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Eur J Biochem. 1999 Mar;260(3):661-6.

Examining the efficiency of receptor/G-protein coupling with a cleavable beta2-adrenoceptor-gsalpha fusion protein.

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1
Howard Hughes Medical Institute, Stanford University Medical, California, USA.

Abstract

Reconstitution of high-affinity agonist binding at the beta2-adrenoceptor (beta2AR) expressed in Sf9 insect cells requires a large excess of the stimulatory G-protein of adenylyl cyclase, Gsalpha, relative to receptor [R. Seifert, T. W. Lee, V. T. Lam & B. K. Kobilka, (1998) Eur. J. Biochem. 255, 369-382]. In a fusion protein of the beta2AR and Gsalpha (beta2AR-Gsalpha), which has only a 1 : 1 stoichiometry of receptor and G-protein, high-affinity agonist binding and agonist-stimulated GTP hydrolysis, guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding and adenylyl cyclase (AC) activation are more efficient than in the nonfused coexpression system. In order to analyze the stability of the receptor/G-protein interaction, we constructed a fusion protein with a thrombin-cleavage site between beta2AR and Gsalpha (beta2AR-TS-Gsalpha). beta2AR-TS-Gsalpha efficiently reconstituted high-affinity agonist binding, agonist-stimulated GTP hydrolysis, GTP[S] binding and AC activation. Thrombin cleaves approximately 70% of beta2AR-TS-Gsalpha molecules in Sf9 membranes. Thrombin cleavage did not impair high-affinity agonist binding and GTP[S] binding but strongly reduced ligand-regulated GTPase activity and AC activity. We conclude that fusion of the beta2AR to Gsalpha promotes tight physical association of the two partners and that this association remains stable for a single activation/deactivation cycle even after cleavage of the link between the receptor and G-protein. Dilution of Gsalpha in the membrane and release of activated Gsalpha into the cytosol can both prevent cleaved beta2AR-TS-Gsalpha from undergoing multiple activation/deactivation cycles.

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