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

J Biol Chem. 2003 Oct 24;278(43):42578-87. doi: 10.1074/jbc.M306165200. Epub 2003 Aug 14.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Cell Line
  • Dimerization
  • GTP-Binding Protein alpha Subunits, Gq-G11 / genetics
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
  • GTP-Binding Proteins / biosynthesis
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism*
  • Guanosine Triphosphate / metabolism
  • Humans
  • Mice
  • Receptors, Adrenergic, alpha-1 / genetics
  • Receptors, Adrenergic, alpha-1 / physiology
  • Receptors, G-Protein-Coupled / agonists
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / physiology*
  • Receptors, Histamine H1
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transcriptional Activation*

Substances

  • ADRA1B protein, human
  • Adra1b protein, mouse
  • Receptors, Adrenergic, alpha-1
  • Receptors, G-Protein-Coupled
  • Receptors, Histamine H1
  • Recombinant Fusion Proteins
  • Guanosine Triphosphate
  • GTP-Binding Proteins
  • GTP-Binding Protein alpha Subunits, Gq-G11