Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M702542200 on September 13, 2007

J. Biol. Chem., Vol. 282, Issue 45, 33000-33008, November 9, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/45/33000    most recent
M702542200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brock, C.
Right arrow Articles by Pin, J.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brock, C.
Right arrow Articles by Pin, J.-P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Activation of a Dimeric Metabotropic Glutamate Receptor by Intersubunit Rearrangement*Formula

Carsten Brock1, Nadia Oueslati, Stéphan Soler, Laure Boudier, Philippe Rondard, and Jean-Philippe Pin2

From the University of Montpellier 1 and 2, CNRS UMR5203, Institute of Functional Genomics, Montpellier F-34094, France and INSERM, U661, Montpellier F-34094, France

Received for publication, March 23, 2007 , and in revised form, July 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs),3 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 (1). 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 (2–4). In particular, movement of TM6 likely opens a crevice allowing interaction with the C terminus of the G protein {alpha}-subunit, triggering its activation (5).

Many GPCRs have been shown to form dimers, but the functional role of this remains elusive (6–8). Although a GPCR monomer is sufficient to activate a G protein (8–12), 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 (13–15).

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 (16). 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 (17). 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) (18, 19), which is both necessary (20) and sufficient (21) 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 (22, 23). But agonist binding may also induce a relative reorientation of the two VFT domains (Fig. 1) (18), and a second model (Fig. 2B) thus proposes that this may in turn yield an activating rearrangement of the two TMDs (24, 25). Recent fluorescence resonance energy transfer (FRET) studies are indeed consistent with a glutamate-induced reorientation of the two TMDs within an mGluR dimer (26). 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) (19), 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 (27–30).


Figure 1
View larger version (78K):
[in this window]
[in a new window]

  		FIGURE 1.
Different conformations of an mGluR VFT domain dimer determined by x-ray crystallography. The conformations observed under various crystallization conditions are represented in the same orientation: lobes I of both protomers on top and lobes II on bottom. Chain A (yellow) is in front, and chain B (blue) is in back. Agonist binding stabilizes a closed conformation of the VFT domain. In the case of the mGlu1 VFT domain dimer, it also induces a relative reorientation of the two VFT domains, bringing their lobes II closer together. No such reorientation is observed in the crystal structure of the extracellular domain of mGlu3 (Protein Data Bank codes 1ewt, 1ewk, 1isr, and 2e4u).

 


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2.
Intersubunit rearrangement and activation of a dimeric mGluR. A, the VFT domain in its agonist-bound conformation could directly stabilize the activated conformation of its associated TMD (intrasubunit transduction). The observed agonist-induced intersubunit rearrangement could be a simple side effect of these conformational changes. B, the agonist-induced intersubunit rearrangement could represent the mechanism through which agonist binding to a VFT domain is transduced into TMD activation. In this case, agonist binding to one VFT domain should activate not only the TMD of the same subunit (cis-activation), but also that of the other subunit (trans-activation).

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Plasmids for mGlu5 were either as described previously (31–34) or, based on plasmids described in these references, newly constructed by standard molecular biology techniques (PCR, site-directed mutagenesis, subcloning) as described (35). Compared with the previously described C2 constructs (31), 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 (32). 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 (36). 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.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 3.
Controlling the subunit composition of a dimeric mGluR. A, schematic representation. Part of the C-terminal tail of mGlu5 was replaced with part of the C-terminal tail of either subunit of the heterodimeric GABAB receptor, containing notably the CC domain, followed by an intracellular retention signal (RSR or KKXX). Heterodimerization of the two constructs permits specific interaction of the two CC domains, masking the adjacent retention signals. Thus, monomeric or homodimeric mGlu5-C1 or mGlu5-C2KKXX is retained inside the cell, and only the heterodimer reaches the cell surface. B, cell-surface expression of the different constructs. ELISA was performed using anti-HA antibody on intact (non-permeabilized) cells expressing the different mGlu5 constructs, each carrying an HA tag at their extracellular N termini. Mcps, million counts/s. C, heterodimerized mGlu5 is functional. Cells from the same transfections as described for B were stimulated with various concentrations of the glutamate analog Quis, and the resulting Ca2+ response was measured as the fluorescence increase in the Ca2+-sensitive dye Fluo-4. D, heterodimerized mGlu5 is symmetric. Shown is the Quis-stimulated Ca2+ response of an mGlu5-C1/C2KKXX heterodimer carrying the G protein-uncoupling mutation F767S (x) in both subunits ({circ}), only in the C1 subunit ({triangleup}), or only in the C2KKXX subunit ({blacktriangleup}). All constructs carried an HA tag at their extracellular N termini, and similar cell-surface expression was verified by ELISA on cells from the same transfection. Anti-HA ELISA signals were within a range of 2.2 million counts/s ± 15%. a. u., absorbance units; WT, wild-type.

