Identification of a GABAB Receptor Subunit, gb2, Required for Functional GABAB Receptor Activity

G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant γ-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situhybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor. G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant γ-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situhybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor. γ-aminobutyric acid G protein-coupled receptor inwardly rectifying K+ channel polymerase chain reaction Metabotropic GABAB1 receptors were first distinguished pharmacologically by Hill and Bowery (1Hill D.R. Bowery N.G. Nature. 1981; 290: 149-152Crossref PubMed Scopus (911) Google Scholar). Kaupmann et al. (2Kaupmann K. Huggle K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (870) Google Scholar) recently cloned two alternatively spliced forms of the GABAB receptor, termed gb1a and gb1b, which belong to the G protein-coupled receptor superfamily and are most closely related to the metabotropic glutamate receptors. Although native GABAB receptors are reported to activate inwardly rectifying K+ channels (Kir) (3Misgeld U. Bijak M. Jarolimek W. Prog. Neurobiol. 1995; 46: 423-462Crossref PubMed Scopus (419) Google Scholar), recombinant gb1a receptors coexpressed with Kir channels in Xenopus oocytes failed to be functionally active (2Kaupmann K. Huggle K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (870) Google Scholar, 4Couve A. Filippov A.K. Connolly C.N. Bettler B. Brown D.A. Moss S.J. J. Biol. Chem. 1998; 273: 26361-26367Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 12Jones K.A. Borowsky B. Tamm J.A. Dai M. Yao W.-J. Johnson M. Huang L.-Y. Chang H.-Y. Laz T.M. Tang C. Shen Q. Nagarathnam D. Noble S.A. Branchek T.A. Gerald C. Society for Neuroscience Abstracts, Los Angeles, November 7–12, 1998 Abstr. 795.9. Neuroscience Society, Washington, D. C.1998Google Scholar). A recent report has shown that recombinant GABAB receptors fail to couple to effector pathways in a variety of non-neuronal and neuronal cell types, suggesting that additional cellular component(s) are required (4Couve A. Filippov A.K. Connolly C.N. Bettler B. Brown D.A. Moss S.J. J. Biol. Chem. 1998; 273: 26361-26367Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Failure to show GABAB receptor function coupled with previous reports that GPCRs can undergo dimerization (5Ng G.Y.K. O'Dowd B.F. Chung H.T. Lee S.P. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Comm. 1996; 227: 200-204Crossref PubMed Scopus (240) Google Scholar, 6Hébert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 272: 29229-29327Google Scholar, 7Romano C. Yang W. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 8Bai M. Trivedi S. Brown E.M. J. Biol. Chem. 1998; 273: 23605-23610Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), suggested that heterodimerization may be important for GABAB receptor function. Furthermore, receptor heterodimerization appears to rescue function of mutated or chimeric muscarinic and adrenergic receptors (9Maggio R. Vogel Z. Wess J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3103-3107Crossref PubMed Scopus (295) Google Scholar). We report here that coexpression of gb1 and gb2 receptors are necessary for the formation of functional GABAB receptors and result in heterodimerization. The murine gb1a (mgb1a) cDNA was constructed from two expressed sequence tags (IMAGE Consortium clone identification numbers 472408 and 319196), combined by standard PCR methods, and subcloned into the pCINeo (Stratagene) and pcDNA3.1 (Invitrogen) vectors. 2R. Sullivan, F. L. Kolakowski, Jr., M. P. Johnson, A. Chateauneuf, G. P. O'Neill, and G. Y. K. Ng, manuscript in preparation. The rat gb1a receptor was obtained by PCR of rat brain cDNA using oligonucleotide primers based on the published sequence (Ref. 2Kaupmann K. Huggle K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (870) Google Scholar; GenBankTM accession number Y10369) and subcloned into pcDNA3.1. Two independently derived cDNAs of human gb2 (GenBankTMaccession numbers AF069755 and AF056085, the former having previously been called GPR51 (10Ng G.Y.K. MacDonald T. Bonnert T. Rigby M. Heavens R. Whiting P. Chateauneuf A. Coulombe N. Kargman S. Caskey T. Evans J. O'Neill G. Liu Q. Genomics. 1999; 56: 288-295Crossref PubMed Scopus (17) Google Scholar)) were used to make expression constructs. A N-terminal FLAG-tagged hgb2/pcDNA3.1 construct encoding a modified influenza hemaglutinin signal sequence (MKTIIALSYIFCLVFA) followed by an antigenic FLAG (DYKDDDDK) epitope was generated by PCR (10Ng G.Y.K. MacDonald T. Bonnert T. Rigby M. Heavens R. Whiting P. Chateauneuf A. Coulombe N. Kargman S. Caskey T. Evans J. O'Neill G. Liu Q. Genomics. 1999; 56: 288-295Crossref PubMed Scopus (17) Google Scholar). The hgb2 construct used for expression in 293 cells was the coding sequence from GenBankTM accession number AF056085 with the C-terminal splice variant of GenBankTM accession numberAF095723 3J. Clark, É. Mezey, A. S. Lam, and T. I. Bonner, manuscript in preparation. inserted into pcDNA3.1 in a manner similar to that for the rat gb1a. Adjacent coronal rat brain sections were hybridized with labeled antisense and sense riboprobes directed against rgb2 (GenBankTM accession number AF058795) or rgb1 as described previously (11Bradley D.J. Towle H.C. Young W.S. J. Neurosci. 1992; 12: 2288-2302Crossref PubMed Google Scholar). 4http://intramural.nimh.nih.gov/lcmr/snge/Protocol.html. Probes were generated by amplification of rgb2 with JC216 (T3 promoter followed by bases 1172–1191) and JC217 (T7 promoter and the complement of bases 1609–1626) or with JC218 (T3 promoter and bases 2386–2405) and JC219 (T7 promoter and the complement bases 2776–2793) or by amplification of rgb1a with JC160 (T3 promoter and bases 631–648) and JC161 (T7 promoter and the complement of bases 1024–1041). For colocalization experiments, probes were either labeled with digoxigenin (rgb1a) or35S (rgb2). Detection of the radiolabeled rgb2 probe was performed using emulsion after detection of the digoxigenin-labeled rgb1 probe on the same brain slices. Growth of Xenopus laevis melanophores and fibroblasts and DNA transfections by electroporation were performed as described previously (13Kolakowski Jr., L.F. O'Neill G.P. Howard A.D. Broussard S.R. Sullivan K.A. Feighner S.D. Sawzdargo M. Nguyen T. Kargman S. Shiao L.-L. Hreniuk D.L. Tan C.T. Evans J. Abramovitz M. Chateauneuf A. Coulombe N. Ng G. Johnson M.P. Tharian A. Khoshbouei H. George S.R. Smith R.G. O'Dowd B.F. J. Neurochem. 1998; 71: 2239-2251Crossref PubMed Scopus (162) Google Scholar). To monitor the efficiency of transfection an internal control GPCR was used (pcDNA1-cannabinoid 2). For Gi-coupled responses (pigment aggregation), cells were preincubated in the presence of 100 μl/well of 70% L-15 medium containing 2.5% fibroblast-conditioned growth medium, 2 mm glutamine, 100 μg/ml streptomycin, 100 units/ml penicillin, and 15 mm HEPES, pH 7.3, for 30 min to induce pigment dispersion. Absorbance readings at 600 nm were measured using a Bio-Tek Elx800 Microplate reader (ESBE Scientific) before and after 1.5 h incubation with the ligands. Hgb2 and rgb1a cDNAs in pcDNA3.1 were used to transfect HEK-293 cells. Stably expressing cells were identified after selection in geneticin (0.375 mg/ml) by dot blot analysis. For coexpression experiments the stable cell lines hgb2-42 and rgb1a-50 were transiently transfected with hgb2 or rgb1a, and cells were assayed for cAMP responses. Wild-type HEK-293 cells or HEK-293 cells stably and transiently expressing the hgb2 and rgb1a receptors were lifted in 1× phosphate-buffered saline, 2.5 mm EDTA, counted, pelleted, and resuspended at 1.5 × 105 cells/100 μl in Krebs-Ringer-HEPES medium (14Blakely R.D. Clark J.A. Rudnick G. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar), 100 μm Ro 20-1724 (RBI) and assayed for agonist-mediated inhibition of forskolin-stimulated cAMP synthesis using a modified version of an assay previously described by Kaupmann et al.(2Kaupmann K. Huggle K. Heid J. Flor P.J. Bischoff S. Mickel S.J. McMaster G. Angst C. Bittiger H. Froestl W. Bettler B. Nature. 1997; 386: 239-246Crossref PubMed Scopus (870) Google Scholar). cAMP determinations were made using a solid phase modification (15Maidment N.T. Brumbaugh D.R. Rudolph V.D. Erdelyi E. Evans C.J. Neuroscience. 1989; 33: 549-557Crossref PubMed Scopus (130) Google Scholar) of the cAMP radioimmunoassay described by Brooker et al. (16Brooker G. Harper J.F. Teresaki W.L. Moylan R.D. Adv. Cyclic Nucleotide Res. 1979; 10: 1-33PubMed Google Scholar) and previously reported (17Clark J.A. Bonner T.I. Kim A.S. Usdin T.B. Mol. Endocrinol. 1998; 12: 193-206Crossref PubMed Google Scholar). Xenopusoocytes were isolated as described (18Hébert T.E. Monette R. Drapeau P. Dunn R.J. Proc. R. Soc. Lond. B Biol. Sci. 1994; 256: 253-261Crossref Scopus (9) Google Scholar). cDNA constructs for human Kir 3.1, Kir 3.2 channel isoforms (gifts from Dr. Hubert Van Tol, University of Toronto), Giα1 (a generous gift of Dr. Maureen Linder, Washington University), and cDNAs of mgb1a or FLAG-hgb2 (subcloned into pT7TS, a gift of Dr. Paul Krieg, University of Texas) were linearized and transcribed using T7 RNA polymerase and the mMessage mMachine (Ambion). Individual oocytes were injected with 5–10 ng (in 25–50 nl) of Kir3.1 and Kir3.2 constructs with mRNAs for mgb1a or FLAG-hgb2 receptors with and without Giα1. Recordings were made after 48 h as described (18Hébert T.E. Monette R. Drapeau P. Dunn R.J. Proc. R. Soc. Lond. B Biol. Sci. 1994; 256: 253-261Crossref Scopus (9) Google Scholar). Standard recording solution was KD-98, 98 mm KCl, 1 mm MgCl2, 5 mm K-HEPES, pH 7.5, unless otherwise stated. Data collection and analysis were performed using pCLAMP v6.0 (Axon Instruments) and Origin v4.0 (MicroCal) software. For subtraction of endogenous and leak currents, records were obtained in ND-96, 96 mm NaCl, 2 mm KCl, 1 mmMgCl2, 5 mm Na-HEPES, and these were subtracted from recordings in KD-98 before further analysis. COS-7 cells (ATCC) were cultured and transiently transfected with FLAG-hgb2 and mgb1a receptor DNAs alone and in combination using LipofectAMINE reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Membranes prepared from these cells were digitonin-solubilized, and mgb1a/FLAG-gb2 heterodimers were immunoprecipitated with either a mouse anti-FLAG M2 antibody (Kodak IBI) targeting the FLAG-gb2 receptor or anti-gb1 receptor rabbit polyclonal antibodies 1713.1–1713.22 using previously described conditions (6Hébert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 272: 29229-29327Google Scholar). The immunoprecipitates were then washed and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with either the anti-FLAG or anti-gb1 antibodies using previously reported conditions (6Hébert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 272: 29229-29327Google Scholar) and as described below. Northern blot analysis revealed that many brain regions, including cortex, show overlapping expression patterns for gb2 and gb1 receptor mRNA (10Ng G.Y.K. MacDonald T. Bonnert T. Rigby M. Heavens R. Whiting P. Chateauneuf A. Coulombe N. Kargman S. Caskey T. Evans J. O'Neill G. Liu Q. Genomics. 