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Previous Article | Next Article
The Journal of Neuroscience, September 1, 2002, 22(17):7352-7361
No Ligand Binding in the GB2 Subunit of the GABAB Receptor Is Required for Activation and Allosteric Interaction between the Subunits
Julie Kniazeff1, Thierry Galvez1, Gilles Labesse2, and Jean-Philippe Pin1 1 Centre National de la Recherche Scientifique (CNRS)-Unité Propre de Recherche 9023, Mécanismes Moléculaires des Communications Cellulaires, 34094 Montpellier Cedex 5, France, and 2 CNRS-Unité Mixte de Recherche 5048, Centre de Biochimie Structurale, Faculté de Pharmacie, 34060 Montpellier Cedex, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The GABAB receptor plays important roles in the tuning of many synapses. Although pharmacological differences have been observed between various GABAB-mediated effects, a single GABAB receptor composed of two subunits (GB1 and GB2) has been identified. Although GB1 binds GABA, GB2 plays a critical role in G-protein activation. Moreover, GB2 is required for the high agonist affinity of GB1. Like any other family 3 G-protein-coupled receptors, GB1 and GB2 are composed of a Venus Flytrap module (VFTM) that usually contains the agonist-binding site and a heptahelical domain. So far, there has been no direct demonstration that GB2 binds GABA or another endogenous ligand. Here, we have further refined the GABA-binding site of GB1 and characterized the putative-binding site in the VFTM of GB2. None of the residues important for GABA binding in GB1 appeared to be conserved in GB2. Moreover, mutation of 10 different residues, alone or in combination, within the possible binding pocket of GB2 affects neither GABA activation of the receptor nor the ability of GB2 to increase agonist affinity on GB1. These data indicate that ligand binding in the GB2 VFTM is not required for activation. Finally, although in either GB1 or the related metabotropic glutamate receptors most residues of the binding pocket are conserved from Caenorhabditis elegans to human, no such conservation is observed in GB2. This suggests that the GB2 VFTM does not constitute a binding site for a natural ligand.
Key words: ligand recognition; evolution; three-dimensional modeling; dimerization; GPCR; allostery; baclofen
INTRODUCTION
In addition to the ligand-gated channels, GABA also activates a G-protein-coupled receptor (GPCR), the GABAB receptor (Bettler et al., 1998
; Couve et al., 2000
In contrast to many other GPCRs, the GABAB receptor requires two distinct subunits, GB1 and GB2, to activate G-proteins (Marshall et al., 1999
The GB1+GB2 heteromer appears to be the only GABAB receptor in the brain, because the knock-out of the GB1 subunit is enough to suppress all studied GABAB-mediated effects (Prosser et al., 2001
In the present study, we refined the analysis of GABA binding on GB1 and used this information to examine whether or not ligand binding in the GB2 VFTM would be required for GABAB receptor activation or modulation.
MATERIALS AND METHODS
Materials. GABA, baclofen, 3-aminopropylphosphinic acid (APPA), 3-{1-(R)-[2-(S)-hydroxy-3-(hydroxy-{5-[3-(4-hydroxy-3-iodo-phenyl)-propionylamino]-pentyl}-phosphinoyl)-propylamino]-ethyl}-benzoic acid (CGP64213), and [125I]CGP64213 were gifts from Drs. W. Froestl and K. Kaupman (Novartis Pharma, Basel, Switzerland). Fetal bovine serum, culture media, and other solutions used for cell culture were from Invitrogen (Cergy Pontoise, France). myo-[3H]inositol (23.4 Ci/mol) was purchased from PerkinElmer Life Sciences (NEN) (Paris, France). All other reagents used were of molecular or analytical grade where appropriate.
Sequence comparison and molecular modeling. Protein sequence database searches were performed with the Position-Specific Iterated-Basic Local Alignment Search Tool program, version 2.0.5 (Altschul et al., 1997
-helix and
-strand) of GB1 and GB2 VFTMs were predicted using Jpred2 (Cuff et al., 1998
Plasmids and site-directed mutagenesis. The plasmids encoding the wild-type GB1a and GB2 subunits epitope tagged with the hemagglutinin (HA) at their N-terminal ends [pRK-GB1a-XXX (HA) and pRK-GB2-HA], under the control of a cytomegalovirus promoter, were described previously (Galvez et al., 2001
Mutant subunits, carrying single or multiple mutations, were obtained using the Quick-Change strategy (Stratagene, La Jolla, CA). Briefly, cDNA fragments encoding part of wild-type GB1a (EcoRI-XbaI) and GB2 (EcoRI-BamHI) were cloned into the pBluescript SK(
) vector. For each mutagenesis, two complementary 27 mer primers (Genaxis Biotechnologie, Nîmes, France) were designed to contain the desired mutation. To allow a rapid screening of mutated clones, primers carried an additional silent mutation introducing (or removing) a restriction site. The presence of the desired mutations and the absence of additional ones were confirmed by DNA sequencing. For multiple mutants, several Quick-Change reactions were performed successively. Finally, short fragments surrounding the mutations were subcloned in the place of corresponding wild-type fragments of pRK-GB1a-HA (PshAI-BamHI) or pRK-GB2-HA (EcoRI-BamHI).
