Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M611071200 on February 19, 2007

J. Biol. Chem., Vol. 282, Issue 16, 12154-12163, April 20, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/16/12154    most recent
M611071200v1
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 Binet, V.
Right arrow Articles by Prézeau, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Binet, V.
Right arrow Articles by Prézeau, L.
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?

Common Structural Requirements for Heptahelical Domain Function in Class A and Class C G Protein-coupled Receptors*

Virginie Binet{ddagger}§, Béatrice Duthey{ddagger}, Jennifer Lecaillon{ddagger}, Claire Vol{ddagger}, Julie Quoyer{ddagger}, Gilles Labesse, Jean-Philippe Pin{ddagger}, and Laurent Prézeau{ddagger}1

From the {ddagger}Département de Pharmacologie Moléculaire, Institut de Génomique Fonctionnelle, CNRS-UMR 5203, INSERM U661, Universités Montpellier 1 et 2, 34094 Montpellier Cedex 5, §Centre Hospitalo-Universitaire de Montpellier, 34000 Montpellier, and Centre de Biochimie Structurale, CNRS UMR 5048, INSERM U554, Universités Montpellier 1 et 2, 34000 Montpellier, France

Received for publication, December 1, 2006 , and in revised form, January 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the {gamma}-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of {gamma}-aminobutyric acid type B2 can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs)2 are encoded by one of the most important gene families in mammalian genomes (1). These membrane proteins play a critical role in transducing extracellular signals into the cell and constitute the major target for drug development. Although they are important molecules, little is known about their activation mechanism (2, 3). All these receptors possess a heptahelical domain (HD), the structure of which has been solved for rhodopsin only (4). This available structure is in a fully inactive state, as it is stabilized by the covalently linked inverse agonist cis-retinal. Thus, our actual knowledge on the active conformation relies mostly on functional and biophysical analysis of a variety of mutated receptors and their coupling to G proteins and their effectors (2, 3).

Four main classes of GPCRs have been defined in mammals based on sequence analysis (1, 5, 6), with the rhodopsin-like class A receptors being the largest and the most studied. Class A receptor activation is associated with a movement of TM6 relative to TM3, leading to the opening of a cavity between the intracellular loops 2 and 3 connecting TM3 to TM4 and TM5 to TM6, respectively (2, 7). The inactive state is stabilized by a network of interactions between residues at the cytoplasmic end of the TMs (4). This network includes ionic interactions that involve the highly conserved class A residues (D/E)RY at the cytoplasmic end of TM3. In many class A GPCRs, the Arg of the (D/E)RY motif makes an ionic interaction with a conserved acidic residue (D/E) of TM6 (3). Mutation of these residues leads either to a loss or a gain of function, consistent with this motif being involved in a lock that can control the conformational state of class A GPCRs (3, 8, 9). Alternatively, the Arg of the (D/E)RY motif has also been proposed to play a direct role in receptor G protein interaction and activation for some class A receptors (8).

The (D/E)RY motif is not found in the GPCRs from the other classes. This is the case for the class C receptors that are activated by the two main neurotransmitters, glutamate and {gamma}-aminobutyrate (GABA), by Ca2+ ions, taste compounds, and pheromones (10). The sequence divergence between the HDs of class A and class C GPCRs questioned whether both get activated the same way. In addition, class C receptor general functioning appears quite different from that of class A receptors despite their likely common dimeric organization (11). Although conformational changes within the HD of class A GPCRs is regarded as the main step in receptor activation, activation of class C receptors is assumed to result from a change in the relative position of the subunits in the dimer. Indeed, the crystal structure of the large dimeric extracellular domain of mGlu1, with and without bound agonist, revealed a major change in their relative position resulting from agonist binding (12). Moreover, fluorescence resonance energy transfer analysis in living cells with cyan fluorescent protein and yellow fluorescent protein fused at various places in the intracellular segments of each of the subunits is consistent with a relative movement of the HDs during class C receptor dimer activation (13). However, a number of modeling and mutagenesis studies are consistent with the HDs of class C GPCRs being structurally similar to rhodopsin (10, 14, 15). Thus, besides the putative movement between the HDs (13), a change in the conformation of the HDs themselves is likely also involved, as indicated by the effect of allosteric regulators interacting directly in the HD (15-18). But are any of the structural requirements involved in such a conformational change similar in both class A and class C GPCRs?

In this study, we aimed at identifying key residues involved in the activation of class C GPCRs. Using the GABAB receptor as a model system, we thus examined the functional consequences resulting from the mutation of such residues. This leads us to the observation that a network of interactions involving TM3 and TM6 plays a critical role in the activation process, illustrating some similarity in the structural determinants controlling G protein activation by class A and class C GPCRs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—GABA was purchased from Sigma. [3H]CGP54626, purchased from Tocris (Bristol, UK), had a specific activity of 40 Ci·mmol-1. CGP54626 was purchased from Tocris (Fisher-Bioblock, Illkrich, France). Fetal bovine serum, culture media, and other solutions used for cell culture were from Invitrogen. [3H]Myoinositol (23.4 Ci/mol) was purchased from PerkinElmer Life Sciences. All other reagents used were of molecular or analytical grade where appropriate.

Plasmids and Site-directed Mutagenesis—The plasmids encoding the wild-type and chimeric GABAB1a and GABAB2 subunits epitope-tagged at their N-terminal ends (pRK-GABAB1a-HA, pRK-GABAB2-cMyc), under the control of a cytomegalovirus promoter, were described previously (19, 20). Site-directed mutagenesis was performed on a pRK-GABAB1a-HA or a pRK-GABAB2-cMyc vector using a QuikChange® strategy (Stratagene). Briefly, the mutations were generated by using two complementary 30- to 40-mer oligonucleotides designed to contain the desired mutation, and the sequence of the constructs was confirmed by DNA sequencing (Genome Express, France).

Cell Culture and Transfection—Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transfected by electroporation as described elsewhere (17). Unless stated otherwise, 107 cells were transfected with plasmid DNA containing the coding sequence of the receptor subunits and completed to a total amount of 10 µg of plasmid DNA with pRK6 plasmid. For determination of inositol phosphate accumulation, the cells were also tranfected with the chimeric G{alpha}qi9 G protein, which allows the coupling of the recombinant heteromeric GABAB receptor to phospholipase C (20).

