Functional significance of G protein carboxymethylation.
Heterotrimeric G proteins are isoprenylated and methylated on their gamma subunits. Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of control of G protein activity. A method for selectively demethylating isoprenylated and methylated G proteins is reported here using retinal transducin (T) as a model system. It was found that pig liver esterase is capable of completely hydrolyzing T beta gamma, but not T alpha beta gamma, to its unmethylated form. This allows for the direct determination of the activities of methylated and unmethylated T beta gamma. The activities of the T beta gamma s were determined by measuring their abilities to stimulate 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. Therefore, methylation does not affect the intrinsic ability of T beta gamma to functionally interact with T alpha and R*. However, in disk membranes an approximate 2-fold effect was observed, with the methylated T beta gamma being more efficient. Therefore, isoprenylated protein methylation may play a quantitative role in signal transduction, even though the intrinsic activities of the methylated subunits in detergent may be no different from their unmethylated counterparts. Finally, the use of pig liver esterase to demethylate isoprenylated proteins should allow for a clarification of the physiological role(s) of isoprenylated protein carboxymethylation in general.
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
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D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004950:Esterases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008745:Methylation; D010777:Photochemistry; D012160:Retina / Q000737:chemistry; D012243:Rhodopsin / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine; D015293:Transducin / Q000378:metabolism
D046911:Macromolecular Substances; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D004950:Esterases; D019204:GTP-Binding Proteins; D015293:Transducin