Immunological characterization of guanine nucleotide-binding proteins: effects of a monoclonal antibody against the gamma subunit of transducin on guanine nucleotide-binding protein-receptor interactions.
Guanine nucleotide-binding proteins (G proteins) transduce signals from agonist- and light-sensitive receptors. In the visual excitation system, the photon receptor rhodopsin is coupled to the G protein Gt (transducin). Gt is composed of alpha, beta, and gamma subunits; the alpha subunit binds guanine nucleotide, whereas the beta and gamma subunits, which are tightly associated, appear to facilitate interaction of alpha with receptor and pertussis toxin-catalyzed ADP-ribosylation of alpha. To study the function of transducin, monoclonal antibodies were developed against the purified protein. Monoclonal antibody 2H3 reacted with Gt gamma but not G gamma from bovine brain or rabbit liver. In the absence of photolyzed rhodopsin, both intact 2H3 and Fab fragments of 2H3 were able to inhibit completely, in a concentration-dependent manner, ADP-ribosylation of transducin by pertussis toxin 2H3 had no effect on ADP-ribosylation in the presence of photolyzed rhodopsin. The GTPase activity of transducin, which is dependent on rhodopsin, was inhibited only 50% by 2H3. These data are consistent with the hypotheses that an epitope recognized by 2H3 may be important in the formation of the alpha beta gamma complex or that interaction of 2H3 with gamma may alter conformation of the latter and, thereby, inhibit complex formation. Further, reactions of gamma with 2H3 appear to be prevented by interaction with rhodopsin, suggesting that its interaction either shields or alters the epitope recognized by 2H3.
Laboratory of Cellular Metabolism National Institutes of Health, Bethesda, Maryland 20892.
J L
JL
Halpern
J
J
Moss
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D002417:Cattle; D000939:Epitopes / Q000276:immunology; D019204:GTP-Binding Proteins / Q000276:immunology*; D006868:Hydrolysis; D051379:Mice; D011487:Protein Conformation; D011817:Rabbits; D012243:Rhodopsin / Q000494:pharmacology; D015293:Transducin / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D015293:Transducin