric proteins between Gα15 and Gα16 at the α4-α5 region were constructed by swapping corresponding portions using silent restriction sites or by introducing point mutations. b, immunoblot analysis of Gα15 and Gα16 proteins and the chimeras and the point mutations expressed in HEK293 cells, stained with the anti-Gα16 antibodies. c, differential coupling of the Gα15, Gα16, and their reciprocal chimeras to mGlu2 and mGlu8 receptors. Basal (open bars) and 1 mm glutamate-induced (dark bars) IP formations were determined in HEK 293 cells coexpressing the mGlu2 (a) or mGlu8 (b) receptors with the G-protein α subunits and their chimeras. The new constructs of the Gα16 proteins with augmented expression levels were used. Data represent the radioactivity in the IP fraction divided by the total radioactivity in the membranes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4a, chimeric proteins between Gα15 and Gα16 at the αN−β1 region were constructed by swapping corresponding portions using silent restriction sites. b, immunoblot analysis of Gα15 and Gα16 proteins and the chimeras and the point mutations expressed in HEK293 cells, stained with the anti-Gα16antibodies. c, differential coupling of the Gα15, Gα16, and their reciprocal chimeras to mGlu2 and mGlu8 receptors. Basal (open bars) and 1 mm glutamate-induced (dark bars) IP formations were determined in HEK 293 cells coexpressing the mGlu2 (a) or mGlu8 (b) receptors with the G-protein α subunits and their chimeras. The new constructs of the Gα16 proteins with augmented expression levels were used. Data represent the radioactivity in the IP fraction divided by the total radioactivity in the membranes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The mGlu2 and mGlu8 receptors were tagged with MRGS-His6 epitope at the C termini as in our previous studies (14Blahos J., II Mary S. Perroy J. de Colle C. Brabet I. Bockaert J. Pin J.P. J. Biol. Chem. 1998; 273: 25765-25769Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Briefly, the pRK5 plasmid containing the sequence encoding the epitope MRGS-His6flanked by a unique Nhe I site at its 5′ end, and an in-frame TAA stop codon at its 3′ end was used. The receptors coding sequences were then introduced by polymerase chain reaction using Nhe I restriction site so that the stop codon was replaced by alanine followed by serine and the MRGS-His6 epitope (where MRGS indicates: methionine, arginine, glycine, serine). The three-dimensional model of the Gα15 subunit was constructed by homology using the coordinates of different G-protein α subunits in their GDP form obtained by x-ray crystallography. These include transducin in its GDP plus Mg2+ form, the αt/αi chimeric protein in its heterotrimeric form with βtγt (35Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1046) Google Scholar), the αi1 in its GDP plus Mg2+ form (36Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (267) Google Scholar), and in its heterotrimeric complex with β1γ2 (37Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1008) Google Scholar) (Protein Data Bank accession numbers 1TAG, 1GOT, 1GDD, and 1GP2, respectively). Some structural elements were deleted due to their inappropriate folding in the expected heterotrimeric form bound to the receptor. These deletions include the N-terminal α helix (the 24 N-terminal residues of the resolved structure) and the extreme C terminus (4 residues) of the αi1 in its GDP plus Mg2+form. Some constraints were also imposed during the modeling process of Gα15: an α helical secondary structure was imposed to residues 7–39, 305–325, 330–334, and 349–370. The sequence alignment and three-dimensional models were generated using the program Modeler (38Sali A. Blundell T. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10447) Google Scholar) in the Insight-II environment (Molecular Simulation Inc., San Diego, CA) on a Silicon Graphic R10000 O2 work station. A statistical evaluation of the three-dimensional model was performed using the Verify 3D algorithm (39Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2539) Google Scholar) and the Verify 3D Structure Evaluation Server. The model giving the best one-dimensional/three-dimensional scores was selected and subjected to energy minimization using the program Discover 2.9.7 (Molecular Simulation Inc.) and the CVFF force field. The extreme C terminus of the Gα15 subunit was modeled manually according to the resolved structure of the 10 C-terminal residues of transducin bound with rhodopsin (40Kisselev O.G. Kao J. Ponder J.W. Fann Y.C. Gautam N. Marshall G.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4270-4275Crossref PubMed Scopus (163) Google Scholar). The β1γ2 subunits were added after superposition of the Gα15 model with the Gαi1 subunit in its heterotrimeric complex. The mGlu2 and mGlu8 receptors couple to the Gi type of G-proteins and do not activate PLC pathway when expressed alone in HEK 293 cells. Because Gα15 and Gα16 activate PLCs, their efficient coupling to these mGlu receptors can easily be assayed by measuring the capability of glutamate to activate the PLC pathway in cells expressing both the receptor and one of these G-proteins. As mentioned in the introduction, we reported previously that glutamate activated PLC in HEK 293 cells coexpressing mGlu2 receptors and either Gα15 or Gα16. In contrast, glutamate activated IP formation in cells expressing mGlu8 receptors and Gα15 but not in those coexpressing this receptor with Gα16. To conclude that Gα16can be activated by mGlu2 but not mGlu8 receptors, it was necessary to verify that both G-proteins were expressed at similar