SELECTIVITY OF THE BETA-ADRENERGIC-RECEPTOR AMONG GS, GI, AND GO - ASSAY USING RECOMBINANT ALPHA-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 beta-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein beta-gamma-subunits, and individual recombinant G-protein alpha-subunits that were expressed in and purified from Escherichia coli. Receptor and beta-gamma-subunits were incorporated into phospholipid vesicles, and the a subunits bound to the vesicles stoichiometrically with respect to beta-gamma. Efficient regulation of alpha-subunit by receptor required the presence of beta-gamma. Regulation of G proteins was measured according to the stimulation of the initial rate of GTP-gamma-S 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 alpha-s (long or short form) was stimulated by receptor to approximately the extent described previously for natural G(s). Both alpha-i,1 and alpha-i,3 were regulated with 25-33% of that efficiency. Stimulation of alpha-o and alpha-i,2 was weak, and stimulation of alpha-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.