BETA-ADRENERGIC-RECEPTOR-MEDIATED DISSOCIATION AND MEMBRANE RELEASE OF THE GS-PROTEIN IN S49 LYMPHOMA-CELL MEMBRANES - DEPENDENCE ON MG2+ AND GTP

We reported [Ransnas, Svoboda, Jasper & Inset (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7900 7903] that in intact S49 lymphoma cells the beta-adrenergic-receptor agonist isoprenaline dissociates the stimulatory guanine-nucleotide-binding protein, G(s), into its alpha(s) and beta-gamma-subunits, leading to redistribution of alpha(s) from plasma membranes to the cytoplasm. In the present studies we investigated the kinetics of G(s) dissociation and membrane release in plasma membranes from S49 lymphoma cells. We analysed cholate extracts of membranes for alpha(s) levels by a competitive e.l.i.s.a. with a polyclonal antibody that selectively recognizes monomeric alpha(s) and we assayed supernatant fractions using both competitive e.l.i.s.a. and immunoblotting. The plasma membranes contained 19.3 +/- 1.4 pmol of alpha(s)/mg of membrane protein and lacked significant dissociation of G(s) and activity of adenylate cyclase in the absence of guanine nucleotides. Mg2+ ions were obligatorily required for isoprenaline-induced dissociation of G(s) in plasma membranes and for membrane release of alpha(s). At a physiological concentration of free Mg2+ ions (100-mu-M), 100-mu-M-GTP induced a slow first-order (k = 0.038 +/- 0.004 min-1) dissociation of 17.8 +/- 1.2 pmol of G(s)/mg of membrane protein. A substantial increase in the dissociation rate of G(s) was achieved by addition of 1-mu-M-isoprenaline and 100 -mu-M-GTP; 18.4 +/- 0.9 pmol of G(s)/mg of membrane protein was dissociated, with a k of 1.49 +/- 0.22 min-1. The effect of isoprenaline on the dissociation rate and on membrane release of G(s) was completely blocked by the beta -adrenergic receptor antagonist propranolol. The concentration response relationship for isoprenaline-induced dissociation during the first 1 min after addition of hormone yielded a k(act.) of 16 +/- 5 nM, whereas the k(act) for isoprenaline-induced membrane release was 10 nm. We conclude that release of alpha(s) from plasma membranes is likely to accompany G(s)-subunit dissociation and constitutes a potentially important facet of G(s) action.