Reconstitution of β2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells -: High coupling efficiency in a β2-adrenoceptor-Gsα fusion protein

In most studies, coupling of the beta(2)-adrenoceptor (beta(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 mode! system in which beta(2)AR-G, interactions can be studied directly at the level of the G-protein. We expressed the beta(2)AR alone, in combination with the alpha-subunit of G(s) (G(s alpha)), and as fusion protein with G(s alpha) (beta(2)AR-G(s alpha)) in Sf9 insect cells. The beta(2)AR expressed alone couples poorly to the endogenous G(s alpha)-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. beta(2)AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta(2)AR co-expressed with a large excess of G(s alpha). In membranes expressing beta(2)AR-G(s alpha) 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 beta(2)AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta(2)AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta(2)AR-G(s)alpha fusion protein provides a very sensitive model system to study the regulation of G(s) function by beta(2)AR agonists and inverse agonists directly at the level of the G-protein.