The Intracellular Loops of the GB2 Subunit Are Crucial for G-Protein Coupling of the Heteromeric -Aminobutyrate B Receptor

The -aminobutyrate B (GABAB) receptor is the first discovered G-protein-coupled receptor (GPCR) that needs two subunits, GB1 and GB2, to form a functional receptor. The GB1 extracellular domain (ECD) binds GABA, and GB2 contains enough molecular determinants for G-protein activation. The precise role of the two subunits in G-protein coupling is investigated. GB1 and GB2 are structurally related to the metabotropic glutamate, Ca -sensing and other family 3 GPCRs in which the second (i2) as well as the third (i3) intracellular loop play important roles in G-protein coupling. Here, the role of the i2 loops of GB1 and GB2 in the GABAB receptor ability to activate G proteins is investigated. To that aim, the i2 loops were swapped between GB1 and GB2 heptahelical domains (HDs), either in the wild-type subunits or in the chimeric subunits GB1/2 that contain the ECD of GB1 and the HD of GB2. The effect of an additional mutation within the i3 loop of GB2 that prevents coupling of the heteromeric receptor was also examined. Combinations of interest were found to be correctly addressed at the cell surface and to assemble into heteromers. Taken together our data revealed the following new information on the G-protein coupling of the heteromeric GABAB receptor: 1) the i2 loop of GB2 within the GB2 HD is required for the heteromeric GABAB receptor to couple to G-proteins, whereas the i2 loop of GB1 is not; 2) the presence of the i2 loop of GB2 within the GB1 HD is not sufficient to allow coupling of GB1; 3) the GB2 HD activates the Gqi9 protein whether it is associated with the GB2 or GB1 ECD; 4) in the combination with two GB2 HDs, each is able to couple to G-proteins; and finally, 5) the use of mutations in i2, i3, or both within the GB2 HD brings evidence for the absence of domain swapping enabling the exchange of region including i2 and i3 between the subunits. The main inhibitory neurotransmitter -aminobutyrate (GABA) activates two types of receptors, the ligand-gated Cl -channel receptors (GABAA and GABAC receptors) and the metabotropic GABAB receptor. The later belongs to a large family of receptors coupled to G-proteins. GABAB receptors modulate synaptic transmission by regulating the activity of Ca or K channels (Sodickson and Bean, 1996). GABAB receptors also inhibit the activity of adenylyl cyclase (for a review see Kerr and Ong, 1995). Most of these actions of the GABAB receptors are mediated by pertussis toxinsensitive G-proteins, Go or Gi. Our understanding of GPCR coupling to G-proteins has always been based on the assumption of a monomeric form of the receptor. However, recent data suggest that at least some GPCRs may function in a dimeric (or multimeric) form (Angers et al., 2002). Moreover, mGlu and Ca -sensing receptors have been shown to function as homodimers (Bai et al., 1998). The functional importance of this dimerization is still not fully elucidated. Dimerization of unrelated receptors can lead to a novel receptor with unique pharmacological properties, profile of G-protein coupling, and association with other signaling pathways (Bouvier, 2001). These observations raise a number of questions about the respective roles of the two subunits in a dimeric GPCR. The structure of the GABAB receptor was elusive until This work was supported by grants from the Grant Agency of the Czech Republic (GACR 301/00/0654 to J.B.); Grant Agency of the Ministry of Healthcare of Czech Republic (NL 6114-3/00, NF 6704-3/01); 5th Framework of the European Commission (QLG3-CT-2001-00929 “Epileptosome” to J.B.); Vyzkumny zamer (J13/98 11120005 to J.B.); Centre National de la Recherche Scientifique (CNRS), the Program “Physique et Chimie du Vivant” from the CNRS and Institut National de la Santé et de la Recherche Médicale (PCV00134 to J.-P.P); the Action Concertée Incitative Molécules et Cibles Thérapeutique (J.-P.P); and CisBio International (DIVT2035-CNRS751869/00) (Marcoule, France). ABBREVIATIONS: GABA, -aminobutyrate; GPCR, G-protein-coupled receptors; mGlu receptors, metabotropic glutamate receptors; ECD extracellular domain; HD, heptahelical domain; i2 loop, second intracellular loop; i3 loop, third intracellular loop; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; IP, inositol phosphates; XL665, allophycocyanin. 