1999; 56: 288-295Crossref PubMed Scopus (17) Google Scholar). In situ hybridization in adjacent coronal sections of rat parietal cortex using 35S-labeled antisense riboprobes indicates that mRNAs for both gb1 and gb2 receptors are coexpressed in this brain region (Fig. 1,A and B). No hybridization signal was detected with the control 35S-labeled sense gb1 and gb2 riboprobes (data not shown). To examine expression at the cellular level, digoxigenin-labeled gb1 and radiolabeled gb2 riboprobes were hybridized to the same sections (Fig. 1, C and D). Overlay of the gb1 and gb2 hybridization signals revealed that messages for both receptors are generally expressed in the same neurons (Fig.1 E). In other major brain regions, including the hippocampus, thalamus, and cerebellum,3 gb2 and gb1 receptor mRNAs are colocalized with ≥95% of gb2 expressing cells also expressing gb1. This suggests that their coexpression may be required for activity in these neurons. In melanophores transiently cotransfected with the mgb1a and hgb2 receptors, GABA mediated a dose-dependent pigment aggregation response with an IC50 value of 3–7 μM (n = 3), which is absent in mock-transfected cells and cells transfected with the mgb1a or hgb2 alone (Fig. 2). The GABA-mediated inhibitory activity represented 42–56% (n = 3) of a control Gi-coupled CB2 cannabinoid receptor response (Fig.2, inset). GABA activity could be inhibited by the CGP71872 antagonist (n = 3), indicating that this was GABAB receptor-specific (Fig. 2). To confirm the observations that functional GABAB receptor results from the coexpression of gb1a and gb2 receptors and that it is Gi-coupled, we directly examined modulation of cAMP levels in HEK-293 cells. In cell lines stably expressing the individual receptors we observed small and inconsistent responses in assays to examine agonist-mediated modulation of cAMP synthesis (Fig.3). However, transient hgb2 transfection of HEK-293 cells stably expressing rgb1a (rgb1a-50) and transient rgb1a transfection of HEK-293 cells stably expressing hgb2 (hgb2-42) significantly enhanced the ability of baclofen and GABA to inhibit forskolin-stimulated cAMP synthesis. Hgb2-42/rgb1a and rgb1a-50/hgb2 cells exhibited 28 and 34% reductions in forskolin-stimulated cAMP synthesis with 30 μm baclofen and 40 and 43% decreases with 30 μm GABA, respectively (Fig. 3). Although inhibition of cAMP synthesis was sometimes observed with rgb1a-50/rgb1a and hgb2-42/hgb2, these effects were relatively small (0–20% inhibition; Fig. 3) when compared with those observed with the coexpressing cells. Neither baclofen nor GABA in the absence of forskolin had any effect on cAMP synthesis (Fig. 3). In addition, wild-type HEK-293 cells did not exhibit baclofen- or GABA-mediated inhibition of forskolin-stimulated cAMP synthesis (Fig. 3). These data demonstrate that the functional GABAB receptor requires both the gb1 and gb2 receptors for signaling via adenylyl cyclase. Native functional GABAB receptors have been reported to couple to Kirs (3Misgeld U. Bijak M. Jarolimek W. Prog. Neurobiol. 