Coordinated Agonist Regulation of Receptor and G Protein Palmitoylation and Functional Rescue of Palmitoylation-deficient Mutants of the G Protein G sub(11) alpha following Fusion to the alpha sub(1b)-Adrenoreceptor: PALMITOYLATION OF G sub(11) alpha IS NOT REQUIRED FOR INTERACTION WITH beta super(.) gamma COMPLEX

Transfection of either the alpha sub(1b)-adrenoreceptor or G alpha sub(11) into a fibroblast cell line derived from a G alpha sub(q)/G alpha sub(11) double knockout mouse failed to produce elevation of intracellular [Ca super(2+)] upon the addition of agonist. Co-expression of these two polypeptides, however, produced a significant stimulation. Co-transfection of the alpha sub(1b)-adrenoreceptor with the palmitoylation-resistant C9S,C10S G alpha sub(11) also failed to produce a signal, and much reduced and kinetically delayed signals were obtained using either C9S G alpha sub(11) or C10S G alpha sub(11). Expression of a fusion protein between the alpha sub(1b)-adrenoreceptor and G alpha sub(11) allowed [Ca super(2+)] sub(i) elevation, and this was also true for a fusion protein between the alpha sub(1b)-adrenoreceptor and C9S,C10S G alpha sub(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 super(.) gamma -sequestering agent, fully attenuated the Ca super(2+) signal. Both of these fusion proteins and one in which an acylation-resistant form of the receptor was linked to wild type G alpha sub(11) were also targets for agonist-regulated [ super(3)H]palmitoylation and bound [ super(35)S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in an agonist concentration-dependent manner. The potency of agonist to stimulate [ super(35)S]GTP gamma S 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 super(.) gamma complex upon agonist stimulation whether or not the G protein can be palmitoylated. They also demonstrate that Ca super(2+) signaling in EF88 cells by such fusion proteins is mediated via release of the G protein beta super(.) gamma complex.