 
FRET—FRET measurements were conducted as described (37). 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 {Delta}665, 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 (35). 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
"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 {gamma}-aminobutyric acid type B (GABAB) receptor (35, 38–40) to the homodimeric metabotropic glutamate receptor mGlu5 (31). 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 (31) in either the C1 or C2KKXX subunit was similar (Fig. 3D).


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 4.
mGluRs are dimers, not higher order oligomers. A, FRET between donor and acceptor fluorophore-labeled antibodies directed against HA and FLAG tags, respectively, placed at the extracellular N termini of different receptor constructs. Left, the two tags are placed on the two subunits within the same mGlu5-C1/C2KKXX dimer. Right, one tag is placed on one of the mGlu5 dimer subunits, and the other one on a vasopressin V2 receptor (hatched box). Middle, the two tags are placed on one subunit per dimer in two different mGlu5 dimers. They cannot be within the same dimer because these two subunits carry the same CC domain and therefore cannot mutually mask their retention signals. To reach the cell surface (and be recognized by the antibodies), either HA- or FLAG-tagged mGlu5-C2KKXX construct must dimerize with untagged mGlu5-C1. Similar cell-surface expression levels of the different constructs were verified in parallel by anti-HA and anti-FLAG ELISAs on cells from the same transfections. Anti-HA ELISA signals were in a maximal range of ±17%, ~2.7 million counts/s; anti-FLAG ELISA signals were in a maximal range of ±29%, ~2.4 million counts/s. B, functional complementation between an mGlu5 mutant not activated by glutamate and its analogs (mGlu5-YADA) and an mGlu5 mutant incapable of G protein activation (mGlu5-F767S). Coexpression of the two mutants restores a Quis-stimulated Ca2+ response (middle), indicating the formation of functional heterodimers. No such functional complementation is observed upon coexpression of mGlu5-YADA with an mGlu5 dimer in which both subunits carry the F767S mutation (right). All mGlu5-F767S constructs carry an HA tag and mGlu5-YADA carries a FLAG tag at their extracellular N termini, and similar cell-surface expression levels were verified by ELISAs on cells from the same transfections. Anti-HA ELISA signals were within a maximal range of ±8%, ~4.2 million counts/s; anti-FLAG ELISA signals were within a range of 1.4 million counts/s ± 22%. a. u., absorbance units.

 
mGlu5 Is a Dimer, Not a Higher Order Oligomer—At least some GPCRs may form not only dimers, but also higher order oligomers (41, 42). 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 (37). 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 (31), was coexpressed with mGlu5-YADA, which is not activated by glutamate or its analogs due to a mutation in the VFT domain (31). 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 (31), 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.


Figure 5
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 5.
Possible mechanisms for trans-activation within an mGluR dimer. The hypothetical mechanisms in A–C are consistent with the model in Fig. 2A. Only when excluding these three possibilities can trans-activation within an mGluR dimer stand as a proof for the intersubunit rearrangement as the activation mechanism (D), as presented in Fig. 2B.

 
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) (43). 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 (34). 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 (43), 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 (33), 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 (33) 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.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 6.
trans-Activation is not by domain swap. Shown is the Quis-stimulated Ca2+ response (middle) upon coexpression of mGlu5-YADA (not activated by glutamate or Quis) either with mGlu5-F767S (incapable of G protein activation) or with mGlu5-(1–614) (devoid of its TMD). mGlu5-YADA carries an N-terminal FLAG tag, and the two other constructs carry an N-terminal HA tag; efficient heterodimerization was verified by FRET on intact cells from the same transfections (negative control, FLAG-mGlu5-YADA + HA-CD4, {Delta}665 = 674 ± 42) (lower). Similar cell-surface expression levels were verified by ELISAs on cells from the same transfections. Anti-HA ELISA signals were within a maximal range of ±13%, ~4.2 million counts/s; anti-FLAG ELISA signals were within a range of 0.9 million counts/s ± 21%. a. u., absorbance units.

 


Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 7.
Uncoupling of one VFT domain from its TMD within an mGluR dimer does not prevent its activation. Shown is the activation of an mGlu5-C1/C2KKXX dimer carrying the mutation C240E (x; functionally uncoupling the VFT domain from the TMD) in both ({circ}), in none (•), or in only one of the two subunits (mGlu5-C240E-C1 + mGlu5-C2KKXX ({triangleup}) and mGlu5-C1 + mGlu5-C240E-C2KKXX ({blacktriangleup})) as measured by the Quis-stimulated Ca2+ response. All constructs carry an HA tag at their extracellular N termini, and similar cell-surface expression levels were verified by ELISA on cells from the same transfections. Anti-HA ELISA signals were within a range of 1.8 million counts/s ± 25%. a. u., absorbance units.

 
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 (12, 32, 44). 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 (45), but without triggering any G protein activation, due to an additional F767S mutation.


Figure 8
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 8.
trans-Activation is not via the other VFT domain. Shown is the Quis-stimulated Ca2+ response mediated by mGlu5-C1/C2KKXX dimers composed of one subunit carrying the C240E mutation (functionally uncoupling the VFT domain from its TMD; mGlu5-C240E-C1) and one subunit carrying the same mutation ({circ}) or the F767S mutation (uncoupling the TMD from the G protein; {blacktriangleup}) or neither of these mutations ({triangleup}). All constructs carry an HA tag at their extracellular N termini, and similar cell-surface expression levels were verified by ELISA on cells from the same transfections. Anti-HA ELISA signals were within a range of 1.8 million counts/s ± 10%. a. u., absorbance units.

 


Figure 9
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 9.
trans-Activation is not via the other TMD: only one TMD reaches the active state at a time. Shown is the Quis-stimulated Ca2+ response mediated by mGlu5-C1/C2KKXX dimers composed of one subunit carrying the F767S mutation (uncoupling its TMD from the G protein) and the 3Ro triple mutation (rendering it sensitive to the drug Ro 01-6128; mGlu5–3Ro-F767S-C2KKXX) and one subunit carrying (triangles) or not (circles) the C240E mutation (uncoupling the VFT domain from the TMD) in the absence (filled symbols) or presence (open symbols) of 100 µM Ro 01-6128. Ro 01-6128 directly stabilizes the active conformation of the TMD of the mGlu5–3Ro-F767S-C2KKXX subunit, but, due to the F767S mutation, without triggering G protein activation. Accordingly, Ro 01-6128 alone has no effect (not shown). Preincubation with Ro 01-6128 abolishes the Quis-stimulated Ca2+ response mediated by both mGlu5 dimers, demonstrating that the second TMD cannot reach the active state at the same time as the Ro 01-6128-bound TMD. All constructs carry an HA tag at their extracellular N termini, and similar cell-surface expression levels were verified by ELISA on cells from the same transfections. Anti-HA ELISA signals were within a range of 2.7 million counts/s ± 1%. a. u., absorbance units.

 
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).