1995; 46: 423-462Crossref PubMed Scopus (419) Google Scholar). Co-expression of the mgb1a and hgb2 with Kir 3.1/3.2 resulted in a significant stimulation of Kir current in response to GABA (301 ± 20.6% (n = 3) increase over control current) measured at −80 mV, which could subsequently be washed out with control solution (Fig. 4) or blocked with the CGP71872 antagonist (data not shown).Modulation of Kir 3.1/3.2 was not seen in oocytes expressing mgb1a or hgb2 individually even in the presence of Giα1 (Fig. 4). The dependence of functional GABAB receptor activity on the coexpression of gb1a and gb2 suggests that these receptors may undergo heterodimerization. Immunoblot analysis revealed selective expression of mgb1a monomers and homodimers in mgb1a and mgb1a/FLAG-gb2 expressing cells and expression of FLAG-gb2 receptors in FLAG-gb2 and mgb1a/FLAG-gb2 expressing cells (Fig.5, lanes 1–8). To demonstrate the existence of gb1a-gb2 heterodimers, we utilized a differential co-immunoprecipitation and immunoblotting strategy. Anti-gb1 receptor antibodies were used to blot receptors immunoprecipitated with anti-FLAG antibodies (Fig. 5, lanes 9–12). No mgb1a immunoreactivity was detected in samples prepared from mock vector transfected cells, FLAG-gb2 expressing cells, and mgb1a receptor expressing cells as expected because these species could not be immunoprecipitated with the anti-FLAG antibody and detected with the anti-gb1 antibody (Fig. 5, lanes 9–11). Immunoreactive ∼250- (representing the mgb1a-gb2 heterodimer) and ∼130-kDa species (representing the mgb1a monomer) were detected only in cells coexpressing the mgb1a and FLAG-gb2 receptors, demonstrating that gb1a and gb2 can only be co-immunoprecipitated as part of a complex (Fig. 5,lane 12). Similar demonstration of gb1a-gb2 heterodimerization was obtained when coexpressed receptors were immunoprecipitated first with anti-gb1 antibodies followed by immunoblotting with the anti-FLAG antibody (Fig. 5, lane 16). The ∼250-kDa species represents the heterodimer, whereas the ∼130-kDa species represents the FLAG-gb2 monomer. No FLAG-gb2 immunoreactivity was detected in samples prepared from mock vector transfected cells, FLAG-gb2 expressing cells and mgb1a receptor expressing cells as expected because these species could not be immunoprecipitated with the anti-gb1 antibody and detected with the anti-FLAG antibody (Fig. 5, lanes 13–15). The gb1a-gb2 heterodimer, which is stable in SDS, might result from SDS-resistant intermolecular transmembrane interactions as reported for the formation of β2-adrenergic and dopamine D2 receptor homodimers (5Ng G.Y.K. O'Dowd B.F. Chung H.T. Lee S.P. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Comm. 1996; 227: 200-204Crossref PubMed Scopus (240) Google Scholar, 6Hébert T.E. Moffett S. Morello J.P. Loisel T.P. Bichet D.G. Barret C. Bouvier M. J. Biol. Chem. 1996; 272: 29229-29327Google Scholar). The monomer presumably results from partial disruption of protein-protein binding domains (Sushi Repeats) or C-terminal α-helical domains in mgb1a.2 Disulfide bonds may also contribute to dimer formation as has been reported for the structurally related metabotropic glutamate receptor (7Romano C. Yang W. O'Malley K.L. J. Biol. Chem. 1996; 271: 28612-28616Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). We have shown in three disparate expression systems that coexpression of the gb1 and gb2 receptors is required for functional GABAB receptor activity. That the two receptors are co-immunoprecipitated is consistent with the formation of heterodimers. We cannot rule out the possibility that either receptor may function as a monomeric or homodimeric receptor in the appropriate native cells. Indeed there are regions of the brain, such as caudate/putamen, where gb1 mRNA is abundant and gb2 mRNA is undetectable (Fig. 1,A and B). In such regions gb1 would either require a different subunit or function as a monomer or homodimer. However, our in situ hybridization analysis indicates that in brain regions expressing gb2, gb1 is expressed in a majority of the same neurons, suggesting that native receptors are heterodimers in these cells. GPCRs are commonly thought of as monomeric receptors. 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