Abstract

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The β₂-adrenoceptor (β₂AR) fused to the long splice variant of G_sα (G_sαL) exhibits a higher apparent constitutive activity than the β₂-adrenoceptor fused to the short splice variant of G_sα (G_sαS). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the β₂AR fused to G_sαL relative to the β₂AR fused to G_sαS, indicating that the agonist-free β₂AR and the β₂AR occupied by partial agonists promote GDP dissociation from G_sαL more efficiently than from G_sαS. In fact, the GDP affinity of G_sαS fused to the β₂AR is higher than the GDP affinity of G_sαL fused to the β₂AR. We asked the question whether the histamine H₂-receptor (H₂R) exhibits similar coupling to G_sα splice variants as the β₂AR. To address this question, we studied H₂R-G_sα fusion proteins expressed in Sf9 cells. In contrast to β₂AR-G_sα fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H₂R fused to G_sαL and G_sαS as assessed by guanosine-5′-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5′-O-(3-thio)triphosphate binding indicated that G_sαS fused to the H₂R possesses a higher GDP-affinity than G_sαL fused to the H₂R. Our data show that the H₂R fused to G_sαL and G_sαS possesses similar constitutive activity and is insensitive to differences in GDP affinity of G_sα splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.