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• W2068201543 abstract "Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs. Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs. G protein-coupled receptors (GPCRs), 3The abbreviations used are: GPCRs, G protein-coupled receptors; TMD, transmembrane domain; mGluR, metabotropic glutamate receptor; VFT, Venus flytrap; FRET, fluorescence resonance energy transfer; CC, coiled-coil; HA, hemagglutin; ELISAs, enzyme-linked immunosorbent assays; GABAB, γ-aminobutyric acid type B. which compose the largest family of mammalian genes (∼1000 members), are involved in a vast variety of physiological and pathological processes and represent the target of almost 50% of all modern drugs (1Howard A.D. McAllister G. Feighner S.D. Liu Q. Nargund R.P. Van der Ploeg L.H. Patchett A.A. Trends Pharmacol. Sci. 2001; 22: 132-140Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The common structural feature of all GPCRs is a transmembrane domain (TMD) made of seven transmembrane segments (TM1–TM7). Despite the vast variety of ligands and the low sequence similarity between GPCRs from various classes, their activation results from similar conformational changes in their TMDs (2Schwartz T.W. Frimurer T.M. Holst B. Rosenkilde M.M. Elling C.E. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 481-519Crossref PubMed Scopus (349) Google Scholar, 3Sheikh S.P. Vilardarga J.P. Baranski T.J. Lichtarge O. Iiri T. Meng E.C. Nissenson R.A. Bourne H.R. J. Biol. Chem. 1999; 274: 17033-17041Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 4Binet V. Duthey B. Lecaillon J. Vol C. Quoyer J. Labesse G. Pin J.P. Prézeau L. J. Biol. Chem. 2007; 282: 12154-12163Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In particular, movement of TM6 likely opens a crevice allowing interaction with the C terminus of the G protein α-subunit, triggering its activation (5Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (530) Google Scholar). Many GPCRs have been shown to form dimers, but the functional role of this remains elusive (6Angers S. Salahpour A. Bouvier M. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 409-435Crossref PubMed Scopus (517) Google Scholar, 7Milligan G. Mol. Pharmacol. 2004; 66: 1-7Crossref PubMed Scopus (421) Google Scholar, 8Chabre M. le Maire M. Biochemistry. 2005; 44: 9395-9403Crossref PubMed Scopus (193) Google Scholar). Although a GPCR monomer is sufficient to activate a G protein (8Chabre M. le Maire M. Biochemistry. 2005; 44: 9395-9403Crossref PubMed Scopus (193) Google Scholar, 9Bayburt T.H. Leitz A.J. Xie G. Oprian D.D. Sligar S.G. J. Biol. Chem. 2007; 282: 14875-14881Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 10Whorton M.R. Bokoch M.P. Rasmussen S.G. Huang B. Zare R.N. Kobilka B. Sunahara R.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7682-7687Crossref PubMed Scopus (568) Google Scholar, 11Ernst O.P. Gramse V. Kolbe M. Hofmann K.P. Heck M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10859-10864Crossref PubMed Scopus (189) Google Scholar, 12White J.F. Grodnitzky J. Louis J.M. Trinh L.B. Shiloach J. Gutierrez J. Northup J.K. Grisshammer R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12199-12204Crossref PubMed Scopus (131) Google Scholar), it has been proposed that dimerization of GPCRs may facilitate their activation, i.e. that allosteric interactions between the protomers, through changes at the dimerization interface or a larger scale reorientation of the two subunits, may contribute to stabilization of the active conformation (13Breitwieser G.E. Circ. Res. 2004; 94: 17-27Crossref PubMed Scopus (170) Google Scholar, 14Guo W. Shi L. Filizola M. Weinstein H. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17495-17500Crossref PubMed Scopus (252) Google Scholar, 15Ridge K.D. Palczewski K. J. Biol. Chem. 2007; 282: 9297-9301Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). To further elucidate a possible role of an intersubunit rearrangement in the activation of a dimeric GPCR, we chose a metabotropic glutamate receptor (mGluR) as a model. These GPCRs are clearly established as constitutive dimers, the two subunits being linked by a disulfide bridge (16Romano C. Yang W.L. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Moreover, glutamate and its analogs do not bind to the TMD of an mGluR, but to a distinct extracellular domain called the Venus flytrap (VFT) domain (17Pin J.P. Galvez T. Prézeau L. Pharmacol. Ther. 2003; 98: 325-354Crossref PubMed Scopus (555) Google Scholar). This separation may help to dissect the relationship between agonist binding and activation within these GPCRs. Agonist binding stabilizes a closed conformation of the VFT domain (Fig. 1) (18Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1118) Google Scholar, 19Muto T. Tsuchiya D. Morikawa K. Jingami H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3759-3764Crossref PubMed Scopus (313) Google Scholar), which is both necessary (20Bessis A.S. Rondard P. Gaven F. Brabet I. Triballeau N. Prézeau L. Acher F. Pin J.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11097-11102Crossref PubMed Scopus (109) Google Scholar) and sufficient (21Kniazeff J. Saintot P.P. Goudet C. Liu J. Charnet A. Guillon G. Pin J.P. J. Neurosci. 2004; 24: 370-377Crossref PubMed Scopus (76) Google Scholar) for the activation of a class C GPCR. How the VFT domain closure is in turn transduced into TMD activation is, however, yet unknown. Two mechanisms, not necessarily exclusive, have been proposed. The first model (Fig. 2A) proposes that the closed VFT domain directly stabilizes the active conformation of the TMD of the same subunit (22Pin J.P. De Colle C. Bessis A.S. Acher F. Eur. J. Pharmacol. 1999; 375: 277-294Crossref PubMed Scopus (146) Google Scholar, 23Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1233) Google Scholar). But agonist binding may also induce a relative reorientation of the two VFT domains (Fig. 1) (18Kunishima N. Shimada Y. Tsuji Y. Sato T. Yamamoto M. Kumasaka T. Nakanishi S. Jingami H. Morikawa K. Nature. 2000; 407: 971-977Crossref PubMed Scopus (1118) Google Scholar), and a second model (Fig. 2B) thus proposes that this may in turn yield an activating rearrangement of the two TMDs (24Pin J.P. Kniazeff J. Liu J. Binet V. Goudet C. Rondard P. Prézeau L. FEBS J. 2005; 272: 2947-2955Crossref PubMed Scopus (142) Google Scholar, 25Kubo Y. Tateyama M. Curr. Opin. Neurobiol. 2005; 15: 289-295Crossref PubMed Scopus (39) Google Scholar). Recent fluorescence resonance energy transfer (FRET) studies are indeed consistent with a glutamate-induced reorientation of the two TMDs within an mGluR dimer (26Tateyama M. Abe H. Nakata H. Saito O. Kubo Y. Nat. Struct. Mol. Biol. 2004; 11: 637-642Crossref PubMed Scopus (155) Google Scholar). However, this could also simply be a “side effect” not necessarily involved in the activation process. Moreover, in contrast to mGlu1, the relative orientation of the VFT domains of mGlu3 appears not to be influenced by agonist binding (Fig. 1) (19Muto T. Tsuchiya D. Morikawa K. Jingami H. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3759-3764Crossref PubMed Scopus (313) Google Scholar), further putting into question this second proposal (Fig. 2B). Notably, only in the second model (Fig. 2B), but not the first (Fig. 2A), agonist binding to one VFT domain will not only cis-activate the TMD of the same, but also equally well trans-activate the TMD of the other subunit. Using a new system to control the subunit composition and thereby introduce different mutations specifically into either protomer, we demonstrate here that both cis- and trans-activation within an mGluR dimer occur with the same probability. We show moreover that this trans-activation is not indirect via the other VFT domain, via the other TMD, or by a domain “swap” between the two subunits (see also Fig. 5). Our data therefore demonstrate that an agonist-induced intersubunit rearrangement can indeed be responsible for the activation of a dimeric GPCR, a mechanism already well accepted for tyrosine kinase and guanylate cyclase receptors (27Livnah O. Stura E.A. Middleton S.A. Johnson D.L. Jolliffe L.K. Wilson I.A. Science. 