Activation of an alpha2A-adrenoceptor-Galphao1 fusion protein dynamically regulates the palmitoylation status of the G protein but not of the receptor.
Post-translational thio-acylation of a fusion protein between the alpha2A-adrenoceptor and the alpha subunit of the G protein G(o1) is both dynamic and regulated by agonist binding. Incorporation of [3H]palmitate into the fusion protein was reduced substantially in the presence of the agonist adrenaline. This was dependent on the concentration of adrenaline and correlated with occupancy of the ligand binding site. Both the receptor and G-protein elements of the fusion construct incorporated [3H]palmitate but this occurred more rapidly for the G-protein element and regulation of acylation by the agonist occurred only for the G protein. The kinetics of de-palmitoylation of the alpha2A-adrenoceptor-Galpha(o1) fusion were accelerated markedly by agonist. Again, this reflected modulation of the G protein but not of the receptor. Agonist-induced regulation of the kinetics of thio-acylation of the G protein was abolished, however, in a mutant unable to bind guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) in response to adrenaline. Despite the dynamic nature of the post-translational acylation and its regulation by agonist, the ability of adrenaline to activate the G protein, monitored by stimulation of the binding of [35S]GTP[S] to such fusion constructs, was unaffected by the palmitoylation potential of either the receptor or G-protein element.
Barclay
Elaine
E
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
O'Reilly
Mark
M
Milligan
Graeme
G
D000215:Acylation; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D010168:Palmitates / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D010168:Palmitates; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D006160:Guanosine Triphosphate; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D004837:Epinephrine