Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go.alpha. with rhodopsin, anti-Go.alpha. polyclonal antibodies, and a monoclonal antibody against transducin .alpha. subunit and Gi.alpha.

Abstract

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of .alpha., .beta., and .gamma. subunits; the .alpha. subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go.alpha. with .beta..gamma. subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go.alpha. purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt.beta..gamma. or by rabbit liver G.beta..gamma.. Go.alpha. in the presence of G.beta..gamma. is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5''-O-(2-thiodiphosphate). ADP-Ribosylation of Go.alpha. by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go.alpha. is similar to Gt.alpha. and Gi.alpha.; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt.beta..gamma.. Go.alpha. (39K) can be distinguished from Gi.alpha. (41K) but not from Gt.alpha. (39K) by molecular weight. A monoclonal antibody against Gt.alpha. that cross-reacts well with Gi.alpha. but relatively poorly with Go .alpha. and polyclonal antibodies directed to Go.alpha. but not Gi.alpha. and Gt.alpha. can be used to distinguish the two 39-kilodalton proteins.