Measurement of Inositol Phosphate Production—Determination of inositol phosphate (InP) accumulation in transfected cells was performed in 96-well plates (0.2 x 106 cells/well) after overnight labeling with [3H]myoinositols (0.5 µCi/well) as already described (21). The stimulation was conducted for 30 min in a medium containing 10 mM LiCl and the indicated concentration of agonist or antagonist. The reaction was stopped by replacing the medium by 0.1 M formic acid. Supernatants were recovered, and InPs were purified by ion exchange chromatography using Dowex AG1-X8 resin (Bio-Rad) in 96-well filter plates (Millipore, Bedford, MA). Total radioactivity remaining in the membrane fractions was counted after treatment of cells with a solution containing 10% Triton X-100 and 0.1 N NaOH. Radioactivity was quantified using Wallac 1450 MicroBeta liquid scintillation counter. Data were expressed as the amount of total InPs produced over the amount of radioactivity remaining in the membranes plus the produced InP, multiplied by 100. Unless stated otherwise, all data are means ± S.E. of at least three independent experiments. The dose-response curves were fitted using the Graph-Pad (San Diego) PRISM software and the following equation: y = ((ymax - ymin)/(1 + (x/EC50)nH)) + ymin, where the EC50 is the concentration of the compound necessary to obtain 50% of the maximal effect, and nH is the Hill coefficient.

Anti-HA Tag ELISA for Quantification of Cell Surface Expression—Twenty four hours after transfection (107 cells, HA-tagged GABAB1 (2 µg) and c-Myc-tagged GABAB2 (2 µg) subunits), cells were fixed with 4% paraformaldehyde and then blocked with phosphate-buffered saline + 5% fetal bovine serum. After a 30-min reaction with primary antibody (monoclonal anti-HA clone 3F10 (Roche Applied Science) at 0.5 µg/ml) in the same buffer, the goat anti-rat antibody coupled to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA) was applied for 30 min at 1 µg/ml. After intense washes with phosphate-buffered saline, secondary antibody was detected and quantified instantaneously by chemiluminescence using Supersignalp® ELISA femto maximum sensitivity substrate (Pierce) and a Wallac Victor2 luminescence counter.

Ligand Binding on Intact HEK293 Cells—Ligand binding experiments were performed on intact HEK293 cells. The cells were plated after electroporation the day before the experiment. Thus, the cells on ice were washed with ice-cold binding buffer (20 mM Tris-HCl, pH 7.4, 118 mM NaCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.7 mM KCl, 1.8 mM CaCl2) and incubated in the presence of 1.5 nM [3H]CGP54626 with or without unlabeled ligand at the indicated concentration. The incubation was terminated by washing with ice-cold binding buffer. The cells were disrupted with 0.1 nM NaOH (400 µl), and the bound radioactivity was counted in a scintillation beta counter. Displacement curves were fitted with the GraphPad (San Diego) PRISM software, using the equation y = ((ymax - ymin)/(1 + (x/EC50)nH) + ymin, where EC50 is the concentration of cold drug necessary to displace half of the specially bound [3H]CGP54626, and nH is the Hill number.

Structural Biology—Sequence alignment of class C receptors was performed using ClustalW (22) and refined manually. Fold compatibility for truncated sequences corresponding to the HD of various class C receptors was searched using the meta-server @TOME (23 and references therein). Sequence-structure alignments, including various class C receptors and the crystal structures of rhodopsin (Protein Data Bank codes 1LH9, 1U19, and 1GZM (4, 24, 25)), were manually refined with the help of the program ViTO (26). Models of various receptors (mainly mGluRs, T1R2, T1R3, GABAB1, and GABAB2) were performed using SCWRL3.0 (27) and MODELLER7.7 (28). In the absence of tools for structure evaluation of modeled membrane proteins, several rounds of alignment refinement/molecular modeling were performed. Visual inspection of the localization of hydrophobic and/or conserved residues relative to the membrane interface and protein core was performed for each TM helix and then for the whole HD. Strict conservation was computed for each subfamily of class C receptors (e.g. GABAB2) or for the whole superfamily using ViTO. Final three-dimensional models were built for using MODELLER 7.7 with the loop optimization procedure.


Figure 1
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 1.
Alignment of the sequences of class C GPCRs. Three conserved residues at the cytoplasmic face of TM3 and TM6 were chosen, as their positions are close to the positions of the (D/E)RY and D/E motifs in TM3 and TM6 rhodopsin, respectively. The notation of most of the sequences are after the SwissProt data bank notation, with the following accession numbers: MGR5_RAT P31424, MGR1_RAT P23385, MGR1_CAEEL Q09630, MGR_DROME P91685, MGR7_RAT P35400, MGR6_RAT P35349, MGR8_RAT P70579, CASR_MOUSE Q9QY96, TS1R1_RAT Q9Z0R8, GABR1_RAT Q9Z0U4, GABR2_RAT O88871, and OPSD_BOVIN P02699. AAK13420 and AAK13421 sequence, which are GABAB subunits homologues from Drosophila, are notated after their gene bank names.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Conserved Residues at the Cytoplasmic Ends of TM3 and TM6 of Class C GPCRs—Alignment of the sequences of class C receptor HDs revealed several highly conserved residues. Among these are two basic residues located at the cytoplasmic side of TM3, at positions close to that of the (D/E)RY motif in class A receptors (Fig. 1). A second highly conserved position is that of an acidic residue of the cytoplasmic end of TM6 (Fig. 1).


Figure 2
View larger version (55K):
[in this window]
[in a new window]

  		FIGURE 2.
Molecular modeling of GABAB2. A, the side chains of the residues conserved in the GABAB subunits from different species (rat, human, C. elegans, and Drosophila melanogaster) are showed in black. The representation of the model is viewed from the extracellular side, with the helical TMs showed in ribbon representation. B, residues Lys-572 and Arg-575 in TM3 and Asp-688 in TM6 are shown in wire frame and in color according to the atom types. The two cysteines conserved in the class C receptors and corresponding to the conserved cysteines in class A receptors involved in a disulfide bridge at the extracellular face are also shown. The representation of the model is viewed from the side and shows the helical TMs in ribbon representation. Loops are shown in white/gray, and TMs are colored as follows: TM1 in red, TM2 in brown, TM3 in orange, TM4 in green, TM5 in dark green, TM6 in cyan, and TM7 in dark blue. The figures have been made using the program ViTO.

 
To get some possible information on the functional role of these conserved charged residues, three-dimensional models of class C HDs were generated based on rhodopsin structure. A fold recognition approach using the meta-server @TOME confirmed the expected compatibility of sequences from class C receptors with the seven-helix bundle of rhodopsin and provided us with a structural alignment. However, because of the rather low level of sequence identity (~12%), this alignment needed to be refined and critically assessed. The alignment was refined taking into account the following: first the position of the highly conserved residues, considering these are likely buried in the core structure (Fig. 2A); second, the position of the hydrophobic residues likely exposed to the plasma membrane; and third, amino acid hydrophobicity was also used to delimitate helix termini, and large sequence variation (in length and amino acid composition) was clustered in loops. The refinement of the combined alignment was performed by several rounds of manual editing, molecular modeling, and structure analysis using the program ViTO on the @TOME meta-server. This strategy was first applied locally, i.e. on each TM separately, and then validated on the full model.

As illustrated with the GABAB2 HD (Fig. 2), the final models obtained for various class C GPCR HDs revealed consistencies with a rhodopsin-like structure. For example, TM1 showed only a thin patch of conserved residues in agreement with its thin interhelical surface. Also in agreement with TM3 being mostly buried in the core structure, this helix showed the same bias in amino acid composition (enrichment in Ser, Thr, Gly, and Ala) in class A and class C GPCRs. Moreover, the conserved disulfide bridge that links TM3 to TM5 in class A receptor is predicted to be present in class C GPCRs.

The final model obtained is in agreement with those already reported (14, 29-33). Indeed an identical alignment of TM1, TM3, TM6, and TM7 with rhodopsin was even published for the calcium sensing receptor (33). These models were validated by directed mutagenesis of allosteric ligand putative binding sites. Indeed, the HD of class C receptors contains a binding site for allosteric modulators at positions similar to that of the binding site of small ligands interacting with class A receptors (14, 29-33). We further checked that our structural alignment is compatible with these additional restraints by building models for the corresponding receptors (especially GABAB2, mGluR1, mGluR5, CaSR, and T1R3) and assessing the positions of the residues proposed to line the binding pocket (data not shown).


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3.
Expression at the cell surface and functional assay of the receptors containing a mutated GABAB2 subunit. A, cell surface expression of the receptors was analyzed using an ELISA method on intact cells and by detecting the presence of the GABAB1 subunit tagged at its extracellular end by an HA epitope. As GABAB1 reaches the cell surface only when associated with GABAB2, the detection of the GABAB1 at the cell surface indicates that GABAB2 was folded and able to interact correctly with GABAB1 and to take it to the cell surface. The data are the means ± S.E. of at least three independent experiments performed in triplicate. B, in functional assays, the ligand-induced activity of the mutated receptors was assayed by quantifying the accumulating InP second messenger molecules formed upon activation of the receptors by GABA 1 mM. To get coupled to the InP pathway, the GABAB receptor is expressed with the Gqi9 chimeric G protein (see "Experimental Procedures"). The data are the means ± S.E. of at least four independent experiments performed in triplicate. WT, wild type.

 
Using this alignment and the GABAB2 model, we examined the predicted position of the highly conserved residues identified at the cytoplasmic face of TM3 and TM6 of class C GPCRs. The conserved arginine in TM3 (Arg-575 in GABAB2) is shifted by one residue from the arginine of the (D/E)RY motif of class A receptors. However, it is optimally located near the solvent interface where the G proteins are expected to bind. Our model also places the conserved TM3 lysine (Lys-572 in GABAB2) one helical turn above the former arginine in a buried and rather hydrophobic environment. Searching for negatively charged residues in the vicinity of this lysine highlighted solely the highly conserved aspartate of TM6 (Asp-688 in GABAB2). Of interest, this aspartate residue is located one turn of {alpha}-helix above the highly conserved acidic residue at the bottom of TM6 in class A receptors (Glu-247 in rhodopsin). As depicted in Fig. 2B, the three-dimensional model of the class C GABAB2 HD is consistent with the conserved charged residues identified in TM3 and TM6 facing each other. This ménage-à-trois might be involved in a network of ionic interactions mimicking that observed in class A GPCRs involving the (D/E)RY motif.


Figure 4
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 4.
Expression at the cell surface and functional assay of the receptors containing a mutated GABAB1 subunit. A, cell surface expression of the receptors was analyzed using an ELISA method on intact cells as described in Fig. 3. The data are the means ± S.E. of at least three independent experiments performed in triplicate. B, ligand-induced activity of the mutated receptors was measured as described in Fig. 3. The data are the means ± S.E. of at least four independent experiments performed in triplicate. WT, wild type; InP, inositol phosphate; mGlu, metabotropic Glu.

 
Mutated GABAB Subunits Are Correctly Expressed at the Cell Surface—To test the possible involvement of the conserved basic and acidic residues, these were mutated such that the charge is suppressed (alanine), conserved, or inverted. To perform this study, we used the GABAB receptor as a model system. This receptor is a heterodimer composed of two homologous proteins GABAB1 and GABAB2. Although the GABAB1 subunit is responsible for agonist binding (34, 35), the GABAB2 subunit plays a pivotal role in G protein activation (20, 36-38). Of interest, one of the three conserved residues (arginine) is not conserved in the mammalian GABAB1 HD (Fig. 1). As such, analyzing the role of these conserved residues in this receptor heterodimer may also allow a better understanding of the specific role of each HDs in G protein activation.

We first mutated these three conserved residues (Lys-572, Arg-575, and Asp-688) in the GABAB2 subunit and co-expressed these with the wild-type GABAB1 subunit. As shown in Fig. 3A, the mutated GABAB2 subunits allow the correct surface targeting of the N-terminal HA-tagged GABAB1 subunit (between 77 ± 1 and 125 ± 11% of the wild-type GABAB receptor cell surface expression), as revealed by an ELISA performed on intact cells using anti-HA antibodies. Because it is well known that GABAB1 reaches the cell surface when associated with GABAB2 only (19, 39-41), these data indicate that the mutations introduced in the GABAB2 HD affect neither GABAB2 expression nor its ability to form heterodimeric complexes with GABAB1.

Mutations of the corresponding residues in GABAB1 (Lys-683, Trp-686, and Asp-800) were also generated. These HA-tagged mutants were expressed between 33 ± 3 and 107 ± 28% of the wild-type GABAB receptor (Fig. 4A) when co-expressed with GABAB2. Again, these data indicate that the mutations introduced in the HD of GABAB1 do not restrict protein expression nor the correct interaction of the mutant with GABAB2 because interaction is needed for these subunits to reach the cell surface.

As expected by the modular nature of the GABAB heterodimeric receptor, all these mutant dimers were found to bind GABA at the surface of intact cells, as revealed by displacement of bound [3H]CGP54626 by 1 mM GABA (data not shown).

Mutation of the Residues Lys-572, Arg-575, and Asp-688 of GABAB2 Led to Different Phenotypes—As shown in Fig. 3B, the mutations of the three residues Lys-572, Arg-575, and Asp-688 in GABAB2 led to different effects on the activity of the GABAB receptor as measured with an InP accumulation assay. Because the GABAB receptor is coupled to Gi/o G proteins, the GABAB subunits were co-transfected with the chimeric G protein Gqi9 allowing it to activate phospholipase C (42). As reported previously, this approach allowed the measurement of both basal and agonist-induced activities of the receptor (Fig. 3B). As shown in Fig. 3B, the mutation of Arg-575 into any of the tested residues (R575K, R575A, R575D, and R575E) suppressed GABAB receptor activity (both basal and agonist-stimulated). In contrast, the mutation of Asp-688 into either alanine (D688A), lysine (D688K), arginine (D688R), or glutamate (D688E) led to a functional receptor, with the D688E mutant displaying a higher constitutive activity than the wild-type (209 ± 24% of wild type basal activity) and a slightly higher agonist potency (EC50 = 310 ± 70 and 190 ± 10 nM for the wild type and the D688E mutant, respectively) (Fig. 7). The D688R and D688K mutants are less active, with both a lower agonist-induced response and a lower basal activity (Fig. 3B), and a GABA potency similar to or lower than that of the wild-type receptor (EC50 = 640 ± 160 and 410 ± 80 nM for the D688K and D688R mutant, respectively (Fig. 7)).


Figure 5
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 5.
Expression at the cell surface and functional assay of the receptors containing the GABAB2 subunit bearing the double mutation of Lys-572 and Asp-688. A, cell surface expression of the receptors was analyzed using an ELISA method on intact cells as described in Fig. 3. The data are the means ± S.E. of at least three independent experiments performed in triplicate. B, functional responses of mutated receptors. The ligand-induced activity of the mutated receptors was measured as described in Fig. 3. The data are the means ± S.E. of at least three independent experiments performed in triplicate. WT, wild type.

 
In respect to the Lys-572, its mutation into an acidic residue (K572D and K572E) suppressed receptor activity (both basal and agonist-induced; Fig. 3B) despite the good expression level of these mutant subunits (124 ± 22 and 105 ± 15% of wild-type expression, respectively; Fig. 3A) and their ability to bind GABA (Fig. 8). This indicates that a negative charge at this position prevents GABAB receptor function. The mutation of this residue into either alanine or arginine led to functional receptors with lower basal (K572A) and agonist-induced (K572A and K572R) activities than the wild-type combination. The mutation of the corresponding residues in GABAB1 did not affect the functional properties of the GABAB receptor (Fig. 4B; GABA EC50 being 310 ± 70, 490 ± 60, and 640 ± 100 nM for the wild-type, GB1K683D, and GB1D800K mutants, respectively).

Inversion of the Charge of Asp-688 Can Rescue the Function of the Inactive K572D or K572E Mutants—The proposed model for the HD of GABAB2 suggested that Lys-572 and Arg-575 of TM3 are facing Asp-688 of TM6 suggesting the possible formation of an ionic interaction between TM3 and TM6. Therefore, we examined whether the mutation of Asp-688 into either lysine or arginine could rescue the loss of function of the K572D, K572E, R575D, or R575E mutants.

As shown in Fig. 5A, receptors carrying the double mutations K572D/D688K, K572E/D688K, K572D/D688R, and K572E/D688R were all expressed at the cell surface like the wild-type receptor. Of interest, these four mutants could activate Gqi9 upon stimulation with saturating concentrations of GABA, although the responses measured were low compared with those obtained with the wild-type receptor (Fig. 5B). The best responses were obtained with the K572D/D688K and the K572E/D688R mutants. Full dose-response curves of GABA further revealed that the lower response resulted from a lower coupling efficacy, rather than a large decrease in agonist potency (Fig. 7). Indeed, the potency of GABA on K572D/D688K mutated receptor was only 3-fold lower than that measured on the wild-type receptor (EC50 = 880 ± 70 and 310 ± 70 nM, respectively). In contrast, mutating Asp-688 into a basic residue did not restore function of the R575D and R575E mutants (Fig. 6B), even though these double mutants were all found at the cell surface (Fig. 6A). These data highlight that opposite charges at positions 572 of TM3 and 688 of TM6 of GABAB2 are important for G protein activation by this receptor, although not absolutely required because the mutation of Asp-688 into alanine, lysine, or arginine does not prevent function.

The GABAB2 K572D/K572E Mutants Display a High Agonist Affinity—In some cases, mutation of the arginine residue of the (D/E)RY motif of class A GPCRs leads to constitutively active receptors, as observed with the {alpha}1B adrenergic receptor receptors (43). Alternatively, such a mutation can result in a non-functional receptor that shares with constitutively active receptors high agonist affinity, such as for the H2 histamine receptor (44). None of the mutations of Lys-572 or Arg-575 of the GABAB2 lead to constitutive activity. We therefore examined whether the mutation of these residues affects agonist affinity of the GABAB heterodimer. Binding experiments performed using the high affinity GABAB receptor antagonist [3H]CGP54626 revealed that all mutants of Lys-572 and Arg-575 bind the corresponding nonradiolabeled antagonist CGP54626 with the same Ki value as on the wild-type receptor (Ki = 1.22 ± 0.22, 1.58 ± 0.31, 1.40 ± 0.24, and 1.57 ± 0.23 nM for K572D, K572E, R575D, and wild-type receptors, respectively). Analysis of the affinity (Ki) of the agonist GABA revealed that, although not functional (Fig. 7), the K572D and K572E displayed a higher agonist affinity than the wild-type receptor or the K572A mutant (Ki = 2.3 ± 0.3, 2.1 ± 0.3, 6.7 ± 0.4, and 5.8 ± 0.3 µM, respectively) (Fig. 8A). In contrast, the R575D mutant displayed the same affinity for GABA as the wild-type receptor (Ki = 6.8 ± 0.8 µM) (Fig. 8A). The same analysis was also performed on the D688E mutant that displays a higher constitutive activity than the wild-type receptor. As shown in Fig. 8B, this constitutively active mutant also displays a higher affinity for GABA (Ki = 3.7 ± 0.5 and 6.7 ± 0.4 µM for the D688E mutant and the wild-type, respectively). As a control, the affinity of the antagonist CGP54626 was not altered by the mutation (1.28 ± 0.37 and 1.57 ± 0.23 nM for D688E and wild-type, respectively). The increase in agonist affinity is thus consistent with the conformation of the K572D/K572E mutated GABAB2 HD resembling that of the active state.


Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 6.
Expression at the cell surface and functional assay of the receptors containing the GABAB2 subunit bearing the double mutation of Arg-575 and Asp-688. A, cell surface expression of the receptors was analyzed using an ELISA method on intact cells as described in Fig. 3. The data are the means ± S.E. of at least three independent experiments performed in triplicate. B, functional responses of mutated receptors. The ligand induced activity of the mutated receptors was measured as described in Fig. 3. The data are the means ± S.E. of at least three independent experiments performed in triplicate. WT, wild type.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we identified two basic residues located in the cytoplasmic end of TM3 and one acidic residue located in the cytoplasmic end of TM6 highly conserved among class C GPCRs. We show that these residues (Lys-572, Arg-575, and Asp-688 in GABAB2) play an important role in GABAB receptor activation.


Figure 7
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 7.
Dose-response curves of the receptor in a functional InP assay. The mutated receptor activity was measured in the presence of increasing concentrations of GABA. The indicated GABAB2 subunits were expressed together with the wild-type GABAB1 subunit and the Gqi9 chimeric G protein. See "Experimental Procedures" for details. This experiment is representative of three independent experiments performed in triplicate.

 


Figure 8
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 8.
Displacement curves of the radioligand [3H]CGP54626 by increasing concentrations of GABA on wild-type and mutated GABAB receptor. The indicated GABAB2 subunits were expressed together with the wild-type (WT) GABAB1 subunit. See "Experimental Procedures" for details. A, displacement curves on the wild-type GABAB receptor and on receptors bearing the mutations K572D, K572E, or K572A and R575D. This experiment is representative of three independent experiments performed in triplicate. B, displacement curves on the wild-type GABAB receptor and on receptors bearing the mutation D688E. This experiment is representative of three independent experiments performed in triplicate.

 
The most conserved residue of class A GPCRs is the arginine of the (D/E)RY motif at the cytoplasmic side of TM3. Indeed, among the 3% mammalian rhodopsin-like GPCRs that lack this residue, none have been shown to activate G proteins (45). Moreover, for the receptors lacking this basic residue, introduction of an arginine enables them to activate G proteins, as shown for example for the C5A-binding protein C5L2 (46) or the olfactory receptor 912-93 (47). In the inactive state of rhodopsin, this arginine residue makes an ionic interaction with the preceding glutamate of the ERY motif and makes hydrogen bonds with Glu-247 of TM6 (conserved in many GPCRs) (4, 25) and possibly Thr-251 as shown in some models of rhodopsin (4). Disrupting this interaction network has been shown to either prevent G protein activation or to stabilize the receptor in an active state. Indeed, protonation of the acidic residue of the ERY motif allows the arginine side chain to move and to make new contacts, and this could be a molecular step in the process leading to rhodopsin activation (Ref. 48 and reference therein). Moreover, neutralization of this acidic residue in many GPCRs results in their constitutive activation reinforcing the idea of its role in stabilizing the inactive state of the receptor (44, 49-52). In contrast, mutation of the arginine residue leads to various phenotypes, either a loss (53-58) or a gain of function (43, 59-61). In many cases, arginine mutations leading to loss of function are associated with a receptor conformation resembling that of the active state, as observed with the {alpha}1b adrenergic, the M1 muscarinic, and the H2 histamine receptors (43, 55, 62, 63). This may be the consequence of constitutive desensitization of the receptor as reported for a mutant of the V2 vasopressin receptor (64). Finally, the possible ionic interactions between this arginine and a conserved acidic residue of TM6 has been shown to maintain the beta2-adrenergic, the {alpha}1b, and the luteinizing hormone/human chorionic gonadotropin receptors in their inactive state (52, 65-67). The structure of an active state of rhodopsin has been solved recently and revealed only small conformational changes in the protein (68). However, it cannot be excluded that such limited conformational changes result from constraints from the crystal lattice or from the absence of transducin. Using three-dimensional modeling of the rhodopsin-transducin complex, it has also been proposed that the arginine of the ERY motif directly contacts the G protein (69), therefore playing a direct role in G protein activation. Accordingly, two roles, not necessarily exclusive, have been proposed for the arginine of the (D/E)RY motif of class A GPCRs as follows: 1) stabilization of the inactive state through an ionic network involving TM6 residues, or 2) a direct role in G protein interaction and activation.

Although the (D/E)RY motif is not found in class C GPCRs, two basic residues are highly conserved at an equivalent position (at the cytoplasmic end of TM3) in these receptors. Moreover, like arginine of the (D/E)RY motif, three-dimensional models suggest that their side chains are directed toward or facing TM6 where a conserved acidic residue is located. Mutation of the more C-terminal basic residue Arg-575 in GABAB2 resulted in a loss of function and suppression of the constitutive activity of the receptor, even though the cell surface expression of the mutants was normal. The loss of function of the R575D/R575E mutants cannot be restored by replacing Asp-688 of TM6 by a basic residue. Finally, agonist affinity is not affected by such mutation, indicating that arginine mutation does not convert the receptor into an active-like conformation uncoupled to G proteins. Accordingly, this arginine appears necessary for G protein activation, but the exact reason for this is not clear. As in our GABAB2 model, the arginine residue Arg-575 is oriented toward the G protein, and it is possible that this arginine is required for the proper interaction with the G protein as proposed for Arg-135 of rhodopsin (69). In agreement with this possibility, arginine 575 points toward the cytoplasm, in a cavity between the intracellular loops 2 and 3 (Fig. 2B) known to contact the G{alpha} protein in class C GPCRs (70-72). However, this arginine is not conserved in some class C GPCRs such as the taste T1Rs and mGlu3, despite their known capacity to activate G proteins.

The other conserved basic residue in TM3 of class C GPCRs is a lysine that is located one turn of the {alpha}-helix above the arginine. Replacement of the lysine by an acidic residue resulted in a total loss of function that could be partly restored by mutating Asp-688 of TM6 into arginine or lysine. This likely results from an ionic interaction between the two mutated residues. This is surprising when considering that the K572A and the four mutants of Asp-688 are functional indicating that an ionic interaction between these two positions is not absolutely required for function. However, this does not exclude an ionic interaction between Lys-K572 and Asp-688, because other interactions between TM3 and TM6 may still maintain the correct relative positioning of these helices in the absence of Asp-688. Of interest, equivalent mutations of the corresponding acidic residue in TM6 in the calcium-sensing receptor lead to a nonfunctional receptor (73, 74). If Lys-572 and Asp-688 are facing each other, mutation of Lys-572 into an acidic residue may result in a repulsion of TM3 and TM6, resulting in a change in conformation of the GABAB2 HD. Consistent with this proposal, K572D/K572E mutants display a higher affinity for GABA. Of interest, increasing the length of the acidic side chain of TM6 by replacing Asp-688 by glutamate also increases agonist affinity, consistent with the high agonist affinity state resulting from a movement of TM6 relative to TM3. It is surprising that the mutation of Asp-688 into a basic residue did not have the same effect despite the presence of positive charges in both TM3 and TM6. However, the length of the side chains of lysine and arginine is such that the repulsion of the charges may not necessarily affect the relative positions of the helices.

Although the higher agonist affinity state of the D688E and K572D/K572E mutants may possibly correspond to an active state, only the D688E mutant displays a higher constitutive activity, with the K572D/K572E mutants being inactive. The loss of function of the K572D/K572E does not result from a constitutive desensitization of the GABAB receptor, as reported for a mutant of the V2 vasopressin receptor (64), for several reasons. First, this mutant is still expressed normally at the cell surface, indicating that internalization cannot be responsible for the loss of function. Indeed, the GABAB receptor internalizes neither in HEK293 cells nor in neurons even after long term activation by agonist (75, 76). Second, desensitization of the GABAB receptor has been observed only in the presence of GRK4 that is not expressed in HEK293 cells (75). Moreover, the basal activity of a fully constitutively active GABAB receptor can easily be measured in these cells with our assay (77). Accordingly, the K572D/K572E mutants may be in a specific inactive conformation that is associated, however, with higher agonist affinity. Alternatively, this mutant may be in an active conformation but unable to activate G proteins. As shown above, Arg-575 is crucial for G protein activation by this receptor. This arginine may well interact with the acidic residue at position 572, one turn of the {alpha}-helix above, in the K572E mutant (Figs. 2 and 9), therefore preventing G protein activation. In agreement with this proposal, the double mutation inverting the charges at the bottom of TM3 and TM6 (the K572D/K572E and D688K/D688R) restores receptor function. In this mutant, the reintroduction of the ionic interaction between these TMs likely allows Arg-575 to play its own role in G protein activation (Fig. 9).


