Synergistic activation of a G protein-coupled receptor kinase by G protein beta gamma subunits and mastoparan or related peptides.

Muscarinic acetylcholine receptors (mAChR, m2 subtype) are phosphorylated in an agonist-dependent manner by a G protein-coupled receptor kinase (GR kinase) at sites located in the central part of mAChR. The GR kinase phosphorylated a glutathione S-transferase fusion protein containing the m2 phosphorylation sites (13-GST). Phosphorylation of 13-GST was markedly stimulated by mastoparan: the Km for 13-GST decreased from 7.3 to 0.17 microM in the presence of 100 microM mastroparan and the Vmax increased 23-fold. beta gamma Subunits of G proteins synergistically stimulated the phosphorylation of 13-GST in the presence of 1-10 microM mastoparan by increasing the affinity of mastoparan for the GR kinase. The phosphorylation of mAChR by the GR kinase was also synergistically stimulated by 1-10 microM mastoparan in the presence of beta gamma subunits, but was inhibited by 100 microM mastoparan. Similar stimulatory and inhibitory effects on the phosphorylation of 13-GST and mAChR were observed using synthetic peptides of 15-20 amino acid residues corresponding to sequences of intracellular segments adjacent to the transmembrane segments of mAChR. These results are consistent with and support the idea that the GR kinase is synergistically activated by both G protein beta gamma subunits and agonist-bound receptors.