Cell culture and expression in human embryonic kidney 293. Human embryonic kidney (HEK) 293 cells were cultured and transfected by electroporation as described previously using 2 µg of plasmid DNA containing wild-type or mutated receptor for 10 × 106 cells, unless otherwise indicated. In the case of the mutants GB2-F208A and D256W and those bearing the D256Y mutation, which were expressed at a low level, 4 µg of plasmid DNA were used. For all functional studies, both GB1 and GB2 constructs were transfected together with the chimeric G
Western blotting. After transfection (48 hr), cells were washed with ice-cold PBS and scraped in lysis buffer (Tris-EDTA). Membranes were then pelleted and solubilized in buffer containing 0.1% SDS and 2% Triton X-100 at a final concentration of 10 µg/µl. Membrane (10 µg) was loaded on a 7.5% tricine-SDS-polyacrylamide gel and transferred on a nitrocellulose membrane. After overnight incubation in Tris-buffered saline-Tween 20 (TBST: 10 mM Tris, 150 mM NaCl, and 0.1% Tween 20)-milk 5%, the membranes were incubated for 2 hr at room temperature with monoclonal anti-HA antibody (1/3000 in TBST-milk 2%). After washing, the membranes were incubated for 1 hr at room temperature with the anti-mouse HRP antibody (1/5000). The signal was revealed using an enhanced chemiluminescence assay.
Immunohistochemistry. Twenty-four hours after transfection, HEK 293 cells were plated onto glass coverslips, washed twice with PBS, and incubated for 90 min at 37°C with monoclonal mouse 12CA5 at 1.3 µg/ml in PBS/gelatin (0.2%), as described previously (Ango et al., 1999
Ligand-binding assay. A ligand-binding assay on intact HEK 293 cells was performed as described previously using 0.1 nM [125I]CGP64213 (Galvez et al., 2001
Determination of inositol phosphate accumulation. Determination of inositol phosphate (IP) accumulation in transfected cells was performed after labeling the cells overnight with myo-[3H]inositol (23.4 Ci/mol) as described previously (Brabet et al., 1998
Construction of the evolutionary trees. The sequences of the VFTMs of GB1 from human (Swissprot accession number Q9UBS5), rat (Swissprot accession number Q9Z0U4), mouse (Swissprot accession number Q9WV18), Drosophila melanogaster (GenBank accession number AAK13420), and Caenorhabditis elegans (from the cosmid Y41G9A) and those of the GB2 VFTMs from human (Swissprot accession number O75899), rat (Swissprot accession number O88871), D. melanogaster (GenBank accession number AAK13421), and C. elegans (from the cosmid ZK180) were aligned using Clustal W (version 1.6) (Thompson et al., 1994
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Figure 1. Alignment of the GB1 and GB2 VFTMs with those of mGlu1 (1EWK), LIVBP (2LIV), AmiC (1PEA), NPRA (1DP4), and NPRC (1JDN). Highlighted in black and gray are the
RESULTS
Functional studies suggested that GB2 may be activated by GABA and baclofen (Kaupmann et al., 1998
Several studies have examined in detail the agonist-binding domain of GB1. Although all three-dimensional models described a bilobate VFTM-like structure with GABA interacting primarily with lobe-I, they differ in the region contacting the amino group of GABA. Indeed, some authors proposed that Asp471 plays such a role (Galvez et al., 2000a
Three-dimensional modeling and identification of the GABA-binding site of GB1
A structural alignment of the VFTM of LIVBP (pdb code: 2liv), AmiC (pdb code: 1pea), mGlu1 (pdb code: 1ewk), NPRA (pdb code: 1dp4), and NPRC (pdb code: 1jdn) was deduced from the superposition of their structure. The sequence of the GB1 VFTM was then aligned on top of this alignment as described in Materials and Methods (Fig. 1), and three-dimensional models were generated using Modeler (see Materials and Methods). The best model was selected according to the energy of constraint violation in Modeler, the pseudo-energy computed by PROSA II (Sippl, 1993
In this new model, the acidic moiety of Asp471 of GB1 is not part of the GABA-binding site but rather is involved in a network of H-bonds stabilizing lobe-I (Fig. 2a). This Asp side-chain points toward a
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Figure 2. Details of the three-dimensional model of the GB1 VFTM. A, View of the hydrogen network leading from the common Asp (Asp471 in GB1) to a buried Arg (Arg284 in GB1) lying below the ligand-binding loop
Other residues (Tyr395, Asp397, Asn398, and Glu465) were predicted to be part of the ligand-binding site, and some side-chain conformations of both Tyr395 and Glu465 allow close contacts with the ligand (primarily hydrogen bonding). The N group of GABA can be modeled at contact distance of the side-chains of Glu465 from lobe-I and Tyr395 from lobe-II (Fig. 2b). To validate these possibilities, new GB1 mutants were generated, in which Tyr395, Asp397, and Asn398 were mutated into Ala, and Glu465 was replaced by either Ala, Ser, or Asp. The binding and functional properties of these mutants were then analyzed and compared with those of the wild-type and S246A mutant GB1 coexpressed with GB2.
