EFFECTS OF CARBOXYL-TERMINAL TRUNCATION ON THE STABILITY AND G-PROTEIN COUPLING ACTIVITY OF BOVINE RHODOPSIN

A number of studies have suggested that G protein-coupled receptors po ssess domains within the carboxyl terminus that are important for the catalytic activation of G proteins. To define these regions, truncatio n mutants were generated in the cDNA of bovine rhodopsin, the receptor responsible for visual signal transduction in the retinal rod cell. T he mutants were expressed in HEK-293 cells and analyzed for their abil ity to bind the chromophore, 11-cis-retinal, and for activating G(t) t he G protein of the rod cell regulated by rhodopsin. Removal of 38 car boxyl-terminal amino acids resulted in the production of a mutant (K31 1stop) that does not bind 11-cis-retinal, has an abnormal pattern of g lycosylation, and does not catalyze light-dependent binding of GTP gam ma S to G(t), suggesting that it is unable to fold properly during bio genesis. However, a truncation mutant with only five additional amino acids (C316stop) coupled normally to G(t), using membranes from transf ected cells, despite the fact that it lacked the ''fourth cytoplasmic loop'' formed by palmitoylation of cysteines-322 and -323. When C316st op is extracted from the membrane with detergent, only a fraction is a ble to bind 11-cis-retinal, but the fraction that binds retinal activa tes G(t) normally. In contrast, detergent-solubilized wild-type rhodop sin and K325stop (a truncation mutant with the longest carboxyl termin us) both bind retinal and activate G(t) normally. These data suggest t hat the proximal region of the carboxyl terminus is critical for the p roper folding and stability of the rhodopsin molecule and that amino a cids Cys316 to Ala348 are not necessary for the activation of G(t).