0026-895X/02/6202-343–350$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 2 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1406/997368 Mol Pharmacol 62:343–350, 2002 Printed in U.S.A. 343 at A PE T Jornals on D ecem er 5, 2017 m oharm .aspeurnals.org D ow nladed from recently when the cloning of two GABAB receptor cDNAs, GB1 (Kaupmann et al., 1997) and GB2, was reported (Jones et al., 1998; Kaupmann et al., 1998; White et al., 1998). Coassembly of both GB1 and GB2 proteins was found to be essential for an efficient coupling of the receptor to G-proteins. Both proteins are related to the “family 3” of GPCRs represented by the mGlu, Ca -sensing, some taste, and putative vomeronasal receptors (Bockaert and Pin, 1999). A unique feature of family 3 receptors is their large N-terminal extracellular domain (ECD), which is structurally related to bacterial periplasmic binding proteins and constitute their ligand recognition domain (Galvez et al., 1999; Malitschek et al., 1999). Ligand binding into this Venus flytrap like module causes activation of the heptahelical domain (HD). How the activation of extracellular domain further transmitted to the HD is not known yet. Anyhow, mGlu receptors are homodimers, and it has been proposed that the dimeric structure of the receptor is crucial for activation of the receptor (Kunishima et al., 2000). Recently, several studies revealed some specific roles of each subunit of the GABAB receptor. GB1 subunit contains the extracellular N-terminal region responsible for ligand binding, but the corresponding region of GB2 subunit, although it is unlikely to bind GABA, is required for high affinity binding of agonists on GB1 (Galvez et al., 2000). Moreover, GB2 by interacting with GB1 at the level of its C-terminal tail, masks an endoplasmic reticulum retention signal such that only heterodimeric receptors are correctly inserted in the plasma membrane (Margeta-Mitrovic et al., 2000; Calver et al., 2001; Pagano et al., 2001). Finally, it has been shown recently that the GB2 HD contains enough molecular determinants for coupling to G-proteins, because a G-protein activation can be detected with a GABAB receptor combination containing GB2 HDs only (Galvez et al., 2001). However, the exact role of each subunit in G-protein recognition and activation by the heterodimeric GABAB receptor is still unknown. Within the family 3 GPCRs, the second intracellular (i2) loop plays a critical role for the selective interaction with G-proteins, whereas the i3 loop is important for coupling efficacy (Pin et al., 1994; Gomeza et al., 1996; Francesconi and Duvoisin, 1998; Chang et al., 2000). The homodimeric structure of mGlu receptors results in receptor complexes in which each unit contains pairs of identical intracellular loops. However, in the case of the GABAB receptor, the two i2 loops (that of GB1 and that of GB2) differ substantially from each other. This situation offers a nice opportunity to identify the role of two i2 loops in a dimeric receptor. Are both i2 loops required for the recognition and activation of the G-protein? Do they direct the coupling to different G-proteins? To that end, several chimeric proteins were constructed in which the i2 loops between GB1 and GB2 were swapped. To investigate the roles of other intracellular portions, the GB2 L686P mutant recently described was used (Duthey et al., 2002). These chimeric proteins were functionally expressed in a heterologous system with wild-type subunits. Construct GB1/2, in which the ECD of GB1 subunit is connected to the HD of GB2, and mutants of this chimera were employed to dissect further the functional role of each HD. The present data demonstrate first that within the wildtype heterodimer receptor complex, the two subunits do not play the same role in activation of G-proteins. Our data show that the i2 loop of GB2 is required for coupling of the heterodimeric receptor to G-proteins, whereas that of GB1 is not. Second, within the recombinant receptor that includes HD from GB2 only, it seems that each HD activates Gproteins. The HD of GB2 that is capable of coupling to Gproteins has to include both the second and the third intracellular loops intact. By introduction of the i2 loop from GB1 or by mutating the i3 loop, the subunit capability of coupling is lost. Interestingly, mutated region cannot be supplied from other functional subunit by mechanism of domain swapping. These observations shed more light on the role of GB2 and on the role of each HD in a dimeric receptor. Thus, it will be useful for other studies aimed at explaining the nature of dimerization of GPCRs. Experimental Procedures Materials. Chemicals including GABA were obtained from Sigma-Aldrich Chimie SARL (L’Isle d’Abeau Chesnes, France) unless otherwise indicated. Serum, culture media, and other solutions used for cell culture were from Invitrogen SARL (Cergy Pontoise, France). The plasmids expressing GABAB receptor subunits and their chimeras were described previously (Galvez et al., 2001; Duthey et al., 2002). The G qi9 and G qo proteins (Liu et al., 1995) were kindly provided by Dr. Bruce Conklin (The Gladstone Institute, San Francisco, CA). The mutagenesis was done by introducing silent restriction sites by quick exchange technology (Stratagene, Amsterdam, The Netherlands) at both predicted ends of the second intracellular loops of GB1 and GB2. Swapping of the loop was done using the sites NruI in the N-terminal portion and HindIII at the C-terminal portion of the i2 loops (see Fig. 1). Culture and Transfection of Human Embryonic Kidney (HEK 293) cells. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen SARL) supplemented with 10% fetal calf serum and antibiotics (penicillin and streptomycin, 100 U/ml final). Electroporation was performed in a total volume of 300 l with 6 g of carrier DNA, GABAB-subunit plasmid DNA (2 g), G -subunit plasmid DNA (2 g), and 10 million cells in electroporation buffer (50 mM K2HPO4, 20 mM CH3COOK, 20 mM KOH, pH 7.4). After electroporation (260 V, 1 mF, Bio-Rad Gene Pulser electroporator; Bio-Rad Laboratories, Hercules, CA), cells were resuspended in DMEM supplemented with 10% fetal calf serum and antibiotics, and split in 12-well clusters (Falcon, Paris, France) (10 million cells per 12 wells) previously coated with poly-L-ornithine (15 g/ml; Mr 40,000; Sigma-Aldrich Chimie SARL) to favor adhesion of the cells. Determination of Inositol Phosphates Accumulation. The procedure used for the determination of IP accumulation in transfected cells was adapted from previously published methods (Berridge and Irvine, 1984). Cells were washed 2 to 3 h after electroporation and incubated for 14 h in DMEM (Invitrogen SARL) containing 0.4 Ci/ml [myo-H]inositol (23.4 Ci/mol; PerkinElmer Life Sciences, Paris, France). Cells were then washed two times with HEPES-buffered saline (146 mM NaCl, 4.2 mM KCl, 0.5 mM MgCl2, 0.1% glucose, 20 mM HEPES, pH 7.4), and LiCl was added to a final concentration of 10 mM. The agonist was applied 5 min later and left for 30 min at 37°C. Replacing the incubation medium with 0.5 ml of perchloric acid (5%) stopped the reaction, and the clusters were kept on ice for 30 min. Supernatants were recovered, and the IPs were purified on Dowex columns (Bio-Rad) (Berridge and Irvine, 1984) Total radioactivity remaining in the membrane fraction was counted after treatment with 10% Triton X-100, 0.1 N NaOH for 30 min (room temperature) and used as standard. Results are expressed as the amount of IP produced over the radioactivity present in the membranes. The dose-response curves were fitted according to the equation y [(ymax ymin)/1 (x/EC50) ] ymin using the KaleidaGraph program (Abelbeck Software, Reading, PA). 344 Havlickova et al. at A PE T Jornals on D ecem er 5, 2017 m oharm .aspeurnals.org D ow nladed from Ligand Binding of Receptors on the Cell Surface. Binding assay was done on intact cells to measure properties of receptors that pass the plasmalemma of HEK 293 cells. Cells were plated in 24-well plates after transfection (done as in previous experimental procedure) in density about 10 million cells per plate. On the next day, they were put on ice and washed three times with binding buffer (20 mM Tris-HCl, pH 7.4, 118 mM NaCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.7 mM KCl, and 1.8 mM CaCl2). They were incubated in the presence of 0.1 nM I-CGP64213 with or without competitiveunlabeled ligand for 3 h at 4°C. The compound I-CGP64213 does not pass the cell membrane. The incubation was terminated by washing three times with ice-cold binding buffer. Cells were then disrupted by 0.1 M NaOH and bound radioactivity was counted. Nonspecific binding was determined in the presence of GABA (1 mM). Measurement of Inhibition of cAMP Formation. Production of cAMP was measured as follow. The cells were treated and electroporated as described above. The agonist was applied after two washes with Krebs buffer for 30 min at 37°C. The stimulation of receptors was then stopped by lysis of the cells by 0.5% Triton X-100 in distilled water for 30 min at room temperature. The supernatant was recovered and competitive immunoassay using labeled anticAMP antibodies (with cryptate), and cAMP labeled with XL665 as competitor was carried for 1 h at 4°C using the cAMP kit (CIS Bio International, Paris, France). The transfer of energy between the cryptate and the XL665 was counted using the RUBYstar reader (BMG Labtechnologies, Durham, NC).