Role of isoprenoid lipids on the heterotrimeric G protein gamma subunit indetermining effector activation

Post-translational prenylation of heterotrimeric G protein gamma subunits i s essential for high affinity alpha-beta gamma and alpha-beta gamma-recepto r interactions, suggesting that the prenyl group is an important domain in the beta gamma dimer. To determine the role of the prenyl modification in t he interaction of beta gamma dimers with effecters, the CAAX (where A indic ates alipathic amino acid) motifs in the gamma(1), gamma(2), and y(11) subu nits were altered to direct modification with different prenyl groups. Six recombinant beta gamma dimers were overexpressed in baculovirus-infected Sf 9 insect cells, purified, and examined for their ability to stimulate three phospholipase C-beta isozymes and type II adenylyl cyclase, The native bet a(1)gamma(2) dimer (gamma subunit modified with geranylgeranyl) is more pot ent and effective in activating phospholipase C-beta than either the beta(1 )gamma(1) (farnesyl) or the beta(1)gamma(11) (farnesyl) dimers. However, fa rnesyl modification of the gamma subunit in the beta(1)gamma(2) dimer (beta (1)gamma(2-L71S)) caused a decrement in its ability to activate phospholipa se C-beta. In contrast, both the beta 1 gamma(1-S74L) (geranylgeranyl) and the beta(1)gamma(11-S73L) (geranylgeranyl) dimers were more active than the native forms. The beta(1)gamma(2) dimer activates type II adenylyl cyclase about 12-fold; however, neither the beta(1)gamma(1) nor the beta(1)gamma(1 1) dimers activate the enzyme. As was the case with phospholipase C-P, the beta(1)gamma(2-L71S) dimer was less able to activate adenylyl cyclase than the native beta(1)gamma(2) dimer. Interestingly, neither the beta(1)gamma(1 -S74L) nor the beta(1)gamma(11-S73L) dimers stimulated adenylyl cyclase. Th e results suggest that both the amino acid sequence of the gamma subunit an d its prenyl group play a role in determining the activity of the beta gamm a-effector complex.