Figure 10
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 10.
Equal cis- and trans-activation of a TMD by either VFT domain within an mGluR dimer. Shown is the Quis-stimulated Ca2+ response mediated by mGlu5-C1/C2KKXX dimers carrying the G protein-uncoupling mutation F767S in one subunit (mGlu5-F767S-C2KKXX) and the mutation C240E (functionally uncoupling the VFT domain from the TMD) in the same subunit ({triangleup}), in the other subunit ({blacktriangleup}), or in neither of the subunits (•). All constructs carry an HA tag at their extracellular N termini, and similar cell-surface expression levels were verified by ELISA on cells from the same transfections. Anti-HA ELISA signals were within a range of 3.2 million counts/s ± 10%. a. u., absorbance units.

 
No Preferential Intrasubunit Transduction—As stated above, the two models of intramolecular signal transduction from the VFT domains to the TMDs of an mGluR dimer (Fig. 2, A versus B) are not mutually exclusive. Thus, the above demonstration that the agonist-induced intersubunit rearrangement indeed activates the receptor does not yet exclude that, additionally, the agonist-bound VFT domain could also activate its associated TMD through an intrasubunit transduction. This can be tested by comparing the cis- and trans-activation within the dimer. The intersubunit rearrangement induced by agonist binding to one VFT domain should always cis- and trans-activate equally well both TMDs in the dimer. In contrast, in the case of an additional intrasubunit transduction, the cis-activation should be more efficient than the trans-activation. This is not the case because there was no significant difference between the Quis-stimulated Ca2+ responses mediated by mGlu5 carrying the C240E and F767S mutations in the same subunit (only cis-activation can generate a response) or either one of these mutations in each subunit (only trans-activation) (Fig. 10). This demonstrates that the agonist-induced intersubunit rearrangement is the only mechanism of intramolecular signal transduction and activation of a dimeric mGluR, and there is no additional intrasubunit transduction from the VFT domain to the TMD of the same subunit.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we examined a possible role of an intersubunit rearrangement in the activation of a dimeric mGluR. Two models, not mutually exclusive, have been proposed to explain how agonist binding in the VFT domain leads to activation of the TMD of these receptors. The first model proposes an intrasubunit activation in which the VFT domain directly activates the TMD of the same subunit (Fig. 2A) (22, 23). The second model proposes that TMD activation may result from an agonist-induced intersubunit rearrangement (Fig. 2B) (24, 25). To experimentally test these models, we first developed a system to perfectly control the composition of an mGluR dimer of two defined protomers each carrying or not different mutations. In particular, we took advantage of a point mutation that functionally disconnects the VFT domain from its TMD (33). Our data show that in an mGluR dimer carrying this mutation in a single subunit, agonist binding in one subunit activates equally well either of the two TMDs. This is compatible only with the second model, but not the first. Thus, an intersubunit rearrangement indeed plays a role and is even crucial for the activation of an mGluR.

Intramolecular Signal Transduction within an mGluR Dimer—For most GPCRs, agonists bind directly to their TMD, thereby stabilizing its active conformation (23). For class C GPCRs such as the mGluRs, where agonists bind to an extracellular domain, it has been proposed that this domain in its agonist-bound conformation could in turn interact with the TMD in a way stabilizing its active conformation (Fig. 2A) (22, 46). In line with this proposal, we have reported recently that a disulfide bridge linking the VFT domain of an mGluR to the rest of the receptor is crucial for intramolecular signal transduction (33). However, we have demonstrated here that there is no intrasubunit signal transduction within an mGluR dimer because agonist stimulation results in the activation of either TMD with the same efficiency, even when only one of the two subunits has this disulfide bond. These data can be explained only by an agonist-induced intersubunit rearrangement as the mechanism transducing agonist binding to a VFT domain into activation of a TMD (Fig. 2B).

The role of the crucial disulfide bond linking the VFT domain to the rest of the receptor (33) is therefore not to ensure intrasubunit signal transduction, but to allow the transmission of the agonist-induced rearrangement of the VFT domains to the TMDs. The reduced maximal activity of an mGluR dimer lacking this disulfide bond in one of the two protomers therefore rather reflects a reduced efficiency of this transmission, i.e. either the relative movement is different or the active orientation is less well stabilized.

