Coordinated agonist regulation of receptor and G protein palmitoylation and functional rescue of palmitoylation-deficient mutants of the G protein G11α following fusion to the α1b-adrenoreceptor -: Palmitoylation of G11α is not required for interaction with β-γ complex

Transfection of either the alb-adrenoreceptor or Gall into a fibroblast cell line derived from a G alpha (q)/G alpha (11) double knockout mouse failed to produce elevation of intracellular [Ca2+] 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 G alpha (11) also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G alpha (11) or C10S G alpha (11). Expression of a fusion protein between the alpha (1b)-adrenoreceptor and G alpha (11) allowed [Ca2+](i) elevation, and this was also true for a fusion protein between the alpha (1b)-adrenoreceptor and C9S,C10S G alpha (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 Ca2+ signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G alpha (11) were also targets for agonist-regulated [H-3]palmitoylation and bound [S-35]guanosine 5 ' -3-O-(thio)triphosphate (GTP gammaS) in an agonist concentration-dependent manner. The potency of agonist to stimulate [S-35]GTP gammaS 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 Ca2+ signaling in EF88 cells by such fusion proteins is mediated via release of the G protein beta . gamma complex.