Binding of α‐ and βγ‐subunits of G₀ to β₁‐adrenoceptor in sealed unilamellar lipid vesicles

Abstract

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 11). When the lipid mixture containing dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine at molar ratios of 54:35: 10: 1 was reconstituted with a-and fly-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 P,-adrenoceptor preparation purified from turkey erythrocyte membranes was reconstituted, it was found nearly completely in the smaller heavier vesicle fraction I1 where it was incorporated inside-out. On co-reconstitution of either a. or pi alone with P,-adrenoceptors, some of these subunits appear together with fll-adrenoceptors in the small vesicle fraction 11, but much more a, was bound to the receptor in the presence of fly-subunits.The observations reported are novel and surprising in several respects: firstly, they suggest that &subunits can bind to the non-activated fl,-receptor where they may serve as an anchor for a-subunits. Secondly, the binding of ro-and By-subunits to the B,-adrenoceptors enhances the basal GTPase activity of a,. Thirdly, since the binding domains of the a,-adrenoceptor for G-proteins were facing outwards in our sealed vesicle preparations, it follows that interactions of G-proteins with the P-receptor can occur at the aqueous membrane interface as was postulated originally by M. Chabre [Trends Biochem. Sci. 12, 213 -215 (1987)l for the transducin-rhodopsin interactions. Finally, the binding of Go-subunits from bovine brain to a fl,-adrenoceptor from turkey erythrocytes was not expected, since these polypeptides are not likely to be physiological partners.It has been proposed that the fly-subunits of GTP-binding proteins anchor the cc-subunits to plasma membranes [ 11. The hydrophobic nature of the y-subunit and its post-translational isoprenylation and carboxylmethylation would support such a role [2-71. But Chabre has assumed that transducin is attached to the aqueous cytoplasmic site of the membrane primarily by interactions with membrane-spanning rhodopsin [8]. This suggestion is in agreement with the behaviour of several deletion mutants of the hamster B,-adrenoceptor expressed in mammalian cells [9 -1 I] and with results obtained with site-specific synthethic peptides deduced from the sequences of the P-receptor and a-subunit of G, [12, 131. This study was intended to probe the mutual assistance of z-and pi-subunits in binding to B-adrenoceptors inserted into artificial membranes. The CI-and By-subunits of GTP-binding proteins purified from bovine brain and rod outer segment membranes, trimeric G, from...

Identification of Gβγ Binding Sites in the Third Intracellular Loop of the M3-muscarinic Receptor and Their Role in Receptor Regulation

“…Additional data indicate that even within the context of G-protein heterotrimers, a receptor may make contact with all three G-protein subunits (45)(46)(47)(48)(49). If so, then it is unclear whether the G␤␥ binding site identified in the i3 loop of the M 3 -MR in the present study is also involved in the interaction between receptor and G␤␥ in G-protein heterotrimers.…”

Suramin Disrupts Receptor-G Protein Coupling by Blocking Association of G Protein α and βγ Subunits

“…These findings suggest that suramin may function in the same manner as GTP␥S, which promotes the dissociation of G␣ from G␤␥. Suramin has been shown to reduce the affinity of the transducin ␣ subunit for the plasma membrane (Lehmann et al, 2002); this effect can be attributed to disruption of G␣-G␤␥ association because G␤␥ plays a major role in anchoring G␣ to the membrane (Sternweis, 1986;Kurstjens et al, 1991). Moreover, a docking simulation has suggested that the binding site for suramin is in the vicinity of a conserved arginine residue (Arg 201 ) in the switch II region of G␣ (Lehmann et al, 2002); this binding site may extend toward the N terminus in the three-dimensional structure.…”

Overexpression, Solubization and Purification of Rat and Human Olfactory Receptors

“…Such experiments are essential for future functional studies, since seven-transmembrane-domain receptors are known to activate G-protein only when incorporated in a lipid bilayer [20,311. The availability of large amounts of purified, lipid-reconstituted olfactory receptors will greatly facilitate future research into the area of receptor/ligand recognition and transduction mechanisms in the superfamily of olfactory receptors.…”

“…Lipid vesicles were prepared a5 described [20]. A mixture (total 200 mg) of dimyristoylglycerophosphocholine, cholesterol, dipalmitoylglycerophosphoserine and dipalmitoylglycerophosphoethanolamine (Sigma) in a molar ra-…”