title: Influence of gαq on the dynamics of m3-acetylcholine receptor-g-protein-coupled receptor kinase 2 interaction.
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 Gαq 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 Gαq on the interaction of GRK2 with activated Gq-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 Gq-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 Gαq [GRK2(D110A)] and Gβγ [GRK2(R587Q)] were used to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction, and that not only Gβγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq 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 Gαq influences signaling and desensitization.
authors.affiliation: Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany (V.W., C.K., M.B.); and Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (J.B.).
authors.forename: Valerie
authors.initials: V
authors.lastname: Wolters
authors.affiliation: Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany (V.W., C.K., M.B.); and Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (J.B.).
authors.forename: Cornelius
authors.initials: C
authors.lastname: Krasel
authors.affiliation: Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany (V.W., C.K., M.B.); and Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (J.B.).
authors.forename: Jörg
authors.initials: J
authors.lastname: Brockmann
authors.affiliation: Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University Marburg, Marburg, Germany (V.W., C.K., M.B.); and Department of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany (J.B.) moritz.buenemann@staff.uni-marburg.de.
authors.forename: Moritz
authors.initials: M
authors.lastname: Bünemann
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D019393:Arrestin / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D019393:Arrestin; D043587:Receptor, Muscarinic M3; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000109:Acetylcholine