Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (N(i)) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the α (α(T)) and βγ (βγ(T)) subunit components in mediating this inhibition. The inclusion of either a preactivated α(T)·GTPγS (where GTPγS is guanosine 5'-O-(thiotriphosphate)) complex, or the βγ complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (N(s)) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by ~30-60 and 90%, respectively, at 2 mM MgCl₂). The inhibitions by both of these subunit species are specific for the N(s)-stimulated activity with neither α(T)·GTPγS nor βγ(T) having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl₂ concentration in the assay incubations significantly decreases the inhibitions by both α(T)·GTPγS and βγ(T). Similarly, when the pure hamster lung β-adrenergic receptor is included in the lipid vesicles with N(s) and C, the levels of inhibition of the GppNHp-stimulated activity by both α(T)·GTPγS and βγ(T) are reduced compared to those obtained in vesicles containing just N(s) and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the α(T)·GTPγS inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the βγ(T) complex. The ability of the β-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to N(s) (or α(s)) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the βγ subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.