Binding of α‐ and βγ‐subunits of G 0 to β 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 dimyristoylglycero‐phosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoyglycerophosphoethanolamine at molar ratios of 54:35:10:1 was reconstituted with α‐ and γ‐subunits of G 0 ‐proteins purified to homogeneity from bovine brain, the lipid‐rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a β 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 α o or alone with β 1 ‐adrenoceptors, some of these subunits appear together with β 1 ‐adrenoceptors in the small vesicle fraction II, but much more α o was bound to the receptor in the presence of βγ‐subunits. The observations reported are novel and surprising in several respects: firstly, they suggest that βγ‐subunits can bind to the non‐activated β 1 ‐receptor where they may serve as an anchor for α‐subunits. Secondly, the binding of α o ‐ and βγ‐subunits to the β 1 ‐adrenoceptors enhances the basal GTPase activity of α o . Thirdly, since the binding domains of the β 1 ‐adrenoceptor for G‐proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G‐proteins with the β‐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 β 1 ‐adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.