Functional interactions between the α1b-adrenoceptor and Gα11 are compromised by de-palmitoylation of the G protein but not of the receptor

Both the α1b-adrenoceptor and Gα11 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′-[γ-[35S]thio]triphosphate ([35S]GTPγS) binding. In α1b-adrenoceptor-Gα11 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]GTPγS to α1b-adrenoceptor-Gα11 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 β/γ to Gα11 markedly reduced phenylephrine stimulation of [35S]GTPγS binding. Combination of mutations to prevent thio-acylation and β/γ binding did not, however, have an additive effect on [35S]GTPγS binding. These results indicate that the thio-acylation status of the α1b-adrenoceptor does not regulate G protein activation whereas thio-acylation of Gα11 plays a key role in activation by the receptor beyond providing membrane association and proximity.