Dimers of class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins

The histamine H1 receptor and the alpha(1b)-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of G(q)/G(11). Assessed by coimmunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homodimers. The addition of the G protein G(11)alpha to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5'-3-O-([S-35] thio) triphosphate ([S-35] GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG(11)alpha into the fusions. Mutation of a highly conserved leucine in intracellular loop 2 of each receptor also eliminated agonist function but not binding. Co-expression of the two non-functional but complementary fusion constructs reconstituted agonist-mediated binding of [S-35] GTPgammaS in membranes of HEK293 cells and elevation of [Ca2+](i) in mouse embryo fibroblasts lacking both G(q) and G(11). Co-expression of the histamine H1 receptor- and the alpha(1b)-adrenoreceptor-G(11)alpha fusions allowed detection of functional hetero- dimeric complexes, whereas co-expression of histamine H1 receptor-G(11)alpha with increasing amounts of L151Dalpha(1b)-adrenoreceptor resulted in decreasing levels of histamine-stimulated [S-35] GTPgammaS binding. Co-expression of the alpha(1b)-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha(1b)-adrenoreceptor and G(11)alpha did not result in agonist activation of the G protein but did indicate a role for transmembrane helix 1 in dimerization. These data demonstrate that dimers of these class A receptors function via trans-activation of associated G proteins.