In line with our results, the crystal structure of the mGlu1 VFT domain dimer revealed that agonist binding indeed induces a relative orientation of the two VFT domains (18). When taking into account the positions of the cysteine implicated in the above-mentioned disulfide bond as well as the C terminus of the VFT domain, which both connect this domain to the rest of the subunit, this agonist-induced reorientation of the two VFT domains is indeed expected to induce also a relative movement of the two TMDs within the dimer (18, 33). This is also consistent with the observed glutamate-induced changes in the FRET between cyan fluorescent protein- and yellow fluorescent protein-labeled TMDs of an mGlu1 dimer, indeed suggesting an agonist-induced rearrangement of the two TMDs (26). However, because this green fluorescent protein labeling impairs receptor function (G protein activation), these observed FRET changes may not necessarily reflect the "natural" rearrangements within the receptor. Moreover, none of these previous studies addressed the question of whether the agonist-induced intersubunit rearrangement indeed plays an active role in mGluR activation or whether it is simply a side effect of the mGluR activation. We have demonstrated here that the intersubunit rearrangement does indeed play an active role in and is even crucial for mGluR activation.

However, in contrast to what was previously reported for mGlu1 (18), it has very recently been reported that the relative orientation of the two VFT domains in the crystallized extracellular domains of mGlu3 is not altered by five different agonists (although they do induce VFT domain closure) (Fig. 1) (19). This is not consistent with our data. We speculate that the crystallization conditions or the absence of the TMDs may have prevented the mGlu3 VFT domain dimer from adopting the correct active orientation.

Absence of Higher Order Oligomerization—Another intriguing observation with the crystals of the mGlu3 extracellular domains is that the dimers may associate into larger oligomers (19). If this is also the case for the full-length mGluR at the cell surface, this could lead to an alternative and maybe more complex activation mechanism. Indeed, higher order oligomers have been observed recently for rhodopsin (41) and {alpha}-adrenergic receptors (42). Here, using fluorophore-labeled antibodies, we have shown that no significant FRET could be measured between mGluR dimers, whereas a high FRET signal was measured between the subunits within a dimer. Because the antibodies used were labeled with three to six fluorophores, the absence of FRET is very unlikely due to an orthogonal orientation of the donor and acceptor fluorophores. Moreover, absence of FRET due to a too large distance of the fluorophores within the oligomers is also very unlikely. Indeed, with the R0 being 65 Å, absence of FRET would mean a distance greater than 100 Å, which is incompatible with a direct association between the dimers, especially when considering the size of the antibodies. Thus, mGluR dimers are not in contact with each other at the surface of human embryonic kidney 293 cells. Moreover, we did not observe any trans-activation between two nonfunctional dimers. Thus, if larger mGluR oligomers can form, these likely require other partners than just the receptor subunits themselves, such as intracellular scaffolding proteins, and these are not required for the correct activation of the receptor. This makes the mGluR dimers the functional unit.

Role of an Intersubunit Rearrangement in the Activation of Other GPCRs?—The activation by intersubunit rearrangement described here could represent a particularity of class C GPCRs, where this mechanism could represent primarily a means to intramolecularly transmit the signal from the VFT domain to the TMD level. Indeed, dimerization appears to be at least not generally required for GPCR function because at least some GPCRs can also function as monomers (8–12). Nonetheless, it has been speculated that the dynamic interplay between the two subunits of a GPCR dimer, through changes at the dimerization interface or a larger scale reorientation of the two protomers relative to each other, could at least contribute to the stabilization of the active conformation (13, 15). It is exciting that recent studies nicely demonstrated that activation of the homodimeric dopamine D2 receptor alters its dimerization interface and that, conversely, stabilization of this altered interface by chemical cross-linking results in activation of the receptor even in the absence of agonist (14, 47). This demonstrates that also a class A GPCR dimer can indeed be activated by an intersubunit rearrangement. However, the artificial cross-linking approach used in those studies did not permit the authors to conclude whether this may indeed also play a role in the natural activation of this receptor. Our study now provides the first demonstration that an agonist-induced rearrangement may indeed play an important role also in the natural activation mechanism of a dimeric GPCR. Of note, activation by agonist-induced intersubunit rearrangement is a mechanism widely used by other types of transmembrane receptors, viz. receptors with associated or intrinsic intracellular tyrosine kinase or guanylate cyclase activity (27–30).


   FOOTNOTES
 
* This work was supported in part by CNRS; INSERM; the Ministère de l'Éducation Nationale; the Ministère de l'Enseignement Supérieur et de la Recherche; Grant ACI-BCMS328 from the program "Actions Concertées Incitatives"; Grants ANR-05-PRIB-02502, ANR-BLAN06–3-135092, and ANR-05-NEUR-0121-04 from the Agence Nationale de la Recherche; a grant from the Fondation de France Comité Parkinson; and Grants STREP-GPCR and LSHB-CT-2003-503337 from the Sixth Framework Program of the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A–D. Back

1 Supported by a grant from the Fondation Recherche Médicale. Back

2 To whom correspondence should be addressed: Inst. de Génomique Fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier, France. Tel.: 33-4-6714-2988; Fax: 33-4-6754-2432; E-mail: jppin{at}igf.cnrs.fr.

3 The 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, {gamma}-aminobutyric acid type B. Back


   ACKNOWLEDGMENTS
 
We thank C. Vol and F. Malhaire for expert technical assistance, Dr. D. Maurel for help with FRET experiments, L. Albizu for the generous gift of the FLAG-V2 plasmid, Dr. B. Schwappach for the generous gift of a CD4 plasmid, Dr. A. E. Brady for help with supplemental Fig. A, Drs. J. Perroy and T. Durroux for critical reading of the manuscript, C. Vol and Dr. L. Prézeau for taking care of the Pharmacology Platform "Criblage Interactome," and Dr. C. Goudet for support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Howard, A. D., McAllister, G., Feighner, S. D., Liu, Q., Nargund, R. P., Van der Ploeg, L. H., and Patchett, A. A. (2001) Trends Pharmacol. Sci. 22, 132-140[Medline] [Order article via Infotrieve]
  2. Schwartz, T. W., Frimurer, T. M., Holst, B., Rosenkilde, M. M., and Elling, C. E. (2006) Annu. Rev. Pharmacol. Toxicol. 46, 481-519[CrossRef][Medline] [Order article via Infotrieve]
  3. Sheikh, S. P., Vilardarga, J. P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041[Abstract/Free Full Text]
  4. Binet, V., Duthey, B., Lecaillon, J., Vol, C., Quoyer, J., Labesse, G., Pin, J.-P., and Prézeau, L. (2007) J. Biol. Chem. 282, 12154-12163[Abstract/Free Full Text]
  5. Bourne, H. R. (1997) Curr. Opin. Cell Biol. 9, 134-142[CrossRef][Medline] [Order article via Infotrieve]
  6. Angers, S., Salahpour, A., and Bouvier, M. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 409-435[CrossRef][Medline] [Order article via Infotrieve]
  7. Milligan, G. (2004) Mol. Pharmacol. 66, 1-7[Abstract/Free Full Text]
  8. Chabre, M., and le Maire, M. (2005) Biochemistry 44, 9395-9403[CrossRef][Medline] [Order article via Infotrieve]
  9. Bayburt, T. H., Leitz, A. J., Xie, G., Oprian, D. D., and Sligar, S. G. (2007) J. Biol. Chem. 282, 14875-14881[Abstract/Free Full Text]
  10. Whorton, M. R., Bokoch, M. P., Rasmussen, S. G., Huang, B., Zare, R. N., Kobilka, B., and Sunahara, R. K. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 7682-7687[Abstract/Free Full Text]
  11. Ernst, O. P., Gramse, V., Kolbe, M., Hofmann, K. P., and Heck, M. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 10859-10864[Abstract/Free Full Text]
  12. White, J. F., Grodnitzky, J., Louis, J. M., Trinh, L. B., Shiloach, J., Gutierrez, J., Northup, J. K., and Grisshammer, R. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 12199-12204[Abstract/Free Full Text]