levels. We generated a new polyclonal antibody, called anti- Gα15/16AB that recognizes equally well both Gα15 and Gα16 (Fig. 1). The antiserum was raised in rabbits immunized with a peptide corresponding to the extreme C-terminal region (10 amino acid residues) common to both proteins (Figs. 1 and 2). On Western blots, anti-Gα15/16AB recognized a single major band that migrated at a velocity appropriate for these G-proteins (Fig.1). This band was detected only in lanes where membrane proteins from HEK 293 cells expressing proteins possessing C termini of Gα15 or Gα16 were separated. No additional major band was detected, and no bands were detected in lanes where extracts from cells expressing other Gα-protein subunits were loaded (data not shown). To verify that anti-Gα15/16AB was recognizing both G-proteins at the same extent, we expressed chimeric proteins corresponding to the HA-tagged Gαq protein with its last 51 C-terminal residues replaced by their corresponding 63 residues of either Gα15 or Gα16 (chimeras GαqX5 and GαqX6, see Fig. 1). Those were expressed in HEK 293 cells at the same levels, as shown with the HA specific monoclonal antibody. When blots were reprobed with the new antibody anti-Gα15/16AB, the bands corresponding to chimeras bearing Gα15 and Gα16 C termini were stained with similar intensity (Fig. 1). Employing the anti- Gα15/16AB antibody on immunoblots, we noticed that the level of expression of Gα16 was lower than that of Gα15 in cells transfected with the original vectors (data not shown). We therefore replaced the 5′-untranslated region of Gα16 cDNA, including the Kozak sequence with corresponding sequence from of Gα15 coding cDNA (see ”Experimental Procedures“). This modification was found to raise the expression of Gα16 in transiently transfected HEK 293 cells to levels comparable with those of Gα15 (Figs. 1and 3 b). Using the expression plasmids described above, we examined the coupling of mGlu2 and mGlu8 receptors to both Gα15 and the modified Gα16. As shown in Fig. 3 a, glutamate stimulated IP formation in cells coexpressing mGlu2 receptors with either Gα15 or Gα16 , an effect that is dose-dependent (Fig.6). Using the new Gα16 vector, the maximal glutamate effect was found to be slightly lower than that obtained with Gα15 in cells expressing mGlu2 receptors, whereas it was 50% smaller when the original plasmid was used (29Gomeza J. Mary S. Brabet I. Parmentier M.-L. Restituito S. Bockaert J. Pin J.-P. Mol. Pharmacol. 1996; 50: 923-930PubMed Google Scholar, 30Parmentier M.L. Joly C. Restituito S. Bockaert J. Grau Y. Pin J.P. Mol. Pharmacol. 1998; 53: 778-786Crossref PubMed Scopus (70) Google Scholar). This is in agreement with the increased protein expression levels of Gα16 that resulted from the modification of the coding plasmid. In cells expressing mGlu8 receptors, a large increase in basal and glutamate-induced IP formation was detected when coexpressed with Gα15, but no change was observed with Gα16. All the glutamate effects were dose-dependent and the calculated EC50 values (for mGlu2 receptor-Gα15 pair the EC50 for glutamate was 8.2 ± 0.8 μm; mGlu2 receptor with Gα16 results in EC50 8.0 ± 0.3 μm glutamate, and mGlu8 receptor coexpressed with Gα15 determined EC50 for glutamate was 9.3 ± 1.6 μm) were close to those determined with these receptors using other assays (41Pin J.-P. de Colle C. Bessis A.-S. Ascher F. Eur. J. Pharmacol. 2000; 394: 17-26Crossref PubMed Scopus (30) Google Scholar). It was important to establish that the expression levels of both the receptors and the G-proteins are similar at their different combinations. At the receptor level this was confirmed by using His6-tagged receptors (data not shown). These data further confirm the discriminative coupling of Gα16 to mGlu2 and mGlu8 receptors. To identify the putative regions on the G-protein that cause the differential coupling of Gα15 and Gα16 to mGlu8 receptor, we constructed a series of Gα15/Gα16 chimeras (see panel a in Figs. Figure 3, Figure 4, Figure 5). When transiently transfected into HEK 293 cells, all chimeras were expressed at levels similar to those of the original proteins as revealed by the antiserum anti- Gα15/16AB (panel b in Figs. Figure 3, Figure 4, Figure 5). Moreover, all constructs did couple well to mGlu2 receptor, as they all allowed an effective coupling of this receptor to IP formation upon glutamate application, showing that they were all functional. Coupling of each single constructs with the receptors are shown in Figs. Figure 3, Figure 4, Figure 5(panel c). Chimera Gα15/16C1, which corresponds to Gα15 with the C-terminal 44 residues from Gα16 (Fig. 3 a), showed a smaller response to glutamate as compared with wild-type Gα15 (Fig.3 c). This indicates that the C-terminal portion of Gα16 contains a site that decreases G-protein coupling efficacy to mGlu8 receptors (Fig. 3 c). Progressive exchange of Gα15 C terminus with corresponding sequences of Gα16, as in chimera Gα15/16C2, resulted in a more pronounced decrease in coupling efficiency to mGluR8 (Fig.3 c). The converse chimera Gα15/16C3, which corresponds to Gα16 with the C-terminal 44 residues of Gα15, was activated by mGlu8 receptors, although the glutamate response was smaller than that obtained with Gα15 (Fig. 3 b). The module in Gα16 responsible for discriminating between the two receptors is most likely a region that includes α4 helix, L9 loop, the β6 sheet, and the α5 (residues 331–354). Residues that are different between Gα15 and Gα16 is these regions are: 10 in the α4 helix and N-terminal part of L9 loop,
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