Functional analysis of GB1 mutants
As shown in Table 1, and in agreement with our modeling studies, [125I]CGP64213 binding on intact cells was not affected by the D397A and N398A mutations, and Ki values for GABA were similar to those measured on the wild-type receptor. These results are in agreement with the modeling of these two residues at the edge of the ligand-binding cleft with their side-chains pointing toward the solvent. In contrast, the mutants S246A, Y395A, E465A, E465D, and E465S no longer bind [125I]CGP64213, although they are correctly expressed as shown by Western blotting (Fig. 3a) and targeted to the cell surface in the presence of GB2, as shown by immunolabeling of intact cells (Fig. 3b and data not shown). This is consistent with these three residues playing an important role in CGP64213 binding.
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Table 1. Effect of mutations of various residues in the GB1 VFTM on the binding and functional properties of the heteromeric GABAB receptor
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Figure 3. Expression and plasma membrane insertion of the wild-type and mutated GB1 and GB2 subunits. a, Immunoblot obtained with membrane proteins prepared from cells expressing the wild-type GB1 (WT) or the E465A, E465D, E465S, Y395A, D397A, or N398A GB1 mutants and labeled with the HA antibody. b, Immunolabeling with the HA antibody of intact cells [or cells permeabilized with 0.05% Triton X-100 (perm)] expressing the indicated subunits. Because the HA epitope is fused at the N-terminal extracellular end of the subunits, labeling of intact cells is indicative of a plasma membrane insertion of the subunit. Note that GB1-HA can be detected in permeabilized cells only when expressed alone and in intact cells when coexpressed with GB2.
The possible activation of these mutant receptors by GABA was also analyzed by measuring IP formation after coexpression with the wild-type GB2 and the chimeric G-protein G
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Figure 4. S246 and E465 of GB1 are critical for agonist potency at the heteromeric GABAB receptor. The effect of increasing concentrations of GABA on IP formation in cells coexpressing the wild-type (WT; ), S246A (
), E465A (
), E465D (
), or E465S (
) GB1 subunit with the wild-type GB2 and G
Characterization and functional importance of the putative ligand-binding site of GB2
Based on the alignment of GB2 and GB1 VFTM sequences and of the above described structural alignment (Fig. 1), a three-dimensional model for the GB2 VFTM was generated (Fig. 5). This model satisfies both statistical (Verify 3D) and energetic (PROSA II) criteria for a correctly folded protein. Interestingly, among the three residues identified in GB1 that likely interact with GABA (Ser246, Tyr366, and Glu465), none are conserved in GB2 (the homologous residues being Pro136, Asp256, and Phe354, respectively). Moreover, the residue Ser269 of GB1 that has been shown to be responsible for the increased GABA affinity in the presence of Ca2+ (Galvez et al., 2000b
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Figure 5. Three-dimensional model of the GB2 VFTM. Lobe-I is in orange, and lobe-II is in cyan. Indicated are residues that have been mutated in the present study and the Cys residues involved in putative intramolecular disulfide bonds.
To examine the possible importance of this putative-binding site of GB2, the residues homologous to those responsible for GABA binding in GB1 were mutated into residues with very different properties (Table 2). For example, the acidic residue Asp256 was replaced either by the neutral residue Ala or the aromatic residues Tyr or Trp, and Phe354 was replaced by a Glu. Additional residues that are part of the binding pocket and that may possibly form an H-bond or ionic interaction with a ligand were also mutated into Ala (Thr160, Ser178, Asp179, Arg207, Phe208, and Lys353). GB2 receptor mutants simultaneously carrying two to five of these mutations were also generated, in such a way that the putative GB2-binding site is highly modified.
