Receptor and G beta gamma isoform-specific interactions with G protein-coupled receptor kinases

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme, Previous in vivo studies with purified proteins have suggested that this translocation mag be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free beta gamma subunits of heterotrimeric G proteins (G beta gamma). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of G beta gamma with receptors and GRKs, Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2, This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G beta gamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta(1) gamma(2), activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G beta gamma complex. That G beta gamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G beta gamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergie but not thrombin receptors, In contrast to GRK2, GRK3 Terms a G beta gamma complex after stimulation of all three GPCRs, This G beta gamma binding specificity of the GRKs is also reflected at the level of the purified proteins, Thus the GRK2 carboxyl terminus binds G beta(1) and G beta(2) but not G beta(3) while the GRK3 fusion protein binds all three G beta isoforms, This study provides a direct demonstration of a role for G beta gamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.