Selectivity of the β-Adrenergic Receptor among G_s, G_i’s, and G_o: Assay Using Recombinant α Subunits in Reconstituted Phospholipid Vesicles

The selective regulation of G_s (long and short forms), G_i's (1, 2, and 3), and G_o by the β-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein βγ subunits, and individual recombinant G-protein α subunits that were expressed in and purified from Escherichia coli. Receptor and βγ subunits were incorporated into phospholipid vesicles, and the α subunits bound to the vesicles stoichiometrically with respect to βγ. Efficient regulation of α subunit by receptor required the presence of βγ. Regulation of G proteins was measured according to the stimulation of the initial rate of GTPγS binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted α_s (long or short form) was stimulated by receptor to approximately the extent described previously for natural G_s. Both α_i,1 and α_i,3 were regulated with 25-33% of that efficiency. Stimulation of α_o and α_i,2 was weak, and stimulation of α_o was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.