Binding of alpha- and beta gamma-subunits of Go 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 Go-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 Go-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.
Kurstjens
N P
NP
Physiologisch-Chemisches Institut der Universität Würzburg, Federal Republic of Germany.
Fröhlich
M
M
Dees
C
C
Cantrill
R C
RC
Hekman
M
M
Helmreich
E J
EJ
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism*; D014422:Turkeys; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D008081:Liposomes; D046911:Macromolecular Substances; D011943:Receptors, Adrenergic, beta; D010566:Virulence Factors, Bordetella; D009243:NAD; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins