Molecular Analysis of b 2-Adrenoceptor Coupling to Gs-, Gi-, and Gq-Proteins

The b2-adrenoceptor (b2AR) couples to the G-protein Gs to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the b2AR can also interact with G-proteins of the Giand Gq-family. To assess the efficiency of b2AR interaction with various G-protein a-subunits (Gxa), we expressed fusion proteins of the b2AR with the long (GsaL) and short (GsaS) splice variants of Gsa, the Gi-proteins Gia2 and Gia3, and the Gq-proteins Gqa and G16a in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to Gxa because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the b2AR couples to Gs-, Gi-, and Gq-proteins as assessed by ternary complex formation and ligand-regulated guanosine 59-O-(3-thiotriphosphate) (GTPgS) binding. The combined analysis of ternary complex formation, GTPgS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the b2AR with the various classes of G-proteins. Comparison of the coupling of the b2AR and formyl peptide receptor to Gia2 revealed receptor-specific differences in the kinetics of GTPgS binding. We also detected highly efficient stimulation of GTPgS dissociation from GsaL, but not from Gqa and G16a, by a b2AR agonist. Moreover, we show that the 1:1 stoichiometry of receptor to G-protein in fusion proteins reflects the in vivo stoichiometry of receptor/G-protein coupling more closely than was previously assumed. Collectively, our data show 1) that the b2AR couples differentially to Gs-, Gi-, and Gq-proteins, 2) that there is ligandspecific coupling of the b2AR to G-proteins, 3) that receptorspecific G-protein conformational states may exist, and 4) that nucleotide dissociation is an important mechanism for G-protein deactivation. The b2-adrenoceptor (b2AR) is a prototypical G-proteincoupled receptor that interacts with the stimulatory G-protein of adenylyl cyclase, Gs (Gilman, 1987; Kobilka, 1992). Intriguingly, studies of intact cells, cell membranes, and purified proteins have shown that the b2AR can also interact with Gi-proteins (Katada et al., 1982; Asano et al., 1984; Xiao et al., 1995, 1999; Daaka et al., 1997; Pavoine et al., 1999). In addition, the b2AR can activate phospholipase C-b via Gproteins of the Gq-family, e.g., G16a and Gqa (Zhu et al., 1994; Offermanns and Simon, 1995; Wu et al., 1995). In recent studies (Seifert et al., 1998a,b; Wenzel-Seifert et al., 1998b), we analyzed the coupling of the b2AR to Gsa using fusion proteins. In fusion proteins, the receptor C terminus is covalently linked to the N terminus of Gxa. Fusion ensures a defined 1:1 stoichiometry of receptor to G-protein and promotes efficient coupling without altering the fundamental properties of the signaling partners. The fusion protein approach has been successfully applied to various receptors and G-proteins (Seifert et al., 1999c; Milligan, 2000). With the fusion protein approach we could dissect subtle differences in the coupling of the b2AR to GsaS and GsaL (Seifert et al., 1998b). In the latter study, we analyzed receptor-G-protein coupling by measuring ternary complex formation, i.e., the complex of agonist, receptor, and nucleotide-free G-protein displaying high agonist affinity, steady-state GTP hydrolysis, and adenylyl cyclase activation. The goal of our present study was to quantitatively compare the coupling of the b2AR to Gs-, Gi-, and Gq-proteins. To achieve this aim, we needed a system that ensures defined receptor-G-protein stoichiometry and efficient coupling. This work was supported by The New Faculty Award of the University of Kansas and the J.R. and Inez Jay BioMedical Research Award of The Higuchi Biosciences Center of the University of Kansas to R.S. While working in Stanford, R.S. and K.W.S. were supported by a research fellowship of the Deutsche