1999; 283: 987-990Crossref PubMed Scopus (540) Google Scholar, 28Remy I. Wilson I.A. Michnick S.W. Science. 1999; 283: 990-993Crossref PubMed Scopus (539) Google Scholar, 29Moriki T. Maruyama H. Maruyama I.N. J. Mol. Biol. 2001; 311: 1011-1026Crossref PubMed Scopus (277) Google Scholar, 30den Akker F. J. Mol. Biol. 2001; 311: 923-937Crossref PubMed Scopus (57) Google Scholar). Plasmids—Plasmids for mGlu5 were either as described previously (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar, 32Goudet C. Kniazeff J. Hlavackova V. Malhaire F. Maurel D. Acher F. Blahos J. Prézeau L. Pin J.P. J. Biol. Chem. 2005; 280: 24380-24385Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 33Rondard P. Liu J. Huang S. Malhaire F. Vol C. Pinault A. Labesse G. Pin J.P. J. Biol. Chem. 2006; 281: 24653-24661Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 34Liu J. Maurel D. Etzol S. Brabet I. Ansanay H. Pin J.P. Rondard P. J. Biol. Chem. 2004; 279: 15824-15830Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) or, based on plasmids described in these references, newly constructed by standard molecular biology techniques (PCR, site-directed mutagenesis, subcloning) as described (35Brock C. Boudier L. Maurel D. Blahos J. Pin J.P. Mol. Biol. Cell. 2005; 16: 5572-5578Crossref PubMed Scopus (64) Google Scholar). Compared with the previously described C2 constructs (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar), the last 120 amino acid residues were replaced with the sequence KKTN in the C2KKXX constructs, right after the coiled-coil (CC) domain. Similarly, in some of the C1 constructs, we also replaced the last 39 residues with the sequence KKTN, right after the CC domain. However, we found no significant functional differences between the C1 and C1KKXX constructs (supplemental Fig. B). Both types of constructs are therefore collectively referred to as C1 throughout this work. The FLAG-V2 plasmid was a generous gift from L. Albizu. CD4 cDNA was a generous gift from Dr. B. Schwappach. Cell Culture and Transfection—Cell culture and transfection of human embryonic kidney 293 cells were performed as described (32Goudet C. Kniazeff J. Hlavackova V. Malhaire F. Maurel D. Acher F. Blahos J. Prézeau L. Pin J.P. J. Biol. Chem. 2005; 280: 24380-24385Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). To avoid any mGluR activation due to ambient glutamate, a plasmid encoding the high affinity glutamate transporter EAAC1 was always cotransfected, and the cells were incubated in glutamate-free medium for at least 4 h before the experiments. All experiments were carried out 1 day after transfection. Intracellular Ca2+ Release—Intracellular Ca2+ release was measured as described (36Goudet C. Gaven F. Kniazeff J. Vol C. Liu J. Cohen-Gonsaud M. Acher F. Prézeau L. Pin J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 378-383Crossref PubMed Scopus (179) Google Scholar). In brief, cells were preincubated for 1 h with the Ca2+-sensitive Fluo-4 acetoxymethyl ester (Invitrogen). The fluorescence signals (excitation at 485 nm and emission at 525 nm) were then measured for 60 s (Flex-Station, Molecular Devices). Quis (quisqualate; Tocris Bioscience) was added after the first 20 s. The Ca2+ response is given as the Quis-stimulated fluorescence increase. Sigmoidal concentration-response curves were fitted using GraphPad Prism. FRET—FRET measurements were conducted as described (37Maurel D. Kniazeff J. Mathis G. Trinquet E. Pin J.P. Ansanay H. Anal. Biochem. 