The amphibian photoreceptor rod outer segment contains a guanine nucleotide-binding complex which consists of a 39,000-dalton polypeptide that binds guanine nucleotides (G protein), a 36,000-dalton polypeptide (H protein), and an approximately 6,500-dalton polypeptide. Sensitivity to trypsin proteolysis was utilized as a probe of structure-function relationships for these polypeptides. Digestion of the H protein generated fragments of 26,000 and 15,000 daltons whose proteolytic susceptibility was not altered by guanosine triphosphates, light, or membranes. The approximately 6,500-dalton polypeptide was not trypsin sensitive. When the G protein was eluted from illuminated membranes by GTP, trypsin proteolysis cleaved a terminal 1,000-dalton fragment (G1) to yield a 38,000-dalton fragment (G38). With increased digestion time, a 6,000-dalton fragment (G6) was removed from G38 to yield a 32,000-dalton fragment (G32). G32 was subsequently digested to fragments of 23,000 and 12,000 daltons. However, when the G protein was eluted from illuminated membranes by hydrolysis-resistant analogues of GTP, G32 was protected from further digestion. This is consistent with a GTP-induced conformational change in the G protein which is altered by GTP hydrolysis. Proteolysis of the G protein after covalent labeling with a photoaffinity analogue of GTP demonstrated that the analogue is bound to first G38 and then G32, indicating the GTP-binding site is associated with G32. Fragment G6 was cleaved when the G protein was soluble or bound to unilluminated membranes. However, when bound to illuminated membranes, fragments were generated reflecting the loss of 7,500, 9,000, or 11,000 daltons from the G protein. This light-induced alteration in proteolytic susceptibility indicates there is a light-induced conformational change in the G protein. Fragment G1 was not removed from the G protein when it was membrane bound, suggesting G1 is involved in binding to a membrane structure. These data suggest that the light-induced binding of the G protein to illuminated membranes and the reversal of this binding by GTP are mediated through conformational changes in the G protein and that three conformations exist: 1) a basal, inactive conformation; 2) a primed conformation induced by binding to photolyzed rhodopsin, with a high affinity for GTP; and 3) an active conformation, induced by binding of GTP, which activates the catalytic complex of light-activated phosphodiesterase.

The physiological regulation of light-activated cyclic GMP phosphodiesterase (EC 3.1.4.17) in rod outer segments has been shown to depend upon a heat-stable inhibitor and upon the reversal of its effect by a specific GTP/GTP-binding protein complex (Hurley, J. B. (1980) Biochem. Biophys. Res. Commun. 92, 505-510; Yamazaki, A., Bartucca, F., Ting, A., and Bitensky, M. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3702-3706). Washing of illuminated disc membranes with an isotonic buffer released 86% of the peripheral proteins without any release of inhibitor. Subsequent washing with the same isotonic buffer containing GTP released 80% of the inhibitor. When inhibitor was eluted with guanosine-5'-(beta, gamma-imino)triphosphate, it had an apparent molecular weight of 60,000 on Sephadex G-100. The release of inhibitor by guanosine-5'-(beta, gamma-imino)triphosphate was also demonstrated with sucrose density gradient centrifugation. Inhibitor release from the disc membrane by GTP or its analogue was accompanied by the release of the GTP-binding protein and an increased phosphodiesterase activity in the membrane. However, following GTP hydrolysis, both inhibitor and GTP-binding protein returned to the membrane and phosphodiesterase activity in the membrane decreased proportionally. In contrast, incubation of disc membranes with guanosine-5'-(beta, gamma-imino)-triphosphate produced an increase of inhibitor activity in the supernatant and an increase of phosphodiesterase activity in the pellet which remained constant after the initial increase. These data clearly show that the activation of phosphodiesterase by the GTP/GTP-binding protein complex resulted from the release of inhibitor. Hydrolysis of GTP resulted in the reassociation of inhibitor with and concomitant inhibition of disc membrane phosphodiesterase.