Determinants of alpha 2-adrenergic receptor activation of G proteins: evidence for a precoupled receptor/G protein state.

The ability of agonist-occupied alpha 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]GTP gamma S) binding as an endpoint. Epinephrine (EPI) stimulated [35S]GTP gamma S binding in a Mg(2+)-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]GTP gamma S binding and increased the proportion of EPI-stimulated binding. Increasing concentrations of Na+ also reduced basal [35S]GTP gamma S 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]GTP gamma S 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]GTP gamma S binding in the presence of Na+ and increased the affinity of the high affinity component under Na(+)-free conditions. The selective alpha 2-adrenergic antagonist rauwolscine produced rightward shifts of EPI dose-response curves and decreased the basal level of [35-S]GTP gamma S binding across the same range of concentrations. The extent of decrease was dependent upon the alpha 2-adrenergic receptor expression level, indicating that alpha 2-adrenergic receptors contribute to basal G protein activation in the absence of agonist. The ability of rauwolscine to decrease basal [35S]GTP gamma S binding was diminished as the level of Na+ was increased, suggesting that both agents act to reduce receptor/G protein interaction, by distinctive mechanisms. alpha 2-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 alpha 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.