2004; 329: 253-262Crossref PubMed Scopus (115) Google Scholar). In brief, cells were incubated with Eu3+ cryptate pryidine-bipyridine-labeled anti-hemagglutin (HA; donor) and Alexa Fluor 647-labeled anti-FLAG (acceptor) antibodies or with the donor antibody only (negative control). Following excitation at 337 nm, the emission at 665 nm was measured (RUBYstar, BMG Labtech). The FRET signal was calculated as Δ665, i.e. the difference between the emissions at 665 nm in the presence and absence of the acceptor. The fluorophore-labeled antibodies were provided by Cisbio International (Bagnols-sur-Cèze, France). Enzyme-linked Immunosorbent Assays (ELISAs)—ELISAs using anti-HA antibody were performed as described previously (35Brock C. Boudier L. Maurel D. Blahos J. Pin J.P. Mol. Biol. Cell. 2005; 16: 5572-5578Crossref PubMed Scopus (64) Google Scholar). The same protocol was applied for ELISAs using anti-FLAG antibody, but with 1 μg/ml anti-FLAG antibody M2 (Sigma) and 0.5 μg/ml horseradish peroxidase-conjugated anti-mouse antibody (Amersham Biosciences) instead. “Heterodimerization” of an mGluR—To study the intersubunit interactions within a dimeric receptor, one first has to control its subunit composition. To this end, we previously transferred the “quality control” system of the heterodimeric γ-aminobutyric acid type B (GABAB) receptor (35Brock C. Boudier L. Maurel D. Blahos J. Pin J.P. Mol. Biol. Cell. 2005; 16: 5572-5578Crossref PubMed Scopus (64) Google Scholar, 38Margeta-Mitrovic M. Jan Y.N. Jan L.Y. Neuron. 2000; 27: 97-106Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 39Pagano A. Rovelli G. Mosbacher J. Lohmann T. Duthey B. Stauffer D. Ristig D. Schuler V. Meigel I. Lampert C. Stein T. Prézeau L. Blahos J. Pin J.P. Froestl W. Kuhn R. Heid J. Kaupmann K. Bettler B. J. Neurosci. 2001; 21: 1189-1202Crossref PubMed Google Scholar, 40Calver A.R. Robbins M.J. Cosio C. Rice S.Q. Babbs A.J. Hirst W.D. Boyfield I. Wood M.D. Russell R.B. Price G.W. Couve A. Moss S.J. Pangalos M.N. J. Neurosci. 2001; 21: 1203-1210Crossref PubMed Google Scholar) to the homodimeric metabotropic glutamate receptor mGlu5 (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar). Replacing its C-terminal tail with that of the GABAB1 subunit (C1) (supplemental Fig. A) generates chimeric mGlu5-C1, which is retained inside the cell, unless it forms a heterodimer with chimeric mGlu5-C2, carrying the C-terminal tail of the GABAB2 subunit (C2). In fact, the intracellular retention signal (the sequence RSR) of the C1 part becomes masked by the specific interaction of the adjacent CC domain with the CC domain in the C2 part. However, the mGlu5-C2 chimera, which does not carry a retention signal, could still reach the cell surface and function as a homodimer. To solve this problem, we generated an mGlu5-C2KKXX construct, in which we replaced the extreme C-terminal tail of the C2 part with an intracellular retention signal (KKXX), right after the CC domain (supplemental Fig. A). As a consequence, both mGlu5-C1 and mGlu5-C2KKXX are retained inside the cell when expressed alone (Fig. 3). However, both can reach the cell surface when they are coexpressed in the same cells, demonstrating that the CC domain interaction results in efficient mutual masking of the two retention signals within the heterodimer. Notably, this “heterodimerized” mGlu5 is functional because the Ca2+ response evoked by stimulation with the glutamate analog Quis was similar between wild-type mGlu5 and an mGlu5-C1/C2KKXX heterodimer (Fig. 3C). Moreover, the C1/C2KKXX parts did not affect the symmetry within the dimer because the Quis-stimulated Ca2+ response of such heterodimerized receptors carrying the G protein-uncoupling mutation F767S (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar) in either the C1 or C2KKXX subunit was similar (Fig. 3D). mGlu5 Is a Dimer, Not a Higher Order Oligomer—At least some GPCRs may form not only dimers, but also higher order oligomers (41Fotiadis D. Liang Y. Filipek S. Saperstein D.A. Engel A. Palczewski K. Nature. 