  13. Breitwieser, G. E. (2004) Circ. Res. 94, 17-27[Abstract/Free Full Text]
  14. Guo, W., Shi, L., Filizola, M., Weinstein, H., and Javitch, J. A. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 17495-17500[Abstract/Free Full Text]
  15. Ridge, K. D., and Palczewski, K. (2007) J. Biol. Chem. 282, 9297-9301[Abstract/Free Full Text]
  16. Romano, C., Yang, W. L., and O'Malley, K. L. (1996) J. Biol. Chem. 271, 28612-28616[Abstract/Free Full Text]
  17. Pin, J.-P., Galvez, T., and Prézeau, L. (2003) Pharmacol. Ther. 98, 325-354[CrossRef][Medline] [Order article via Infotrieve]
  18. Kunishima, N., Shimada, Y., Tsuji, Y., Sato, T., Yamamoto, M., Kumasaka, T., Nakanishi, S., Jingami, H., and Morikawa, K. (2000) Nature 407, 971-977[CrossRef][Medline] [Order article via Infotrieve]
  19. Muto, T., Tsuchiya, D., Morikawa, K., and Jingami, H. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 3759-3764[Abstract/Free Full Text]
  20. Bessis, A. S., Rondard, P., Gaven, F., Brabet, I., Triballeau, N., Prézeau, L., Acher, F., and Pin, J.-P. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11097-11102[Abstract/Free Full Text]
  21. Kniazeff, J., Saintot, P. P., Goudet, C., Liu, J., Charnet, A., Guillon, G., and Pin, J.-P. (2004) J. Neurosci. 24, 370-377[Abstract/Free Full Text]
  22. Pin, J.-P., De Colle, C., Bessis, A. S., and Acher, F. (1999) Eur. J. Pharmacol. 375, 277-294[CrossRef][Medline] [Order article via Infotrieve]
  23. Bockaert, J., and Pin, J.-P. (1999) EMBO J. 18, 1723-1729[CrossRef][Medline] [Order article via Infotrieve]
  24. Pin, J.-P., Kniazeff, J., Liu, J., Binet, V., Goudet, C., Rondard, P., and Prézeau, L. (2005) FEBS J. 272, 2947-2955[CrossRef][Medline] [Order article via Infotrieve]
  25. Kubo, Y., and Tateyama, M. (2005) Curr. Opin. Neurobiol. 15, 289-295[CrossRef][Medline] [Order article via Infotrieve]
  26. Tateyama, M., Abe, H., Nakata, H., Saito, O., and Kubo, Y. (2004) Nat. Struct. Mol. Biol. 11, 637-642[CrossRef][Medline] [Order article via Infotrieve]
  27. Livnah, O., Stura, E. A., Middleton, S. A., Johnson, D. L., Jolliffe, L. K., and Wilson, I. A. (1999) Science 283, 987-990[Abstract/Free Full Text]
  28. Remy, I., Wilson, I. A., and Michnick, S. W. (1999) Science 283, 990-993[Abstract/Free Full Text]
  29. Moriki, T., Maruyama, H., and Maruyama, I. N. (2001) J. Mol. Biol. 311, 1011-1026[CrossRef][Medline] [Order article via Infotrieve]
  30. van den Akker, F. (2001) J. Mol. Biol. 311, 923-937[CrossRef][Medline] [Order article via Infotrieve]
  31. Kniazeff, J., Bessis, A. S., Maurel, D., Ansanay, H., Prézeau, L., and Pin, J.-P. (2004) Nat. Struct. Mol. Biol. 11, 706-713[CrossRef][Medline] [Order article via Infotrieve]
  32. Goudet, C., Kniazeff, J., Hlavackova, V., Malhaire, F., Maurel, D., Acher, F., Blahos, J., Prézeau, L., and Pin, J.-P. (2005) J. Biol. Chem. 280, 24380-24385[Abstract/Free Full Text]
  33. Rondard, P., Liu, J., Huang, S., Malhaire, F., Vol, C., Pinault, A., Labesse, G., and Pin, J.-P. (2006) J. Biol. Chem. 281, 24653-24661[Abstract/Free Full Text]
  34. Liu, J., Maurel, D., Etzol, S., Brabet, I., Ansanay, H., Pin, J.-P., and Rondard, P. (2004) J. Biol. Chem. 279, 15824-15830[Abstract/Free Full Text]
