BINDING OF ALPHA-SUBUNIT AND BETA-GAMMA-SUBUNIT OF G0 TO BETA-1-ADRENOCEPTOR IN SEALED UNILAMELLAR LIPID VESICLES

First, we describe a preparation of sealed unilamellar lipid vesicles. When this preparation was subjected to sucrose density gradient centrifugation, two rather uniform fractions emerged, one consisting of lighter lipid-rich vesicles with average diameters ranging over 150-200 nm (fraction I), the other consisting of heavier vesicles with average diameters ranging over 30-70 nm (fraction II). When the lipid mixture containing dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with alpha- and beta-gamma-subunits of G(o)-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a beta-1-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction II where it was incorporated inside-out. On co-reconstitution of either alpha-o or beta-gamma alone with beta-1-adrenoceptors, some of these subunits appear together with beta-1-adrenoceptors in the small vesicle fraction II, but much more alpha-o was bound to the receptor in the presence of beta-gamma-subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that beta-gamma-subunits can bind to the non-activated beta-1-receptor where they may serve as an anchor for alpha-subunits. Secondly, the binding of alpha-o-and beta-gamma-subunits to the beta-1-adrenoceptors enhances the basal GTPase activity of alpha-o. Thirdly, since the binding domains of the beta-1-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the beta-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213-215 (1987)] for the transducin-rhodopsin interactions. Finally, the binding of G(o)-subunits from bovine brain to a beta-1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.