G Protein-coupled Receptor Regulation Of Grk Function

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G(( subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current studies have investigated the role of the interaction of GRK2 with the activated (-opioid receptor (DOR) and G(( subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 translocation to the plasma membrane, GRK2 binding to DOR, and DOR phosphorylation in cells expressing the wild type DOR (WT), but not the mutant DOR lacking the carboxyl terminus which contains all three GRK2 phosphorylation sites. DORs with the GRK2-phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3, but not E355Q/D364N. Moreover, overexpression of G(( subunits promoted, whereas overexpression of transducin ??or the carboxyl terminal of GRK2 blocked GRK2 binding to DOR. Further study demonstrated that agonist stimulation induced formation of a complex containing DOR, GRK2, and G(( subunits in the cell and the agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.