Determinants of α₂-adrenergic receptor activation of G proteins: Evidence for a precoupled receptor/G protein state

The ability of agonist-occupied α(2D)-adrenergic receptors to activate G proteins was measured in membranes from PC-12 cells stably expressing the cloned receptor, using guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding as an endpoint. Epinephrine (EPI) stimulated [35S]GTPγS binding in a Mg2+-dependent manner, showing both micromolar and millimolar cation affinities. Prior treatment of cells with pertussis toxin completely eliminated the EPI stimulation. The presence of GDP decreased basal [35S]GTPγS binding and increased the proportion of EPI- stimulated binding. Increasing concentrations of Na+ also reduced basal [35S]GTPγS binding but had less effect on EPI-stimulated binding, such that the agonist response was proportionately greater at higher Na+ levels. In saturation binding studies with [35S]GTPγS only low affinity binding was observed in the presence of 100 mM Na+, whereas in the absence of Na+ a high affinity component was also present, indicating a Na+-regulated receptor/G protein interaction. EPI induced high affinity [35S]GTPγS binding in the presence of Na+ and increased the affinity of the high affinity component under Na+-free conditions. The selective α2-adrenergic antagonist rauwolscine produced rightward shifts of EPI dose-response curves and decreased the basal level of [35S]GTPγS binding across the same range of concentrations. The extent of decrease was dependent upon the α2- adrenergic receptor expression level, indicating that α2-adrenergic receptors contribute to basal G protein activation in the absence of agonist. The ability of rauwolscine to decrease basal [35S]GTPγS binding was diminished as the level of Na+ was increased, suggesting that both agents act to reduce receptor/G protein interaction, by distinctive mechanisms. α2-Adrenergic receptor antagonists reduced basal G protein activation with a rank order for maximal effectiveness that was different from their receptor binding affinities. These results support the existence of precoupling between α(2D)-adrenergic receptors and G proteins; coupling can be diminished by both Na+ and antagonists, whereas agonists increase the efficiency of receptor/G protein coupling.