  35. Brock, C., Boudier, L., Maurel, D., Blahos, J., and Pin, J.-P. (2005) Mol. Biol. Cell 16, 5572-5578[Abstract/Free Full Text]
  36. Goudet, C., Gaven, F., Kniazeff, J., Vol, C., Liu, J., Cohen-Gonsaud, M., Acher, F., Prézeau, L., and Pin, J.-P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 378-383[Abstract/Free Full Text]
  37. Maurel, D., Kniazeff, J., Mathis, G., Trinquet, E., Pin, J.-P., and Ansanay, H. (2004) Anal. Biochem. 329, 253-262[CrossRef][Medline] [Order article via Infotrieve]
  38. Margeta-Mitrovic, M., Jan, Y. N., and Jan, L. Y. (2000) Neuron 27, 97-106[CrossRef][Medline] [Order article via Infotrieve]
  39. Pagano, 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., and Bettler, B. (2001) J. Neurosci. 21, 1189-1202[Abstract/Free Full Text]
  40. Calver, 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., and Pangalos, M. N. (2001) J. Neurosci. 21, 1203-1210[Abstract/Free Full Text]
  41. Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127-128[CrossRef][Medline] [Order article via Infotrieve]
  42. Lopez-Gimenez, J. F., Canals, M., Pediani, J. D., and Milligan, G. (2007) Mol. Pharmacol. 71, 1015-1029[Abstract/Free Full Text]
  43. Jeoung, M., Lee, C., Ji, I., and Ji, T. H. (2007) Mol. Cell. Endocrinol. 260-262, 137-143[CrossRef][Medline] [Order article via Infotrieve]
  44. Damian, M., Martin, A., Mesnier, D., Pin, J.-P., and Baneres, J. L. (2006) EMBO J. 25, 5693-5702[CrossRef][Medline] [Order article via Infotrieve]
  45. Knoflach, F., Mutel, V., Jolidon, S., Kew, J. N., Malherbe, P., Vieira, E., Wichmann, J., and Kemp, J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13402-13407[Abstract/Free Full Text]
  46. Parnot, C., and Kobilka, B. (2004) Nat. Struct. Mol. Biol. 11, 691-692[CrossRef][Medline] [Order article via Infotrieve]
  47. Guo, W., Shi, L., and Javitch, J. A. (2003) J. Biol. Chem. 278, 4385-4388[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
M. Yanagawa, T. Yamashita, and Y. Shichida
Activation Switch in the Transmembrane Domain of Metabotropic Glutamate Receptor
Mol. Pharmacol., July 1, 2009; 76(1): 201 - 207.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Zhang, B. Klebansky, R. M. Fine, H. Xu, A. Pronin, H. Liu, C. Tachdjian, and X. Li
Molecular mechanism for the umami taste synergism
PNAS, December 30, 2008; 105(52): 20930 - 20934.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Damian, S. Mary, A. Martin, J.-P. Pin, and J.-L. Baneres
G Protein Activation by the Leukotriene B4 Receptor Dimer: EVIDENCE FOR AN ABSENCE OF TRANS-ACTIVATION
J. Biol. Chem., July 25, 2008; 283(30): 21084 - 21092.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
X. Rovira, D. Roche, J. Serra, J. Kniazeff, J.-P. Pin, and J. Giraldo
Modeling the Binding and Function of Metabotropic Glutamate Receptors
J. Pharmacol. Exp. Ther., May 1, 2008; 325(2): 443 - 456.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/45/33000    most recent
M702542200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brock, C.
Right arrow Articles by Pin, J.-P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brock, C.
Right arrow Articles by Pin, J.-P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement