Similar apparent constitutive activity of human histamine H-2-receptor fused to long and short splice variants of G(s alpha)

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta (2)-adrenoceptor (beta (2)AR) fused to the lon g splice variant of G(s alpha) (G(s alphaL)) exhibits a higher apparent con stitutive activity than the beta (2)-adrenoceptor fused to the short splice variant of G(s alpha) (G(s alphaS)). Experimentally, this results in highe r efficacy and potency of partial agonists and in higher efficacy of invers e agonists at the beta (2)AR fused to G(s alphaL) relative to the beta (2)A R fused to G(s alphaS), indicating that the agonist-free beta (2)AR and the beta (2)AR occupied by partial agonists promote GDP dissociation from G(s alphaL) more efficiently than from G(s alphaS). In fact, the GDP affinity o f G(s alphaS) fused to the beta (2)AR is higher than the GDP affinity of G( s alphaL) fused to the beta (2)AR. We asked the question whether the histam ine H-2-receptor (H2R) exhibits similar coupling to G(s alpha) splice varia nts as the beta (2)AR. To address this question, we studied H2R-G(s alpha) fusion proteins expressed in Sf9 cells. In contrast to beta (2)AR-G(s alpha ) fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H2R fused to G(s alphaL ) and G(s alphaS) as assessed by guanosine-5'-O-(3-thio)triphosphate bindin g and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that G(s alphaS) fus ed to the H2R possesses a higher GDP-affinity than G(s alphaL) fused to the H2R. Our data show that the H2R fused to G(s alphaL) and G(s alphaS) posse sses similar constitutive activity and is insensitive to differences in GDP affinity of G(s alpha) splice variants. Thus, GDP affinity of G proteins d oes not generally determine constitutive activity of receptors.