G Protein-Coupled Receptor Kinase 2 Selectively Enhances β-Arrestin Recruitment to the D 2 Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation

• