Guanine nucleotide-binding (G) proteins are important in a number of membrane signal-transducing systems, including the hormone-sensitive adenylyl cyclase and the retinal light-sensitive cyclic GMP phosphodiesterase complex. In retinal rods, transducin (or Gt), a G protein, couples the photoreceptor rhodopsin to the cyclic GMP phosphodiesterase. Like all G proteins, transducin is a heterotrimer composed of alpha, beta, and gamma subunits. The alpha subunit binds guanine nucleotides and possesses intrinsic GTPase activity. The beta-gamma subunits, which are isolated as a complex, facilitate the interactions of alpha with receptor. The amino-terminus of transducin alpha (Gt(alpha)) may be involved in its binding to the beta-gamma subunits. To identify domains responsible for functional interactions between the alpha and beta-gamma subunits, monoclonal antibodies against Gt were prepared. One monoclonal, MSN1, inhibited photolyzed rhodopsin-stimulated GTP hydrolysis catalyzed by Gt(alpha) in the presence of Gt(beta-gamma), as well as beta- gamma-stimulated pertussis toxin-catalyzed ADP-ribosylation of Gt(alpha). Proteolytic fragments of Gt(alpha) generated with V8 protease and trypsin, and lacking the amino-terminus, did not react with MSN1. MSN1 failed to cross-react with Gs(alpha), Gi(alpha), or Go(alpha). Both beta-gamma, which is believed to interact with the amino-terminus of alpha, and light-activated rhodopsin, which is thought to interact with the carboxyl-terminus, inhibited the reaction of a with MSN1 on immunoblots; dark-adapted rhodopsin had no effect. Synergistic inhibition of MSN1 binding was observed in the presence of both beta-gamma and photolyzed rhodopsin, probably due to beta-gamma-enhanced binding of alpha to receptor. These results are compatible with the hypothesis that binding of light-activated rhodopsin at the carboxyl-terminus of alpha can influence conformation of the amino-terminus or that the amino- and carboxyl-termini are in close proximity in the native protein, permitting bound rhodopsin to interfere with MSN1 interaction with an epitope near the amino-terminus.