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Table 2. Effect of mutations of various residues in the GB2 VFTM on the binding and functional properties of the heteromeric GABAB receptor When coexpressed with GB1, all of these mutant GB2 subunits were able to target GB1 to the cell surface, as demonstrated by the significant [125I]CGP64213 binding measured on intact cells (Table 2). Moreover, all GB2 mutants were still able to increase GABA affinity like the wild-type GB2, indicating that none of these mutations affect the allosteric effect of GB2 on GB1. Finally, all GB2 mutants formed a functional GABAB receptor when coexpressed with GB1. However, a higher EC50 value for GABA (Table 2), as well as for the other agonists, baclofen or APPA (data not shown), was observed with the GB2-F208A, D256Y, D256W, and multiple mutant receptors bearing the D256Y mutation. Indeed, the higher the agonist EC50 value, the lower the [125I]CGP64213 binding measured on intact cells (Table 2). Was this decrease in agonist potency caused by the lower level of expression only, or does it result from a lower efficacy of GABA to activate the GB2 HD? To answer this question, GABA potency on the wild-type receptor was measured on cells expressing various levels of the GB1+GB2 heteromer. As shown in Figure 6, a 10-fold decrease in CGP64213 binding results in a threefold to fourfold increase in the GABA EC50 value. Interestingly, although most GB2 mutants fit on the curve obtained with the wild-type heteromer, those bearing the D256Y or D256W mutation have a much larger decrease in GABA potency than would be expected if this was attributable to their low level of expression only. Accordingly, these data show that the mutation of Asp256 into the large residues Tyr or Trp decreases the efficacy of the bound GB1 VFTM to activate the G-protein coupling domain of the receptor.
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Figure 6. Influence of receptor expression level on GABA potency (EC50). HEK 293 cells were transfected with 2 µg of pRKGB1 and various concentrations of pRKGB2 (from 0.05 to 2 µg), and the specific [125I]CGP64213 binding (expressed as the percentage of that measured on cells transfected with 2 µg of both pRKGB1 and pRKGB2) and GABA EC50 values were determined. Values obtained are indicated by open circles in this plot. Closed circles correspond to the data obtained with the various GB2 mutants coexpressed with the wild-type GB1 and indicated in Table 2. Numbers indicate the data obtained with the GB2-P136S, T159S, T160S, D256Y, F354E (1); GB2-D256W (2); GB2-P136S, D256Y (3); GB2-P136S, T159S, T160S, D256Y (4); GB2-D256Y (5); and GB2-F208A (6).
We also analyzed the possible role of Asp360, the GB2 homologous residue of Asp471 of GB1. According to our model, this Asp residue is also involved in the same interaction network as that observed in GB1 and may therefore play a role in the correct folding of the lobe-I of GB2 VFTM. In agreement with this proposal, the mutation of Asp360 into Ala is sufficient to prevent GB2 to target GB1 to the cell surface, as shown by the absence of [125I]CGP64213 binding on intact cells expressing both subunits (Table 2), although this GB2 mutant is expressed and reached the cell surface alone, as shown by immunohistochemistry (Fig. 3b).
Evolution analysis of GB1 and GB2 VFTMs
Regions of proteins involved in ligand recognition (the ligand being either another protein or a small molecule) are subjected to a high pressure during evolution and, as such, are more conserved than the other area of the protein. This has been the basis for the generation of the "evolutionary trace" method to identify possible ligand-interacting sites of proteins (Lichtarge et al., 1996a
The sequences of the group-II mGlu receptors, the GB1 and GB2 subunits from C. elegans, D. melanogaster, and various mammalian species, were retrieved from the data bank, and their VFTM sequences were aligned. The deduced phylogenetic tree revealed a similar distance between the C. elegans and mammalian proteins regardless of whether group-II mGlu receptors or GB1 or GB2 subunits were considered (Fig. 7a). The same analysis was then performed taking into account the residues that constitute the binding pocket only. As shown on Figure 7b, the phylogenetic distance between the C. elegans and mammalian binding pockets was much shorter for the GB1 and group-II mGlu receptors than that observed with the entire VFTMs of these proteins. This clearly indicates a high pressure during evolution on these binding pockets. In contrast, the phylogenetic distances between the C. elegans, D. melanogaster, and mammalian GB2 proteins remain the same regardless of which residues constituting the putative-binding pocket, or the entire sequence of the VFTM, are considered (Fig. 7b). This clearly shows that there has been no higher selective pressure on the putative GB2-binding pocket than on the rest of the protein.
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Figure 7. Absence of selective evolutionary pressure of the putative GB2-binding site. a, Evolutionary trees constructed with the sequences of the VFTM of C. elegans (Cel), D. melanogaster (Dro), or various mammalian [human (Hum), rat, or mouse (Mus)] GB1 or GB2 subunits or group-II mGlu receptors. b, Tree obtained with the residues lining the putative-binding pocket (see Materials and Methods). Note that the phylogenetic distances (expressed as the percentage of divergence divided by 100) between the GB2 subunits remain similar regardless of whether the entire VFTM is considered or only the residues that lined the putative-binding pocket. c, Residues close to the putative-binding pocket of the GB1 and GB2 subunits. These residues correspond to the position of those residues located at <8 Å of glutamate in the mGlu1 VFTM. Residues highlighted in black are those that are not conserved during evolution. The boxed residues are conserved in all GB subunits and are likely to be structurally important. Positions in GB2 indicated with an asterisk are those that have been subjected to mutagenesis in the rat GB2 (present study).
Comparison of the residues that constitute the binding pocket of GB1 revealed that only 14 of 46 are not conserved during evolution (Fig. 7c). In contrast, 34 of 46 residues are not conserved in the GB2 pocket (Fig. 7c). As shown above, even when residues conserved in both D. melanogaster and mammalian GB2 sequences and able to form H-bonds with a ligand are mutated (Ser178, Asp179, Arg207), the heteromeric GABAB receptor retains its functional properties (Table 2).
DISCUSSION
The present study shows that mutations of as many as 10 different residues within the putative-binding pocket of GB2 do not prevent GABA from activating the heteromeric GABAB receptor, nor do they inhibit the positive allosteric effect of the GB2 subunit on the agonist affinity of GB1. Moreover, our data revealed that this putative-binding pocket of GB2 has not been subjected to high pressure during evolution, in contrast to the equivalent binding site of GB1 or mGlu receptors.
Thanks to the recent resolution of the structure of various VFTMs, we have been able to further refine our three-dimensional model for the GB1-binding site. In agreement with previous studies, the carboxylic moiety of GABA interacts with Ser246 as well as with backbone atoms of the loop
-carboxylic group of glutamate.
The GB2 VFTM has been reported previously to be required to obtain a functional GABAB receptor (Jones et al., 2000
One may also consider that the GB2 putative-binding site could be occupied by a specific ligand that may be present in the brain under certain circumstances. This may for instance influence the functioning of the GB1 subunit and may possibly explain the different pharmacological properties of GABAB receptors reported in the literature (Bonanno and Raiteri, 1993
If the GB2 VFTM does not bind a ligand, why is it necessary for GABAB receptor activation? Indeed, a GABAB receptor heteromer in which the GB2 VFTM is deleted (Jones et al., 2000
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Figure 8. Schematic representation of the putative activation mechanism of family 3 GPCRs. Top left, Schematic representation of the inactive receptor with both VFTMs in the open state (Op) and in the resting orientation [according to the structure of the free dimeric mGlu1 VFTM (pdb code: 1ewt)] (Kunishima et al., 2000
For most VFTMs, both closed and open conformations have been observed, even in the absence of ligand (Quiocho, 1990
In conclusion, our data are consistent with a single GABA molecule being sufficient to activate the heteromeric GABAB receptor and reveal that the GB2 subunit is unlikely to bind a ligand at the level of its VFTM. The recent determination of the structure of the dimer of mGlu1 VFTMs sheds light on the possible role of the GB2 VFTM in the GABAB receptor activation process.
FOOTNOTES Received April 22, 2002; revised June 6, 2002; accepted June 7, 2002.
This work was supported by the "Action Incitative Physique et Chimie du Vivant" (PCV00-134) from the Centre National de la Recherche Scientifique (CNRS) (J.-P.P.), the program "Molécules et Cibles Thérapeutiques" from Institut National de la Santé et de la Recherche Médicale and CNRS (J.-P.P.), and Novartis Pharma (Basel, Switzerland). We thank Drs. F. Acher, A.-S. Bessis, B. Bettler, L. Prézeau, and P. Rondard for constructive discussions and constant support throughout this work and I. Brabet for her help in some experiments. We also thank Drs. W. Froestl and K. Kaupmann (Novartis) for the supply of GABAB ligands and A. Turner-Madeuf for help with our English.
Correspondence should be addressed to Dr. Jean-Philippe Pin, Centre National de la Recherche Scientifique-Unité Propre de Recherche 9023, Mécanismes Moléculaires des Communications Cellulaires, 141 rue de la Cardonille, 34094 Montepellier Cedex 5, France. E-mail: pin{at}montp.inserm.fr.
