Abstract
Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta 2-adrenoceptor (beta 2AR) fused to the long splice variant of Gsalpha (Gsalpha L) exhibits a higher apparent constitutive activity than the beta 2-adrenoceptor fused to the short splice variant of Gsalpha (Gsalpha S). Experimentally, this results in higher efficacy and potency of partial ...
Abstract
Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta 2-adrenoceptor (beta 2AR) fused to the long splice variant of Gsalpha (Gsalpha L) exhibits a higher apparent constitutive activity than the beta 2-adrenoceptor fused to the short splice variant of Gsalpha (Gsalpha S). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta 2AR fused to Gsalpha L relative to the beta 2AR fused to Gsalpha S, indicating that the agonist-free beta 2AR and the beta 2AR occupied by partial agonists promote GDP dissociation from Gsalpha L more efficiently than from Gsalpha S. In fact, the GDP affinity of Gsalpha S fused to the beta 2AR is higher than the GDP affinity of Gsalpha L fused to the beta 2AR. We asked the question whether the histamine H2-receptor (H2R) exhibits similar coupling to Gsalpha splice variants as the beta 2AR. To address this question, we studied H2R-Gsalpha fusion proteins expressed in Sf9 cells. In contrast to beta 2AR-Gsalpha fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H2R fused to Gsalpha L and Gsalpha S 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 Gsalpha S fused to the H2R possesses a higher GDP-affinity than Gsalpha L fused to the H2R. Our data show that the H2R fused to Gsalpha L and Gsalpha S possesses similar constitutive activity and is insensitive to differences in GDP affinity of Gsalpha splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.
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