Vol. 299, Issue 3, 1013-1020, December 2001

Similar Apparent Constitutive Activity of Human Histamine H₂-Receptor Fused to Long and Short Splice Variants of G_s

Katharina Wenzel-Seifert, Melissa T. Kelley¹ , Armin Buschauer and Roland Seifert

Department of Pharmacology and Toxicology, the University of Kansas, Lawrence, Kansas (K.W.-S., M.T.K., R.S.); and Department of Pharmacy, University of Regensburg, Regensburg, Germany (A.B.)

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