Abstract

G-protein–coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitization of G-protein–coupled receptors. Based on its ability to bind G-protein βγ subunits as well as activated Gα_q subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gβγ together with negatively charged membrane phospholipids. In the current study, we address the role of Gα_q on the interaction of GRK2 with activated G_q-protein–coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)–based assays to study the interaction of GRK2 with the M₃-acetylcholine (M₃-ACh) receptor as well as G_q-protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M₃-ACh receptor stimulation with 10 µM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Gα_q [GRK2(D110A)] and Gβγ [GRK2(R587Q)] were used to determine the specific role of G_q-protein–binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gα_q enhances the extent and stability of the GRK2–M₃-ACh receptor interaction, and that not only Gβγ but also Gα_q can target GRK2 to the membrane. This reveals an important role of Gα_q in efficient recruitment of GRK2 to M₃-ACh receptors. Furthermore, interactions between Gα_q and GRK2 were associated with a prolongation of the interaction between GRK2 and the M₃-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gα_q influences signaling and desensitization.