Role of C-terminal domains of the G protein beta subunit in the activationof effecters

The prenyl group on the G protein gamma subunit is an important determinant of protein-protein interactions between the beta gamma dimer and its targe ts, such as alpha subunits, receptors, and effectors. In an effort to ident ify domains of the beta subunit important for the activation of effecters, we have prepared two types of mutants, one set in the domain suggested to f orm a hydrophobic prenyl-binding pocket for the gamma subunit's prenyl grou p (prenyl pocket mutants) and the other set in a domain between Gly(306) an d Gly(319) in the beta propeller, which undergoes a conformational change w hen the dimer binds to phosducin (conformational change mutants). Recombina nt baculoviruses for each set of mutants were prepared, and the nine mutant beta subunits were overexpressed with either the gamma(2) subunit (modifie d with geranylgeranyl) or the gamma(2-L71S) subunit (gamma(2) with altered CAAX sequence and modified with farnesyl), The purified dimers were tested for their ability to couple G alpha(i1) to the A1 adenosine receptor and to activate phospholipase C-beta or type II adenylyl cyclase, All dimers cont aining mutant beta subunits were indistinguishable from wild-type beta(1)ga mma(2) or beta(1)gamma(2-L71S) in coupling the receptor to G alpha(i1). The prenyl pocket mutants expressed with gamma(2) were 10-fold less potent in activating phospholipase C-beta and adenylyl cyclase than beta(1)gamma(2) a nd had similar activities to beta(1)gamma(2-L71S). The conformational chang e mutants caused a 15- to 23-fold decrease in EC50 values for activation of these two effecters. Overall, the results suggest that the sites in G beta identified by these mutants are very important in the activation of effect ors. Furthermore, the nature of the prenyl group on G gamma may play an imp ortant role in the conformational change leading to the activity of G beta gamma on effecters.