Forschungsgemeinschaft. ABBREVIATIONS: b2AR, b2-adrenoceptor; b2AR-Gia2 (-Gia3, -Gqa, -GsaL, -GsaS, -G16a), fusion proteins consisting of the b2-adrenoceptor and Gia2, Gia3, Gqa, the short splice variant of Gsa, the long splice variant of Gsa, and G16a, respectively; DHA, [ H]dihydroalprenolol; DCI, dichloroisoproterenol; DOB, dobutamine; EPH, (2)-ephedrine; FPR, formyl peptide receptor; FPR-Gia2, fusion protein consisting of the FPR and Gia2; GTPgS, guanosine 59-O-(3-thiotriphosphate); Gxa, nonspecified G-protein a-subunit; ISO, (2)-isoproterenol; ICI, ICI 118,55 ([erythro-DL-1(7methylindan-4-yloxy)-3-isopropylaminobutan-2-ol]); SAL, salbutamol; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; RT, reverse transcription. 0026-895X/00/050954-13$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 5 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 13106/857840 Mol Pharmacol 58:954–966, 2000 Printed in U.S.A. 954 at A PE T Jornals on D ecem er 9, 2017 m oharm .aspeurnals.org D ow nladed from Therefore, we constructed various b2AR-Gxa fusion proteins and analyzed those proteins in Sf9 insect cells. To validate the results obtained with the fusion protein consisting of the b2AR and Gia2 (b2AR-Gia2), we also coexpressed the b2AR with Gia2. Moreover, we compared b2AR-Gia2 coupling with formyl peptide receptor (FPR)-Gia2 coupling in the fused and nonfused state because the FPR is a prototypical Gi-proteincoupled receptor (Gierschik et al., 1991; Wenzel-Seifert et al., 1998a, 1999). Here, we report differential coupling of the b2AR to Gs-, Gi-, and Gq-proteins and differences in the coupling of the b2AR and FPR to Gi-proteins. Experimental Procedures Materials. The cDNAs of Gia2 and Gia3 in pGEM-2 were kindly provided by Dr. R. Reed (Howard Hughes Medical Institute, JohnsHopkins-University, Baltimore, MD) (Jones and Reed, 1987). The cDNA of G16a in pCMV was a gift from Dr. D. Wu (Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY) (Amatruda et al., 1991). The cDNA of Gqa in pVL1392 was kindly provided by Dr. E. M. Ross (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX) (Biddlecome et al., 1996). Recombinant baculovirus encoding the unmodified versions of the G-protein subunits b1g2 was a kind gift of Dr. P. Gierschik (Abteilung für Pharmakologie und Toxikologie, Universität Ulm, Ulm, Germany). The Gia2 baculovirus was kindly provided by Dr. A. G. Gilman (Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX). Antibodies recognizing Gia3 (anti-Gia3, AS 86, C terminal) (Leopoldt et al., 1997), all Gia subunits (anti-Giacommon, AS 266) (Leopoldt et al., 1997), G16a (anti-G16a, AS 339) (Spicher et al., 1994), and Gqa (anti-Gqa, AS 369) (Spicher et al., 1994) were generously provided by Drs. B. Nürnberg and G. Schultz (Institut für Pharmakologie, Freie Universität Berlin, Germany). The antibody recognizing Gia1/2 was from Calbiochem (La Jolla, CA). [ S]Guanosine 59-O(3-thiotriphosphate (GTPgS; 1000–1500 Ci/mmol) was from NEN Life Science Products (Boston, MA). [H]Dihydroalprenolol (DHA; 85–90 Ci/mmol) was from Amersham Pharmacia Biotech (Piscataway, NJ). Unlabeled GTPgS and GDP were obtained from Roche Diagnostics (Indianapolis, IN). ICI 118,55 ([erythro-DL-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol]) (ICI) was from Research Biochemicals International (Natick, MA). The M1 monoclonal antibody (detecting the FLAG epitope), (2)-isoproterenol (ISO), salbutamol (SAL), (2)-ephedrine (EPH), and (6)-alprenolol were from Sigma (St. Louis, MO). Dichloroisoproterenol (DCI) was from Aldrich (Milwaukee, WI). All restriction enzymes, DNA polymerase I, and T4 DNA ligase were from New England Biolabs (Beverly, MA). Glass fiber filters (GF/C) were from Schleicher & Schuell (Dassel, Germany). All other reagents were of the highest purity available and from