Figure 9
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 9.
Putative functional role of the residues Lys-572, Arg-575, and Asp-688 at the interface of the TM3 and TM6 of GABAB2. A, three-dimensional model of the mutated GABAB2. For clarity, only the region of the mutated residues (Lys-572, Arg-575, and Asp-688) is shown. The structure rendering and the color code used are as in Fig. 2. B, schematic representation of the putative effect of the mutation K572D and of the reversing effect of the double mutation K572D/D688K in GABAB2 deduced from the experimental data and the structural analysis.

 
Our data further illustrate the critical role of the GABAB2 HD in G protein activation by the heterotrimeric GABAB receptor. Indeed, consistent with previous studies (36, 38), single point mutations in the GABAB2 HD are sufficient to suppress G protein activation by the heterodimer, whereas the equivalent mutations in the GABAB1 HD have no effect. It is interesting to note, however, that both the lysine and the acidic residues of TM3 and TM6, respectively, are conserved in GABAB1 (Fig. 1A). In contrast, the arginine residue, although conserved in the Caenorhabditis elegans and Drosophila GABAB1 subunit, is replaced by a tryptophan in vertebrates (Fig. 1A). However, replacement of this tryptophan by an arginine was not sufficient to allow this GABAB receptor subunit to activate G proteins in the absence of GABAB2 or in the presence of a nonfunctional GABAB2 subunit.3 This is in agreement with the absence of function of the Drosophila GABAB1 that still possesses the arginine (78), and further indicates that other specific constraints prevent GABAB1 subunit from activating G proteins.

Taken together, these data highlight the importance of charged residues at the cytoplasmic ends of TM3 and TM6 of class C GPCRs in a dynamic network of interactions controlling activation of the receptor, as already established for many class A GPCRs. Indeed, each of the two conserved basic residues of class C TM3 plays one the proposed roles of the arginine of the (D/E)RY motif of class A GPCRs, with the lysine residue being involved in an ionic interaction with TM6, whereas the arginine plays a direct role in G protein activation. These data revealed that despite their modular structure and their constitutive dimeric organization, the HDs of class C GPCRs share more functional similarities with those of class A GPCRs than originally thought.


   FOOTNOTES
 
* This work was supported by grants from the CNRS, Action Concertée Incitative ACI-BCMS 328 Contract 45 491 (to J. P. P.), the French Ministry of Research Grant ANR-05-NEUR-035, the European Community EEC STREP Program GPCR from the 6th PCRDT Grant LSHB-CT-2003-503337 (to J. P. P.), and contracts with Addex Pharmaceuticals. 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

1 To whom correspondence should be addressed: Dépt. de Pharmacologie Moléculaire, Institut de Génomique Fonctionnelle, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France. Tel.: 33-467-14-29-33; Fax: 33-467-54-24-32; E-mail: laurent.prezeau{at}igf.cnrs.fr.

2 The abbreviations used are: GPCR, G protein-coupled receptors; TM, transmembrane; HD, heptahelical domain; GABA, {gamma}-aminobutyric acid; GABAB, {gamma}-aminobutyric acid, type B; HEK, human embryonic kidney; HA, hemag-glutinin; ELISA, enzyme-linked immunosorbent assay; mGlu, metabotropic Glu. Back

3 M. Havlackova, J. Blahos, L. Prézeau, and J.-P. Pin, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank T. Durroux for helpful discussion and reading.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bockaert, J., Claeysen, S., Becamel, C., Pinloche, S., and Dumuis, A. (2002) Int. Rev. Cytol. 212, 63-132[Medline] [Order article via Infotrieve]
  2. Karnik, S. S., Gogonea, C., Patil, S., Saad, Y., and Takezako, T. (2003) Trends Endocrinol. Metab. 14, 431-437[CrossRef][Medline] [Order article via Infotrieve]
  3. Gether, U. (2000) Endocr. Rev. 21, 90-113[Abstract/Free Full Text]
  4. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745[Abstract/Free Full Text]
  5. Fredriksson, R., Lagerstrom, M. C., Lundin, L. G., and Schioth, H. B. (2003) Mol. Pharmacol. 63, 1256-1272[Abstract/Free Full Text]
  6. Foord, S. M., Bonner, T. I., Neubig, R. R., Rosser, E. M., Pin, J. P., Davenport, A. P., Spedding, M., and Harmar, A. J. (2005) Pharmacol. Rev. 57, 279-288[Abstract/Free Full Text]
  7. Lu, Z. L., Saldanha, J. W., and Hulme, E. C. (2002) Trends Pharmacol. Sci. 23, 140-146[CrossRef][Medline] [Order article via Infotrieve]
  8. Flanagan, C. A. (2005) Mol. Pharmacol. 68, 1-3[Abstract/Free Full Text]
  9. Perez, D. M., and Karnik, S. S. (2005) Pharmacol. Rev. 57, 147-161[Abstract/Free Full Text]
  10. Pin, J.-P., Galvez, T., and Prezeau, L. (2003) Pharmacol. Ther. 98, 325-354[CrossRef][Medline] [Order article via Infotrieve]
  11. Pin, J. P., Kniazeff, J., Goudet, C., Bessis, A. S., Liu, J., Galvez, T., Acher, F., Rondard, P., and Prezeau, L. (2004) Biol. Cell 96, 335-342[CrossRef][Medline] [Order article via Infotrieve]
  12. Pin, J. P., Kniazeff, J., Liu, J., Binet, V., Goudet, C., Rondard, P., and Prezeau, L. (2005) FEBS J. 272, 2947-2955[CrossRef][Medline] [Order article via Infotrieve]
  13. 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]
  14. Pagano, A., Ruegg, D., Litschig, S., Stoehr, N., Stierlin, C., Heinrich, M., Floersheim, P., Prezeau, L., Carroll, F., Pin, J. P., Cambria, A., Vranesic, I., Flor, P. J., Gasparini, F., and Kuhn, R. (2000) J. Biol. Chem. 275, 33750-33758[Abstract/Free Full Text]
  15. Goudet, C., Gaven, F., Kniazeff, J., Vol, C., Liu, J., Cohen-Gonsaud, M., Acher, F., Prezeau, L., and Pin, J. P. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 378-383[Abstract/Free Full Text]
  16. Goudet, C., Binet, V., Prezeau, L., and Pin, J.-P. (2004) Drug Discov. Today Ther. Strateg. 1, 125-133[CrossRef]
  17. Binet, V., Brajon, C., Le Corre, L., Acher, F., Pin, J. P., and Prezeau, L. (2004) J. Biol. Chem. 279, 29085-29091[Abstract/Free Full Text]
  18. Ray, K., Tisdale, J., Dodd, R. H., Dauban, P., Ruat, M., and Northup, J. K. (2005) J. Biol. Chem. 280, 37013-37020[Abstract/Free Full Text]
  19. Pagano, A., Rovelli, G., Mosbacher, J., Lohmann, T., Duthey, B., Stauffer, D., Ristig, D., Schuler, V., Heid, J., Meigel, I., Lampert, C., Stein, T., Prézeau, L., Pin, J.-P., Froestl, W., Kuhn, R., Kaupmann, K., and Bettler, B. (2001) J. Neurosci. 21, 1189-1202[Abstract/Free Full Text]
  20. Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prezeau, L., and Pin, J. P. (2001) EMBO J. 20, 2152-2159[CrossRef][Medline] [Order article via Infotrieve]
  21. Liu, J., Maurel, D., Etzol, S., Brabet, I., Pin, J.-P., and Rondard, P. (2004) J. Biol. Chem. 279, 15824-15830[Abstract/Free Full Text]
  22. Higgins, D., Thompson, J., Gibson, T., Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) Nucleic Acids Res. 4673-4680
  23. Douguet, D., and Labesse, G. (2001) Bioinformatics (Oxf.) 17, 752-753[Abstract/Free Full Text]
  24. Okada, T., Sugihara, M., Bondar, A. N., Elstner, M., Entel, P., and Buss, V. (2004) J. Mol. Biol. 342, 571-583[CrossRef][Medline] [Order article via Infotrieve]
  25. Li, J., Edwards, P. C., Burghammer, M., Villa, C., and Schertler, G. F. (2004) J. Mol. Biol. 343, 1409-1438[CrossRef][Medline] [Order article via Infotrieve]
  26. Catherinot, V., and Labesse, G. (2004) Bioinformatics (Oxf.) 20, 3694-3696[Abstract/Free Full Text]
  27. Canutescu, A., Shelenkov, A., and Dunbrack, R. J. (2003) Protein Sci. 12, 2001-2014[CrossRef][Medline] [Order article via Infotrieve]
  28. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779-815[CrossRef][Medline] [Order article via Infotrieve]
  29. Jiang, P., Cui, M., Zhao, B., Liu, Z., Snyder, L. A., Benard, L. M., Osman, R., Margolskee, R. F., and Max, M. (2005) J. Biol. Chem. 280, 15238-15246[Abstract/Free Full Text]
  30. Miedlich, S. U., Gama, L., Seuwen, K., Wolf, R. M., and Breitwieser, G. E. (2004) J. Biol. Chem. 279, 7254-7263[Abstract/Free Full Text]
  31. Malherbe, P., Kratochwil, N., Knoflach, F., Zenner, M. T., Kew, J. N., Kratzeisen, C., Maerki, H. P., Adam, G., and Mutel, V. (2003) J. Biol. Chem. 278, 8340-8347[Abstract/Free Full Text]
  32. Malherbe, P., Kratochwil, N., Zenner, M. T., Piussi, J., Diener, C., Kratzeisen, C., Fischer, C., and Porter, R. H. (2003) Mol. Pharmacol. 64, 823-832[Abstract/Free Full Text]
  33. Petrel, C., Kessler, A., Maslah, F., Dauban, P., Dodd, R. H., Rognan, D., and Ruat, M. (2003) J. Biol. Chem. 278, 49487-49494[Abstract/Free Full Text]
  34. Kniazeff, J., Galvez, T., Labesse, G., and Pin, J. P. (2002) J. Neurosci. 22, 7352-7361[Abstract/Free Full Text]
  35. Galvez, T., Parmentier, M. L., Joly, C., Malitschek, B., Kaupmann, K., Kuhn, R., Bittiger, H., Froestl, W., Bettler, B., and Pin, J. P. (1999) J. Biol. Chem. 274, 13362-13369[Abstract/Free Full Text]
  36. Duthey, B., Caudron, S., Perroy, J., Bettler, B., Fagni, L., Pin, J. P., and Prezeau, L. (2002) J. Biol. Chem. 277, 3236-3241[Abstract/Free Full Text]
  37. Margeta-Mitrovic, M., Jan, Y. N., and Jan, L. Y. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 14649-14654[Abstract/Free Full Text]
  38. Robbins, M. J., Calver, A. R., Filippov, A. K., Hirst, W. D., Russell, R. B., Wood, M. D., Nasir, S., Couve, A., Brown, D. A., Moss, S. J., and Pangalos, M. N. (2001) J. Neurosci. 21, 8043-8052[Abstract/Free Full Text]
  39. Brock, C., Boudier, L., Maurel, D., Blahos, J., and Pin, J. P. (2005) Mol. Biol. Cell 16, 5572-5578[Abstract/Free Full Text]
  40. Couve, A., Filippov, A. K., Connolly, C. N., Bettler, B., Brown, D. A., and Moss, S. J. (1998) J. Biol. Chem. 273, 26361-26367[Abstract/Free Full Text]
  41. Margeta-Mitrovic, M., Jan, Y. N., and Jan, L. Y. (2000) Neuron 27, 97-106[CrossRef][Medline] [Order article via Infotrieve]
