G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D<sub>2</sub> Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation
Marta Sánchez-Soto,
Noelia M. Boldizsar,
Kayla A. Schardien,
Nora S. Madaras,
Blair K. A. Willette,
Laura R. Inbody,
Christopher Dasaro,
Amy E. Moritz,
Julia Drube,
Raphael S. Haider,
R. Benjamin Free,
Carsten Hoffman,
David R. Sibley
Affiliations
Marta Sánchez-Soto
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Noelia M. Boldizsar
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Kayla A. Schardien
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Nora S. Madaras
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Blair K. A. Willette
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Laura R. Inbody
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Christopher Dasaro
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Amy E. Moritz
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Julia Drube
Institut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, D-07745 Jena, Germany
Raphael S. Haider
Institut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, D-07745 Jena, Germany
R. Benjamin Free
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
Carsten Hoffman
Institut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität Jena, Hans-Knöll-Straße 2, D-07745 Jena, Germany
David R. Sibley
Molecular Neuropharmacology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Bethesda, MD 20892, USA
The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R–β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R–β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation.
