FUNCTIONAL-SIGNIFICANCE OF G-PROTEIN CARBOXYMETHYLATION

Heterotrimeric G proteins are isoprenylated and methylated on their ga mma subunits. Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of control of G protein ac tivity. A method for selectively demethylating isoprenylated and methy lated G proteins is reported here using retinal transducin (T) as a mo del system. It was found that pig liver esterase is capable of complet ely hydrolyzing T-beta gamma, but not T-alpha beta gamma, to its unmet hylated form. This allows for the direct determination of the activiti es of methylated and unmethylated T-beta gamma. The activities of the T(beta gamma)s were determined by measuring their abilities to stimula te GTP-gamma-S exchange in the presence of T-alpha and photoactivated rhodopsin (R). It is reported here that, in detergent, unmethylated T -beta gamma was at least as active as its methylated counterpart. Ther efore, methylation does not affect the intrinsic ability of T-beta gam ma to functionally interact with T-alpha and R. However, in disk memb ranes an approximate 2-fold effect was observed, with the methylated T -beta gamma being more efficient. Therefore, isoprenylated protein met hylation may play a quantitative role in signal transduction, even tho ugh the intrinsic activities of the methylated subunits in detergent m ay be no different from their unmethylated counterparts. Finally, the use of pig liver esterase to demethylate isoprenyIated proteins should allow for a clarification of the physiological role(s) of isoprenylat ed protein carboxymethylation in general.