  42. Gomeza, J., Mary, S., Brabet, I., Parmentier, M.-L., Restituito, S., Bockaert, J., and Pin, J.-P. (1996) Mol. Pharmacol. 50, 923-930[Abstract]
  43. Scheer, A., Costa, T., Fanelli, F., De Benedetti, P. G., Mhaouty-Kodja, S., Abuin, L., Nenniger-Tosato, M., and Cotecchia, S. (2000) Mol. Pharmacol. 57, 219-231[Abstract/Free Full Text]
  44. Alewijnse, A. E., Timmerman, H., Jacobs, E. H., Smit, M. J., Roovers, E., Cotecchia, S., and Leurs, R. (2000) Mol. Pharmacol. 57, 890-898[Abstract/Free Full Text]
  45. Rosenkilde, M. M., Kledal, T. N., and Schwartz, T. W. (2005) Mol. Pharmacol. 68, 11-19[Abstract/Free Full Text]
  46. Okinaga, S., Slattery, D., Humbles, A., Zsengeller, Z., Morteau, O., Kinrade, M. B., Brodbeck, R. M., Krause, J. E., Choe, H. R., Gerard, N. P., and Gerard, C. (2003) Biochemistry 42, 9406-9415[CrossRef][Medline] [Order article via Infotrieve]
  47. Gaillard, I., Rouquier, S., Chavanieu, A., Mollard, P., and Giorgi, D. (2004) Hum. Mol. Genet. 13, 771-780[Abstract/Free Full Text]
  48. Menon, S., Han, M., and Sakmar, T. (2001) Pharmacol. Rev. 81, 1659-1688
  49. Capra, V., Veltri, A., Foglia, C., Crimaldi, L., Habib, A., Parenti, M., and Rovati, G. E. (2004) Mol. Pharmacol. 66, 880-889[Abstract/Free Full Text]
  50. Scheer, A., and Cotecchia, S. (1997) J. Recept. Signal. Transduct. Res. 17, 57-73[Medline] [Order article via Infotrieve]
  51. Rasmussen, S. G., Jensen, A. D., Liapakis, G., Ghanouni, P., Javitch, J. A., and Gether, U. (1999) Mol. Pharmacol. 56, 175-184[Abstract/Free Full Text]
  52. Ballesteros, J. A., Shi, L., and Javitch, J. A. (2001) Mol. Pharmacol. 60, 1-19[Abstract/Free Full Text]
  53. Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033[Abstract/Free Full Text]
  54. Acharya, S., and Karnik, S. S. (1996) J. Biol. Chem. 271, 25406-25411[Abstract/Free Full Text]
  55. Scheer, A., Fanelli, F., Costa, T., De Benedetti, P. G., and Cotecchia, S. (1996) EMBO J. 15, 3566-3578[Medline] [Order article via Infotrieve]
  56. Lu, Z. L., Curtis, C. A., Jones, P. G., Pavia, J., and Hulme, E. C. (1997) Mol. Pharmacol. 51, 234-241[Abstract/Free Full Text]
  57. Gruijthuijsen, Y. K., Beuken, E. V., Smit, M. J., Leurs, R., Bruggeman, C. A., and Vink, C. (2004) J. Gen. Virol. 85, 897-909[Abstract/Free Full Text]
  58. Wess, J. (1998) Pharmacol. Ther. 80, 231-264[CrossRef][Medline] [Order article via Infotrieve]
  59. Seibold, A., Dagarag, M., and Birnbaumer, M. (1998) Receptors Channels 5, 375-385[Medline] [Order article via Infotrieve]
  60. Fanelli, F., Menziani, C., Scheer, A., Cotecchia, S., and De Benedetti, P. G. (1999) Proteins 37, 145-156[CrossRef][Medline] [Order article via Infotrieve]
  61. Chen, A., Gao, Z. G., Barak, D., Liang, B. T., and Jacobson, K. A. (2001) Biochem. Biophys. Res. Commun. 284, 596-601[CrossRef][Medline] [Order article via Infotrieve]
  62. Wilbanks, A. M., Laporte, S. A., Bohn, L. M., Barak, L. S., and Caron, M. G. (2002) Biochemistry 41, 11981-11989[CrossRef][Medline] [Order article via Infotrieve]
  63. Jones, P. G., Curtis, C. A., and Hulme, E. C. (1995) Eur. J. Pharmacol. 288, 251-257[CrossRef][Medline] [Order article via Infotrieve]
  64. Barak, L. S., Oakley, R. H., Laporte, S. A., and Caron, M. G. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 93-98[Abstract/Free Full Text]
  65. Greasley, P. J., Fanelli, F., Rossier, O., Abuin, L., and Cotecchia, S. (2002) Mol. Pharmacol. 61, 1025-1032[Abstract/Free Full Text]
  66. Shenker, A., Laue, L., Kosug, S., Merendino, J. J., Minegishi, T., and Cutler, J. B. (1993) Nature 365, 652-654[CrossRef][Medline] [Order article via Infotrieve]
  67. Yano, K., Saji, M., Hidaka, A., Moriya, N., Okuno, A., Kohn, L. D., and Cutler, G. B., Jr. (1995) J. Clin. Endocrinol. Metab. 80, 1162-1168[Abstract]
  68. Salom, D., Lodowski, D. T., Stenkamp, R. E., Trong, I. L., Golczak, M., Jastrzebska, B., Harris, T., Ballesteros, J. A., and Palczewski, K. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 16123-16128[Abstract/Free Full Text]
  69. Filipek, S., Krzysko, K. A., Fotiadis, D., Liang, Y., Saperstein, D. A., Engel, A., and Palczewski, K. (2004) Photochem. Photobiol. Sci. 3, 628-638[CrossRef][Medline] [Order article via Infotrieve]
  70. Pin, J.-P., Joly, C., Heinemann, S. F., and Bockaert, J. (1994) EMBO J. 13, 342-348[Medline] [Order article via Infotrieve]
  71. Havlickova, M., Blahos, J., Brabet, I., Liu, J., Hruskova, B., Prezeau, L., and Pin, J. P. (2003) J. Biol. Chem. 278, 35063-35070[Abstract/Free Full Text]
  72. Gomeza, J., Joly, C., Kuhn, R., Knopfel, T., Bockaert, J., and Pin, J. P. (1996) J. Biol. Chem. 271, 2199-2205[Abstract/Free Full Text]
  73. Francesconi, A., and Duvoisin, R. M. (1998) J. Biol. Chem. 273, 5615-5624[Abstract/Free Full Text]
  74. Chang, W., Chen, T. H., Pratt, S., and Shoback, D. (2000) J. Biol. Chem. 275, 19955-19963[Abstract/Free Full Text]
  75. Perroy, J., Adam, L., Qanbar, R., Chenier, S., and Bouvier, M. (2003) EMBO J. 22, 3816-3824[CrossRef][Medline] [Order article via Infotrieve]
  76. Couve, A., Restituito, S., Brandon, J. M., Charles, K. J., Bawagan, H., Freeman, K. B., Pangalos, M. N., Calver, A. R., and Moss, S. J. (2004) J. Biol. Chem. 279, 13934-13943[Abstract/Free Full Text]
  77. 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]
  78. Mezler, M., Muller, T., and Raming, K. (2001) Eur. J. Neurosci. 13, 477-486[CrossRef][Medline] [Order article via Infotrieve]

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
 J. Biol. Chem.Home page
J. Kniazeff, L. Shi, C. J. Loland, J. A. Javitch, H. Weinstein, and U. Gether
An Intracellular Interaction Network Regulates Conformational Transitions in the Dopamine Transporter
J. Biol. Chem., June 20, 2008; 283(25): 17691 - 17701.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. Brock, N. Oueslati, S. Soler, L. Boudier, P. Rondard, and J.-P. Pin
Activation of a Dimeric Metabotropic Glutamate Receptor by Intersubunit Rearrangement
J. Biol. Chem., November 9, 2007; 282(45): 33000 - 33008.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/16/12154    most recent
M611071200v1
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 Binet, V.
Right arrow Articles by Prézeau, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Binet, V.
Right arrow Articles by Prézeau, L.
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