Binding of alpha- and betagamma-subunits of G0 to beta1-adrenoceptor in sealed unilamellar lipid vesicles

Autor: Margareta Frohlich, Mirko Hekman, Nicol P. Kurstjens, Richard C. Cantrill, Christian Dees, Ernst J.M. Helmreich
Rok vydání: 1991
Předmět:
Zdroj: European Journal of Biochemistry. 197:167-176
ISSN: 1432-1033
0014-2956
DOI: 10.1111/j.1432-1033.1991.tb15895.x
Popis: 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 G0-proteins purified to homogeneity from bovine brain, the lipid-rich lighter vesicle fraction I took up these subunits nearly exclusively. Whereas, when a β1-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 αo or alone with β1-adrenoceptors, some of these subunits appear together with β1-adrenoceptors in the small vesicle fraction II, but much more αo 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 β1-receptor where they may serve as an anchor for α-subunits. Secondly, the binding of αo- and βγ-subunits to the β1-adrenoceptors enhances the basal GTPase activity of αo. Thirdly, since the binding domains of the β1-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 Go-subunits from bovine brain to a β1-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.
Databáze: OpenAIRE
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