Characterization of transducin from bovine retinal rod outer segments. Use of monoclonal antibodies to probe the structure and function of the subunit

A panel of monoclonal antibodies has been developed against the T alpha, T beta and T gamma subunits of bovine transducin. Two anti-T alpha antibodies from this panel (TF15 and TF16) and a third one (4A) against frog T alpha (Witt, P. L., Hamm, H. E., and Bownds, M. D. (1984) J. Gen. Physiol. 84, 251-263) were characterized. Each of these monoclonal antibodies recognizes a different region of T alpha and has a specific effect on the function of transducin. The binding of TF15 is reversibly enhanced by treating T alpha with either 1 M guanidinium chloride or, to a smaller extent, by the removal of bound guanine nucleotide. Its epitope is located in a 12-kDa tryptic fragment containing the binding site for the guanine moiety of GTP. Taken together, these results support previous observations that the conformation of T alpha is modulated by the occupancy of the guanine nucleotide binding site. In contrast to TF15, TF16 recognizes only the native form of T alpha. Its epitope resides within the central portion of the T alpha molecule. While T alpha-bound TF16 does not inhibit either pertussis toxin-catalyzed ADP-ribosylation, rhodopsin binding, or transducin subunit interaction, it blocks both the light-activated uptake of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the GTP-dependent elution of transducin from photolyzed rhodopsin. These effects are unlikely to be caused by the occupation of the guanine nucleotide binding site by TF16 because this antibody quantitatively precipitates T alpha-GTP gamma S. We propose that bound TF16 locks T alpha in a conformation that prevents the entrance of guanine nucleotide and favors T beta gamma association. In contrast to TF16, the epitope of 4A was mapped to the amino-terminal region of T alpha. This monoclonal antibody blocks pertussis toxin-catalyzed ADP-ribosylation, GTP gamma S uptake, and T alpha-T beta gamma association. Moreover, the binding site for 4A becomes inaccessible when transducin binds to photolyzed rhodopsin. These results suggest that the inhibitory effects of 4A are due to a simultaneous steric blockage of both the interaction of T alpha with T beta gamma and their binding to photolyzed rhodopsin. The results obtained from these studies are correlated with the structure and function of T alpha.