title: Isoprenylation/methylation and transducin function.
abstract: Freshly prepared proteolyzed (deprenylated) T beta gamma and material isolated from retina are inert with respect to activating T alpha in the presence of R* in detergent and in disk membranes. In addition, proteolyzed T beta gamma is also incapable of supporting the pertussis toxin-catalyzed ADP ribosylation of T alpha-GDP. These experiments show that isoprenylation/methylation is essential for the fruitful interactions between T alpha and T beta gamma at the membrane. When tested for its ability to support GTP-for-GDP exchange catalyzed by R*, demethylated T beta gamma proved to be approximately 50% as active as methylated T beta gamma in photoreceptor disk membranes (Fig. 3) and in reconstituted liposomes containing rhodopsin. In detergent, no difference was observed between methylated and demethylated T beta gamma, suggesting no role at all for the methyl group in functional interactions between T alpha, T beta gamma, and R*. The twofold activity difference observed in membranes can be accounted for by the twofold lessened affinity of the demethylated T beta gamma, compared with its methylated counterpart, for membranes in the presence of R* and T alpha. It is interesting to note that a substantially larger difference (> 10-fold) in the relative binding of methylated versus demethylated T beta gamma to membranes is observed in the absence of R* and T alpha. However, R* has a substantial affinity for T alpha beta gamma, and the influence of R* and T alpha greatly reduces any differences resulting from the presence or absence of a methyl group on T beta gamma. The results from studies of demethylated T beta gamma demonstrate that specific lipid-receptor interactions are unlikely to play a critical role in the rhodopsin-transducin system, and further show that the effect of methylation is probably due to the increased hydrophobicity of methylated T beta gamma versus its unmethylated counterpart. These studies are, of course, relevant to heterotrimeric G proteins, and specifically to the interactions of receptor (R*) with T alpha and T beta gamma. If a hydrophobic lipid-lipid mechanism is operative, the state of methylation would be expected to have a more profound effect on the membrane-associative properties of farnesylated proteins, but not on those of geranylgeranylated proteins. The increased hydrophobicity of the C20 geranylgeranyl group relative to the C15 farnesyl group will compensate for the loss of the methyl substituent. The results obtained in the transducin-rhodopsin system can be contrasted with the effect of gamma-subunit methylation on effector enzyme activation. In the case of the geranylgeranylated beta 1 gamma 2, methylation proved to have only a small effect on PIPLC beta activation (Fig. 4B). An approximately 25% diminution in efficacy, but not potency, was observed for the demethylated geranylgeranylated beta 1 gamma 2 versus its methylated counterpart. This again shows that specific lipid-protein interactions are unimportant. The effect of methylation on membrane binding would be expected to be small, given that beta 1 gamma 2 is geranylgeranylated. It is of interest to compare these results with those found with methylated and unmethylated T beta gamma as activators of PIPLC beta. In this instance there was a large effect noted, with methylated T beta gamma being at least 10-fold more potent than its unmethylated counterpart with respect to activating either enzyme (Fig. 4A). This result is readily understandable in light of the role of methylation in selectively enhancing hydrophobicity of farnesylated proteins as opposed to geranyl-geranylated proteins. Similar results were obtained for the activation of PI3K, further strengthening the conclusion that it is lipid-lipid interactions that direct beta gamma subunit membrane association. (ABSTRACT TRUNCATED)
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Parish
authors.forename: R R
authors.initials: RR
authors.lastname: Rando
mesh_terms: D000818:Animals; D002417:Cattle; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D008745:Methylation; D017368:Protein Prenylation; D012160:Retina / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D015293:Transducin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin