Abstract
The histamine H1 receptor and the alpha1b-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 G11alpha 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]GTPgammaS) binding to receptor-G protein fusions containing wild type forms of both polypeptides. For both receptors this was abolished by incorporation of G208AG11alpha 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]GTPgammaS 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 alpha1b-adrenoreceptor-G11alpha fusions allowed detection of functional hetero-dimeric complexes, whereas co-expression of histamine H1 receptor-G11alpha with increasing amounts of L151Dalpha1b-adrenoreceptor resulted in decreasing levels of histamine-stimulated [35S]GTPgammaS binding. Co-expression of the alpha1b-adrenoreceptor with a fusion protein incorporating the N-terminal domain and transmembrane helix 1 of the alpha1b-adrenoreceptor and G11alpha 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.
Highlights
The concept that G protein-coupled receptors (GPCRs)1 exist as dimers or higher order oligomers has moved rapidly from hypothesis to being widely accepted [1,2,3,4]
Chimeric class C GPCRs consisting of the extracellular domain of one GPCR and the TM and intracellular elements of a second, closely related GPCR or in which the intracellular loops of dimer partners are exchanged have provided strong evidence that the mechanism of action of the dimer involves transactivation [13,14]; that is, ligand binding to one element of the dimer results in activation of G protein produced by the other GPCR within the dimer
That GPCRs can exist as dimers is widely accepted [1,2,3,4]
Summary
Materials—A fibroblast cell line (EF88) (20 –22) derived from a combined G␣q/G␣11 double knockout mouse [23,24] was the gift of Dr M. In the first step, using the N-terminal primer 5Ј-GATACTGGGCTATCCAAGCTTATGAGCCTCCCCAATTCCTC-3Ј, a HindIII restriction site (underlined) was introduced by PCR upstream of the coding sequence of the human histamine H1 receptor. Using the N-terminal primer 5Ј-TTTCCCAAGCTTATGGAACAAAAACTTATTTCTGAAGAAGATCTGAATCCCGATCTGGACACC-3Ј a HindIII restriction site (underlined) followed by an ATG codon and the c-Myc tag sequence (italics) was introduced by PCR upstream of the coding sequence of the ␣1b-adrenoreceptor. [3H]Mepyramine binding assays, monitoring expression of the histamine H1 receptor-containing constructs were initiated by the addition of 8 g of cell membranes to an assay buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM MgCl2 (pH 7.4) containing [3H]mepyramine (0.1–10 nM). GPCR Co-immunoprecipitation Studies—Co-immunoprecipitation studies using FLAG- and c-Myc-tagged forms of the ␣1b-adrenoreceptor and histamine H1 receptor constructs were performed as in McVey et al [29] and Ramsay et al [30].
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