title: Coordinated agonist regulation of receptor and G protein palmitoylation and functional rescue of palmitoylation-deficient mutants of the G protein G11alpha following fusion to the alpha1b-adrenoreceptor: palmitoylation of G11alpha is not required for interaction with beta*gamma complex.
abstract: Transfection of either the alpha(1b)-adrenoreceptor or Galpha(11) into a fibroblast cell line derived from a Galpha(q)/Galpha(11) double knockout mouse failed to produce elevation of intracellular [Ca(2+)] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the alpha(1b)-adrenoreceptor with the palmitoylation-resistant C9S,C10S Galpha(11) also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S Galpha(11) or C10S Galpha(11). Expression of a fusion protein between the alpha(1b)-adrenoreceptor and Galpha(11) allowed [Ca(2+)](i) elevation, and this was also true for a fusion protein between the alpha(1b)-adrenoreceptor and C9S,C10S Galpha(11), since this strategy ensures proximity of the two polypeptides at the cell membrane. For both fusion proteins, co-expression of transducin alpha, as a beta.gamma-sequestering agent, fully attenuated the Ca(2+) signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type Galpha(11) were also targets for agonist-regulated [(3)H]palmitoylation and bound [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [(35)S]GTPgammaS binding was unaffected by the palmitoylation potential of either receptor or G protein. These studies provide clear evidence for coordinated, agonist-mediated regulation of the post-translational acylation of both a receptor and partner G protein and demonstrate the capacity of such fusions to bind and then release beta.gamma complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca(2+) signaling in EF88 cells by such fusion proteins is mediated via release of the G protein beta.gamma complex.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
authors.forename: P A
authors.initials: PA
authors.lastname: Stevens
authors.forename: J
authors.initials: J
authors.lastname: Pediani
authors.forename: J J
authors.initials: JJ
authors.lastname: Carrillo
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D058646:Adrenergic alpha-1 Receptor Agonists; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D019308:Palmitic Acid / Q000378:metabolism*; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
chemical_list: C547716:ADRA1B protein, human; C547717:Adra1b protein, mouse; D058646:Adrenergic alpha-1 Receptor Agonists; D017931:DNA Primers; D018340:Receptors, Adrenergic, alpha-1; D011993:Recombinant Fusion Proteins; D019308:Palmitic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D002118:Calcium