title: Functional interactions between the alpha1b-adrenoceptor and Galpha11 are compromised by de-palmitoylation of the G protein but not of the receptor.
abstract: Both the alpha1b-adrenoceptor and Galpha11 are targets for post-translational thio-acylation that is regulated by agonist occupancy of the receptor [P.A. Stevens, J. Pediani, J.J. Carrillo, G. Milligan, J. Biol. Chem. 276 (2001) 35883]. In co-expression studies mutation of the sites of thio-acylation in the G protein or treatment of cell membranes with hydroxylamine greatly reduced agonist stimulation of guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTPgammaS) binding. In alpha1b-adrenoceptor-Galpha11 fusion proteins mutation of thio-acylation sites in receptor or G protein did not alter the binding affinity of the antagonist [3H]prazosin or the agonist phenylephrine. Although the potency of phenylephrine to stimulate binding of [35S]GTPgammaS to alpha1b-adrenoceptor-Galpha11 fusion proteins was unaffected by the thio-acylation potential of either element, the maximal effect was reduced by some 50% when the G protein but not the receptor was mutated to prevent thio-acylation. This reflected lack of thio-acylation of the G protein rather than mutation of Cys9 and Cys10 to Ser because treatment with hydroxylamine mimicked this in fusions containing the wild type G protein but was without effect in those mutated to prevent thio-acylation. Mutation to reduce binding of beta/gamma to Galpha11 markedly reduced phenylephrine stimulation of [35S]GTPgammaS binding. Combination of mutations to prevent thio-acylation and beta/gamma binding did not, however, have an additive effect on [35S]GTPgammaS binding. These results indicate that the thio-acylation status of the alpha1b-adrenoceptor does not regulate G protein activation whereas thio-acylation of Galpha11 plays a key role in activation by the receptor beyond providing membrane association and proximity.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
authors.forename: Jiri
authors.initials: J
authors.lastname: Novotny
authors.forename: Dana
authors.initials: D
authors.lastname: Durchankova
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Carrillo
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
authors.forename: Graeme
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000215:Acylation; D001665:Binding Sites / Q000235:genetics; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D009154:Mutation / Q000235:genetics; D010168:Palmitates / Q000378:metabolism*; D011485:Protein Binding; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D010168:Palmitates; D018340:Receptors, Adrenergic, alpha-1; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D043802:GTP-Binding Protein alpha Subunits, Gq-G11