2003; 421: 127-128Crossref PubMed Scopus (661) Google Scholar, 42Lopez-Gimenez J.F. Canals M. Pediani J.D. Milligan G. Mol. Pharmacol. 2007; 71: 1015-1029Crossref PubMed Scopus (153) Google Scholar). If this occurs also for mGluRs, this would substantially complicate our analysis of their intramolecular signal transduction. Our new system to control the subunit composition of cell surface-expressed mGluR dimers (Fig. 3) now permitted us to test this possibility. We first used a FRET approach using donor and acceptor fluorophore-labeled anti-HA and anti-FLAG antibodies, respectively, to test a possible physical interaction between two distinct mGlu5 dimers at the cell surface (37Maurel D. Kniazeff J. Mathis G. Trinquet E. Pin J.P. Ansanay H. Anal. Biochem. 2004; 329: 253-262Crossref PubMed Scopus (115) Google Scholar). We coexpressed mGlu5-C1/C2KKXX “heterodimers” with only the C2KKXX subunits carrying an HA or a FLAG tag at their extracellular N termini (Fig. 4A, middle). To reach the cell surface and become accessible to the antibodies, any tagged C2KKXX subunit must be in a dimer with an untagged C1 subunit (and not with another tagged C2KKXX subunit). Only a very low FRET signal was measured under these conditions (Fig. 4A, middle), similar to that obtained with the vasopressin V2 receptor as a negative control (Fig. 4A, right). A large FRET signal was detected, however, with the positive control with both subunits (C1 and C2KKXX) of the same dimer being tagged (Fig. 4A, left). These differences in FRET were not due to different cell-surface expression levels, which were controlled in parallel by ELISA. Similar results were obtained with the inverse combination of the C1 and C2KKXX subunits (supplemental Fig. D). We next tested a possible functional complementation between two nonfunctional mGlu5 dimers. An mGlu5-C1/C2KKXX dimer with both TMDs carrying the mutation F767S, which abolishes G protein activation (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar), was coexpressed with mGlu5-YADA, which is not activated by glutamate or its analogs due to a mutation in the VFT domain (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar). To reach the cell surface, the C1 and C2KKXX subunits both carrying the F767S mutation must necessarily be part of the same dimer. No functional complementation was observed between this dimer and mGlu5-YADA (Fig. 4B, right), despite the fact that, in line with our previous findings (31Kniazeff J. Bessis A.S. Maurel D. Ansanay H. Prézeau L. Pin J.P. Nat. Struct. Mol. Biol. 2004; 11: 706-713Crossref PubMed Scopus (242) Google Scholar), mGlu5-F767S was principally capable of trans-activating mGlu5-YADA (Fig. 4B, middle). Again, these differences cannot be accounted for by differences in the cell-surface expression levels, which were controlled in parallel by ELISA. Thus, no functional complementation can be observed between two different mGlu5 dimers. In conclusion, these data reveal that mGlu5 dimers neither physically nor functionally associate into larger higher order oligomers. “Direct” trans-Activation?