Reconstitution of β 2 ‐adrenoceptor−GTP‐binding‐protein interaction in Sf9 cells

Abstract

In most studies, coupling of the β 2 ‐adrenoceptor (β 2 AR) to the stimulatory, heterotrimeric GTP‐binding protein of adenylyl cyclase the (G s ) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which β 2 AR‐G s interactions can be studied directly at the level of the G‐protein. We expressed the β 2 AR alone, in combination with the α‐subunit of G s (G sα ), and as fusion protein with G sα (β 2 AR‐G sα ) in Sf9 insect cells. The β 2 AR expressed alone couples poorly to the endogenous G sα ‐like G‐protein of Sf9 cells since no high‐affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high‐affinity GTPase and guanosine 5′‐ O ‐(3‐thiotriphosphate) (GTP[S]) binding were small. β 2 AR‐G sα reconstituted high‐affinity agonist binding and regulated adenylyl cyclase more effectively than the β 2 AR co‐expressed with a large excess of G sα . In membranes expressing β 2 AR‐G sα , highly effective agonist‐ and inverse agonist regulation of high‐affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing β 2 AR and G sα as separate proteins was difficult to detect. Our data show that the β 2 AR interacts with G sα more efficiently when expressed as a fusion protein than when expressed with an excess of non‐fused G sα . The β 2 AR‐G sα fusion protein provides a very sensitive model system to study the regulation of G s function by β 2 AR agonists and inverse agonists directly at the level of the G‐protein.