PHOSPHORYLATION OF THE G-PROTEIN GAMMA(12) SUBUNIT REGULATES EFFECTORSPECIFICITY

Although the G protein beta gamma dimer is an important mediator in ce ll signaling, the mechanisms regulating its activity have not been wid ely investigated. The gamma(12) subunit is a known substrate for prote in kinase C, suggesting phosphorylation as a potential regulatory mech anism. Therefore, recombinant beta(1)gamma(12) dimers were overexpress ed using the baculovirus/Sf9 insect cell system,, purified, and phosph orylated stoichiometrically with protein kinase C alpha. Their ability to support coupling of the G(il) alpha subunit to the A1 adenosine re ceptor and to activate type II adenylyl cyclase or phospholipase C-bet a was examined, Phosphorylation of the beta(1)gamma(12) dimer increase d its potency in the receptor coupling assay from 6.4 to 1 nM, change the K-act for stimulation of type II adenylyl cyclase from 14 to 37 nM , and decreased its maximal efficacy by 50%. In contrast, phosphorylat ion of the dimer had no effect on its ability to activate phospholipas e C-beta. The native beta(1)gamma(10) dimer, which has 4 similar amino acids in the phosphorylation site at the N terminus, was not phosphor ylated by protein kinase C alpha. Creation of a phosphorylation site i n the N terminus of the protein (Gly(4) --> Lys) resulted in a beta(1) gamma(10G4K) dimer which could be phosphorylated. The activities of th is beta gamma dimer were similar to those of the phosphorylated beta(1 )gamma(12) dimer Thus, phosphorylation of the beta(1)gamma(12) dimer o re the gamma subunit with protein kinase C alpha regulates its activit y in an effector-specific fashion. Because the gamma(12) subunit is wi dely expressed, phosphorylation may be an important mechanism for inte gration of the multiple signals generated by receptor activation.