Molecular Analysis of β2-Adrenoceptor Coupling to Gs-, Gi-, and Gq-Proteins
- Howard Hughes Medical Institute, Stanford University Medical School, Stanford, California; and Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, Kansas
Abstract
The β2-adrenoceptor (β2AR) couples to the G-protein Gs to activate adenylyl cyclase. Intriguingly, several studies have demonstrated that the β2AR can also interact with G-proteins of the Gi- and Gq-family. To assess the efficiency of β2AR interaction with various G-protein α-subunits (Gxα), we expressed fusion proteins of the β2AR with the long (GsαL) and short (GsαS) splice variants of Gsα, the Gi-proteins Giα2 and Giα3, and the Gq-proteins Gqα and G16α in Sf9 cells. Fusion proteins provide a rigorous approach for comparing the coupling of a given receptor to Gxα because of the defined 1:1 stoichiometry of receptor and G-protein and the efficient coupling. Here, we show that the β2AR couples to Gs-, Gi-, and Gq-proteins as assessed by ternary complex formation and ligand-regulated guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding. The combined analysis of ternary complex formation, GTPγS binding, agonist efficacies, and agonist potencies revealed substantial differences in the interaction of the β2AR with the various classes of G-proteins. Comparison of the coupling of the β2AR and formyl peptide receptor to Giα2 revealed receptor-specific differences in the kinetics of GTPγS binding. We also detected highly efficient stimulation of GTPγS dissociation from GsαL, but not from Gqα and G16α, by a β2AR 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 β2AR couples differentially to Gs-, Gi-, and Gq-proteins, 2) that there is ligand-specific coupling of the β2AR to G-proteins, 3) that receptor-specific G-protein conformational states may exist, and 4) that nucleotide dissociation is an important mechanism for G-protein deactivation.
Footnotes
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Send reprint requests to: Dr. Roland Seifert, Department of Pharmacology and Toxicology, The University of Kansas, 5064 Malott Hall, Lawrence, KS 66045. E-mail:rseifert{at}falcon.cc.ukans.edu
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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:
- β2AR
- β2-adrenoceptor
- β2AR-Giα2 (-Giα3
- -Gqα, -GsαL, -GsαS, -G16α), fusion proteins consisting of the β2-adrenoceptor and Giα2, Giα3, Gqα, the short splice variant of Gsα, the long splice variant of Gsα, and G16α, respectively
- DHA
- [3H]dihydroalprenolol
- DCI
- dichloroisoproterenol
- DOB
- dobutamine
- EPH
- (−)-ephedrine
- FPR
- formyl peptide receptor
- FPR-Giα2
- fusion protein consisting of the FPR and Giα2
- GTPγS
- guanosine 5′-O-(3-thiotriphosphate)
- Gxα
- nonspecified G-protein α-subunit
- ISO
- (−)-isoproterenol
- ICI
- ICI 118,55 ([erythro-dl-1(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol])
- SAL
- salbutamol
- PCR
- polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- RT
- reverse transcription
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- Received November 18, 1999.
- Accepted July 31, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