—The observed trans-activation between mGlu5-F767S and mGlu5-YADA (Fig. 4B, middle) is perfectly in line with the model of an agonist-induced intersubunit rearrangement as the activating mechanism of an mGluR dimer (Fig. 2B). However, other possibilities also exist. First, this trans-activation could be due to a swap between the VFT domains and the TMDs of the two subunits (Fig. 5A). Second, this trans-activation could be indirect via the other VFT domain, i.e. the mutated VFT domain might nonetheless, through interaction with the agonist-bound VFT domain, become stabilized in an “active” (closed) conformation, in turn activating its TMD (Fig. 5B). Third, the observed trans-activation might also be indirect via the other TMD (Fig. 5C). The aim of the subsequent experiments was to test these possibilities. trans-Activation Is Not by Swap between the VFT Domains and TMDs within an mGluR Dimer—For several glycoprotein hormone GPCRs, it has been demonstrated that a receptor with an inactivated exodomain (extracellular hormone-binding domain) can be trans-activated by a hormone-bound exodomain devoid of the receptor's endodomain (TMD + cytosolic tail) (43Jeoung M. Lee C. Ji I. Ji T.H. Mol. Cell. Endocrinol. 2007; 260 (262): 137-143Crossref PubMed Scopus (24) Google Scholar). This suggests that the hormone-bound “isolated” exodomain may swap with the inactivated one to activate that subunit's endodomain. Could such a swap also occur between the VFT domains and TMDs within an mGluR dimer (Fig. 5A)? To answer this question, we tested whether VFT domain-mutated mGlu5-YADA could also be trans-activated by mGlu5-(1–614), devoid of its TMD (34Liu J. Maurel D. Etzol S. Brabet I. Ansanay H. Pin J.P. Rondard P. J. Biol. Chem. 2004; 279: 15824-15830Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). In this construct, only TM1 was left as a “membrane anchor”, with TM2–TM7 and the cytosolic tail missing. In contrast to TMD-mutated mGlu5-F767S, this TMD-deficient mGlu5-(1–614) did not trans-activate mGlu5-YADA (Fig. 6). This was not due to a lack of heterodimerization, as controlled by FRET (Fig. 6, lower). Thus, in contrast to what has been proposed for the exo- and endodomains of a glycoprotein hormone receptor (43Jeoung M. Lee C. Ji I. Ji T.H. Mol. Cell. Endocrinol. 2007; 260 (262): 137-143Crossref PubMed Scopus (24) Google Scholar), there is no swap between the two VFT domains and TMDs (Fig. 5A), and such a mechanism can therefore not account for the observed trans-activation within an mGluR dimer. Uncoupling a VFT Domain from Its TMD—To further clarify how agonist binding to a VFT domain leads to activation of a TMD of an mGlu5 dimer, our idea was to introduce another mutation, C240E, which functionally uncouples the VFT domain from the TMD (33Rondard P. Liu J. Huang S. Malhaire F. Vol C. Pinault A. Labesse G. Pin J.P. J. Biol. Chem. 2006; 281: 24653-24661Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), into only one of the two subunits. The combination with other mutations should then allow us to study the functional interactions between the different domains within such a dimer. We first verified that an mGlu5-C1/C2KKXX dimer carrying the C240E mutation in only one subunit is still functional (Fig. 7). Indeed, such a receptor was still activated by Quis, although with a reduced maximal response as compared with the receptor not carrying this mutation at similar cell-surface expression levels. Because, at the expression levels used in this study, the maximal Ca2+ response was directly proportional to the amount of mGlu5 at the cell surface (supplemental Fig. C), this reduced maximal response (at similar cell-surface expression levels) of mGlu5 with one “uncoupled” VFT domain therefore indicates a reduced maximal activity. Two different explanations can be proposed. The observed resting activity may simply reflect the activity of the subunit not carrying the C240E mutation, resulting from intrasubunit transduction (Fig. 2A). Alternatively, the decreased maximal response could also reflect that the disulfide bond involving Cys240 (33Rondard P. Liu J. Huang S. Malhaire F. Vol C. Pinault A. Labesse G. Pin J.P. J. Biol. Chem. 2006; 281: 24653-24661Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) is important for transmitting the relative movement of the two VFT domains to the TMDs (Fig. 2B), which may be less efficient when one disulfide bond is missing. Most important, however, the fact that this disulfide bond is not mandatory in both subunits opens the possibility to experimentally refine the route of intramolecular signal transduction within an mGluR dimer. trans-Activation Is Not via the Other VFT Domain—Accordingly, we next tested a possible trans-activation between mGlu5-F767S and mGlu5-C240E. Indeed, Quis stimulation of an mGlu5 dimer composed of these two subunits still elicited a Ca2+ response (Fig. 8). This demonstrates a trans-activation of one TMD (of the C240E mutant) by the VFT domain of the other subunit (the F767S mutant) in a way not involving indirect activation via the other VFT domain (of the C240E mutant) (Fig. 5B). That the maximal response obtained with this combination is lower than that obtained with the heterodimer carrying only the C240E mutation in one subunit is also consistent with the notion that one VFT domain can either cis-activate the TMD of the same subunit or trans-activate that of the other subunit because the cis-activation of the F767S mutant TMD does not yield any G protein activation. trans-Activation Is Not via the Other TMD—The results presented so far do not exclude the theoretical possibility of an indirect activation via the other TMD (Fig. 5C). The VFT domain of the F767S subunit could first activate the TMD of the same subunit (although incapable of G protein activation), in turn trans-activating the TMD of the other subunit. This pathway, anyway, would also imply an intersubunit rearrangement, viz. changes at the interface of the two TMDs. However, this scenario would be in contrast to recent reports indicating that only one TMD within a GPCR dimer can reach the active state at a time (12White J.F. Grodnitzky J. Louis J.M. Trinh L.B. Shiloach J. Gutierrez J. Northup J.K. Grisshammer R. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12199-12204Crossref PubMed Scopus (131) Google Scholar, 32Goudet C. Kniazeff J. Hlavackova V. Malhaire F. Maurel D. Acher F. Blahos J. Prézeau L. Pin J.P. J. Biol. Chem. 2005; 280: 24380-24385Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 44Damian M. Martin A. Mesnier D. Pin J.P. Baneres J.L. EMBO J. 2006; 25: 5693-5702Crossref PubMed Scopus (125) Google Scholar). We nevertheless verified whether this is also true for mGlu5, in particular with one subunit carrying the C240E mutation, as used in our trans-activation experiment in Fig. 8. To this end, we used an mGlu5 subunit with a TMD that, because of a triple mutation (P654S/S657C/L743V, termed 3Ro), can be specifically blocked in its active conformation by the drug Ro 01-6128 (45Knoflach F. Mutel V. Jolidon S. Kew J.N. Malherbe P. Vieira E. Wichmann J. Kemp J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13402-13407Crossref PubMed Scopus (218) Google Scholar), but without triggering any G protein activation, due to an additional F767S mutation. Indeed, pretreatment with this drug completely abolished the Quis-stimulated Ca2+ response mediated by an mGlu5 carrying these mutations (3Ro + F767S) in one of the two subunits (Fig. 9). This demonstrates that, indeed, once the 3Ro-mutated TMD is in the active state (but without triggering G protein activation due to the F767S mutation), the second TMD cannot reach the active state any more. Thus, also in an mGlu5 dimer, only one TMD can reach the active state at a time. Of note, the effect was independent of the absence or presence of the C240E mutation in the other subunit. Thus, the observed trans-activation of one TMD by the VFT domain of the other subunit within an mGluR dimer (Fig. 8) cannot be indirect via activation of the second TMD (Fig. 5C). No Preferential Intrasubunit Transduction" @default.
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• W2068201543 title "Activation of a Dimeric Metabotropic Glutamate Receptor by Intersubunit Rearrangement" @default.
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