Examining the efficiency of receptor/G-protein coupling with a cleavable beta2-adrenoceptor-gsalpha fusion protein.
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.
Seifert
R
R
Howard Hughes Medical Institute, Stanford University Medical, California, USA.
Wenzel-Seifert
K
K
Gether
U
U
Lam
V T
VT
Kobilka
B K
BK
D000262:Adenylyl Cyclases / Q000378:metabolism; D058666:Adrenergic beta-2 Receptor Agonists; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007313:Insecta; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D058666:Adrenergic beta-2 Receptor Agonists; D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D013917:Thrombin; D020558:GTP Phosphohydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases