Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

The histamine H1 receptor and the α1b-adrenoreceptor are G protein-coupled receptors that elevate intracellular [Ca2+] via activation of Gq/G11. Assessed by co-immunoprecipitation and time-resolved fluorescence resonance energy transfer they both exist as homo-dimers. The addition of the G protein G11α to the C terminus of these receptors did not prevent dimerization. Agonists produced a large stimulation of guanosine 5′-3-O-([35S]thio)triphosphate ([35S]GTPγS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11α 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 [35S]GTPγS in membranes of HEK293 cells and elevation of [Ca2+]i in mouse embryo fibroblasts lacking both Gq and G11. Co-expression of the histamine H1 receptor- and the α1b-adrenoreceptor-G11α fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11α with increasing amounts of L151Dα1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPγS binding. Co-expression of the α1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the α1b-adrenoreceptor and G11α 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.