T. Galvez's present address: Department of Molecular Pharmacology, Stanford University Medical Center, Stanford, CA 94304.
REFERENCES
- Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389-3402
[Abstract/Free Full Text] .- Ango F, Albani-Torregrossa S, Joly C, Robbe D, Michel J-M, Pin J-P, Bockaert J, Fagni L (1999) A simple method to transfer plasmid DNA into neuronal primary cultures: functional expression of mGluR5 in cerebellar granule cells. Neuropharmacology 38:793-803[Web of Science][Medline].
- Bernard P, Guedin D, Hibert M (2001) Molecular modeling of the GABA/GABAB receptor complex. J Med Chem 44:27-35[Web of Science][Medline].
- Bessis A-S, Bertrand H-O, Galvez T, De Colle C, Pin J-P, Acher F (2000) Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process. Protein Sci 9:2200-2209[Web of Science][Medline].
- Bettler B, Kaupmann K, Bowery N (1998) GABAB receptors: drugs meet clones. Curr Opin Neurobiol 8:345-350[Web of Science][Medline].
- Billinton A, Ige AO, Bolam JP, White JH, Marshall FH, Emson PC (2001) Advances in the molecular understanding of GABAB receptors. Trends Neurosci 24:277-282[Web of Science][Medline].
- Blahos J, Fischer T, Brabet I, Stauffer D, Rovelli G, Bockaert J, Pin J-P (2001) A novel site on the G
[Abstract/Free Full Text] .- Bonanno G, Raiteri M (1993) Multiple GABAB receptors. Trends Pharmacol Sci 14:259-261[Medline].
- Brabet I, Parmentier M-L, De Colle C, Bockaert J, Acher F, Pin J-P (1998) Comparative effect of L-CCG-I, DCG-IV and
- Calver AR, Robbins MJ, Cosio C, Rice SQ, Babbs AJ, Hirst WD, Boyfield I, Wood MD, Russell RB, Price GW, Couve A, Moss SJ, Pangalos MN (2001) The C-terminal domains of the GABAB receptor subunits mediate intracellular trafficking but are not required for receptor signaling. J Neurosci 21:1203-1210
[Abstract/Free Full Text] .- Colovos C, Yeates TO (1993) Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci 2:1511-1519[Web of Science][Medline].
- Costantino G, Macchiarulo A, Entrena Guadix A, Pellicciari R (2001) QSAR and molecular modeling studies of baclofen analogues as GABAB agonists. Insights into the role of the aromatic moiety in GABAB binding and activation. J Med Chem 44:1827-1832[Web of Science][Medline].
- Couve A, Moss SJ, Pangalos MN (2000) GABAB receptors: a new paradigm in G protein signaling. Mol Cell Neurosci 16:296-312[Web of Science][Medline].
- Cuff JA, Clamp ME, Siddiqui AS, Finlay M, Barton GJ (1998) Jpred: a consensus secondary structure prediction server. Bioinformatics 14:892-893
[Abstract/Free Full Text] .- Duthey B, Caudron S, Perroy J, Bettler B, Fagni L, Pin J-P, Prézeau L (2002) A single subunit (GB2) is required for G-protein activation by the heterodimeric GABAB receptor. J Biol Chem 277:3236-3241
[Abstract/Free Full Text] .- Eisenberg D, Luthy R, Bowie JU (1997) VERIFY3D: assessment of protein models with three-dimensional profiles. Methods Enzymol 277:396-404[Web of Science][Medline].
- Galvez T, Parmentier M-L, Joly C, Malitschek B, Kaupmann K, Kuhn R, Bittiger H, Froestl W, Bettler B, Pin J-P (1999) Mutagenesis and modeling of the GABAB receptor extracellular domain support a Venus flytrap mechanism for ligand binding. J Biol Chem 274:13362-13369
[Abstract/Free Full Text] .- Galvez T, Prézeau L, Milioti G, Franek M, Joly C, Froestl W, Bettler B, Bertrand H-O, Blahos J, Pin J-P (2000a) Mapping the agonist binding site of GABAB type 1 subunit sheds light on the activation process of GABAB receptors. J Biol Chem 275:41166-41174
[Abstract/Free Full Text] .- Galvez T, Urwyler S, Prézeau L, Mosbacher J, Joly C, Malitschek B, Heid J, Brabet I, Froestl W, Bettler B, Kaupmann K, Pin J-P (2000b) Ca2+-requirement for high affinity
[Abstract/Free Full Text] .- Galvez T, Duthey B, Kniazeff J, Blahos J, Rovelli G, Bettler B, Prézeau L, Pin J-P (2001) Allosteric interactions between GB1 and GB2 subunits are required for optimal GABAB receptor function. EMBO J 20:2152-2159[Web of Science][Medline].
- Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18:2714-2723[Web of Science][Medline].
- Hammerland LG, Krapcho KJ, Garrett JE, Alasti N, Hung BC, Simin RT, Levinthal C, Nemeth EF, Fuller FH (1999) Domains determining ligand specificity for Ca2+ receptors. Mol Pharmacol 55:642-648
[Abstract/Free Full Text] .- Hampson DR, Huang XP, Pekhletski R, Peltekova V, Hornby G, Thomsen C, Thogersen H (1999) Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor. J Biol Chem 274:33488-33495
[Abstract/Free Full Text] .- Havlickova M, Prezeau L, Duthey B, Bettler B, Pin J-P, Blahos J (2002) The intracellular loops of the GB2 subunit are crucial for G-protein coupling of the heteromeric GABAB receptor. Mol Pharmacol 62:343-350
[Abstract/Free Full Text] .- He X-L, Chow D-C, Martick MM, Garcia KC (2001) Allosteric activation of a spring-loaded natriuretic peptide receptor dimer by hormone. Science 293:1657-1662
[Abstract/Free Full Text] .- Jones KA, Borowsky B, Tamm JA, Craig DA, Durkin MM, Dai M, Yao W-J, Johnson M, Gunwaldsen C, Huang L-Y, Tang C, Shen Q, Salon JA, Morse K, Laz T, Smith KE, Nagarathnam D, Noble SA, Branchek TA, Gerald C (1998) GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature 396:674-679[Medline].
- Jones KA, Tamm JA, Craig DA, Yao W-J, Panico R (2000) Signal transduction by GABAB receptor heterodimers. Neuropsychopharmacology 23:S41-S49[Web of Science][Medline].
- Kaupmann K, Huggel K, Heid J, Flor PJ, Bischoff S, Mickel SJ, McMaster G, Angst C, Bittiger H, Froestl W, Bettler B (1997) Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386:239-246[Medline].
- Kaupmann K, Malitschek B, Schuler V, Heid J, Froestl W, Beck P, Mosbacher J, Bischoff S, Kulik A, Shigemoto R, Karschin A, Bettler B (1998) GABAB-receptor subtypes assemble into functional heteromeric complexes. Nature 396:683-687[Medline].
- Kerr DIB, Ong J (1995) GABAB receptors. Pharmacol Ther 67:187-246[Web of Science][Medline].
- Kubo Y, Miyashita T, Murata Y (1998) Structural basis for a Ca2+-sensing function of the metabotropic glutamate receptors. Science 279:1722-1725
[Abstract/Free Full Text] .- Kuner R, Kohr G, Grunewald S, Eisenhardt G, Bach A, Kornau HC (1999) Role of heteromer formation in GABAB receptor function. Science 283:74-77
[Abstract/Free Full Text] .- Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka T, Nakanishi S, Jingami H, Morikawa K (2000) Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407:971-977[Medline].
- Labesse G, Mornon J-P (1998) Incremental threading optimization (TITO) to help alignment and modeling of remote homologues. Bioinformatics 14:206-211
[Abstract/Free Full Text] .- Lichtarge O, Bourne HR, Cohen FE (1996a) Evolutionarily conserved G(alpha beta gamma) binding surfaces support a model of the G protein-receptor complex. Proc Natl Acad Sci USA 93:7507-7511
[Abstract/Free Full Text] .- Lichtarge O, Bourne HR, Cohen FE (1996b) An evolutionary trace method defines binding surfaces common to protein families. J Mol Biol 257:342-358[Web of Science][Medline].
- Malitschek B, Schweizer C, Keir M, Heid J, Froestl W, Mosbacher J, Kuhn R, Henley J, Joly C, Pin J-P, Kaupmann K, Bettler B (1999) The N-terminal domain of
[Abstract/Free Full Text] .- Margeta-Mitrovic M, Jan YN, Jan LY (2000) A trafficking checkpoint controls GABAB receptor heterodimerization. Neuron 27:97-106[Web of Science][Medline].
- Margeta-Mitrovic M, Jan YN, Jan LY (2001a) Ligand-induced signal transduction within heterodimeric GABAB receptor. Proc Natl Acad Sci USA 98:14643-14648
[Abstract/Free Full Text] .- Margeta-Mitrovic M, Jan YN, Jan LY (2001b) Function of GB1 and GB2 subunits in G protein coupling of GABAB receptors. Proc Natl Acad Sci USA 98:14649-14654
[Abstract/Free Full Text] .- Marshall FH, Jones KA, Kaupmann K, Bettler B (1999) GABAB receptors-the first 7TM heterodimers. Trends Pharmacol Sci 20:396-399[Medline].
- Martin SC, Russek SJ, Farb DH (1999) Molecular identification of the human GABABR2: cell surface expression and coupling to adenylyl cyclase in the absence of GABABR1. Mol Cell Neurosci 13:180-191[Web of Science][Medline].
- O'Hara PJ, Sheppard PO, Thøgersen H, Venezia D, Haldeman BA, McGrane V, Houamed KM, Thomsen C, Gilbert TL, Mulvihill ER (1993) The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins. Neuron 11:41-52[Medline].
- Okamoto T, Sekiyama N, Otsu M, Shimada Y, Sato A, Nakanishi S, Jingami H (1998) Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1. J Biol Chem 273:13089-13096
[Abstract/Free Full Text] .- 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, Bettler B (2001) C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABAB receptors. J Neurosci 21:1189-1202
[Abstract/Free Full Text] .- Page RDM (1996) TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357-358
[Free Full Text] .- Prosser HM, Gill CH, Hirst WD, Grau E, Robbins M, Calver A, Soffin EM, Farmer CE, Lanneau C, Gray J, Schenck E, Warmerdam BS, Clapham C, Reavill C, Rogers DC, Stean T, Upton N, Humphreys K, Randall A, Geppert M (2001) Epileptogenesis and enhanced prepulse inhibition in GABAb1-deficient mice. Mol Cell Neurosci 17:1059-1070[Web of Science][Medline].
- Quiocho FA (1990) Atomic structures of periplasmic binding proteins and the high-affinity active transport systems in bacteria. Philos Trans R Soc Lond B Biol Sci 326:341-351
[Abstract/Free Full Text] .- Robbins MJ, Calver AR, Filippov AK, Hirst WD, Russell RB, Wood MD, Nasir S, Couve A, Brown DA, Moss SJ, Pangalos MN (2001) GABAB2 is essential for G-protein coupling of the GABAB receptor heterodimer. J Neurosci 21:8043-8052
[Abstract/Free Full Text] .- Sack JS, Saper MA, Quiocho FA (1989) Periplasmic binding protein structure and function. Refined X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine. J Mol Biol 206:171-191[Web of Science][Medline].
- Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-425[Abstract].
- Sali A, Blundell TL (1993) Comparative protein modeling by satisfaction of spatial restraints. J Mol Biol 234:779-815[Web of Science][Medline].
- Saunders R, Nahorski SR, Challiss RAJ (1998) A modulatory effect of extracellular Ca2+ on type 1
- Schuler V, Luscher C, Blanchet C, Klix N, Sansig G, Klebs K, Schmutz M, Heid J, Gentry C, Urban L, Fox A, Spooren W, Jaton A, Vigouret J, Pozza M, Kelly PH, Mosbacher J, Froestl W, Kaslin E, Korn R (2001) Epilepsy, hyperalgesia, impaired memory, and loss of pre- and postsynaptic GABAB responses in mice lacking GABAB1. Neuron 31:47-58[Web of Science][Medline].
- Schwarz DA, Barry G, Eliasof SD, Petroski RE, Conlon PJ, Maki RA (2000) Characterization of gamma-aminobutyric acid receptor GABAB(1e), a GABAB(1) splice variant encoding a truncated receptor. J Biol Chem 275:32174-32181
[Abstract/Free Full Text] .- Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17:355-362[Web of Science][Medline].
- Thompson JD, Higgins DG, Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673-4680
[Abstract/Free Full Text] .- Tsuchiya D, Kunishima N, Kamiya N, Jingami H, Morikawa K (2002) Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+. Proc Natl Acad Sci USA 99:2660-2665
[Abstract/Free Full Text] .- Tuffery P (1995) XmMol: an X11 and motif program for macromolecular visualization and modeling. J Mol Graph 13:67-72[Web of Science][Medline].
- van den Akker F, Zhang X, Miyagi M, Huo X, Misono KS, Yee VC (2000) Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor. Nature 406:101-104[Web of Science][Medline].
- Walmsley AR, Shaw JG, Kelly DJ (1992) Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus. Biochemistry 31:11175-11181[Medline].
- White JH, Wise A, Main MJ, Green A, Fraser NJ, Disney GH, Barnes AA, Emson P, Foord SM, Marshall FH (1998) Heterodimerization is required for the formation of a functional GABAB receptor. Nature 396:679-682[Medline].
- Wolf A, Shaw EW, Nikaido K, Ames GF-L (1994) The histidine-binding protein undergoes conformational changes in the absence of ligand as analyzed with conformation-specific monoclonal antibodies. J Biol Chem 269:23051-23058
[Abstract/Free Full Text] .
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