Role of C-terminal domains of the G protein β subunit in the activation of effectors

The prenyl group on the G protein γ subunit is an important determinant of protein–protein interactions between the βγ dimer and its targets, such as α subunits, receptors, and effectors. In an effort to identify domains of the β subunit important for the activation of effectors, we have prepared two types of mutants, one set in the domain suggested to form a hydrophobic prenyl-binding pocket for the γ subunit's prenyl group (prenyl pocket mutants) and the other set in a domain between Gly(306) and Gly(319) in the β propeller, which undergoes a conformational change when the dimer binds to phosducin (conformational change mutants). Recombinant baculoviruses for each set of mutants were prepared, and the nine mutant β subunits were overexpressed with either the γ(2) subunit (modified with geranylgeranyl) or the γ(2-L71S) subunit (γ(2) with altered CAAX sequence and modified with farnesyl). The purified dimers were tested for their ability to couple Gα(i1) to the A1 adenosine receptor and to activate phospholipase C-β or type II adenylyl cyclase. All dimers containing mutant β subunits were indistinguishable from wild-type β(1)γ(2) or β(1)γ(2-L71S) in coupling the receptor to Gα(i1). The prenyl pocket mutants expressed with γ(2) were 10-fold less potent in activating phospholipase C-β and adenylyl cyclase than β(1)γ(2) and had similar activities to β(1)γ(2-L71S). The conformational change mutants caused a 15- to 23-fold decrease in EC(50) values for activation of these two effectors. Overall, the results suggest that the sites in Gβ identified by these mutants are very important in the activation of effectors. Furthermore, the nature of the prenyl group on Gγ may play an important role in the conformational change leading to the activity of Gβγ on effectors.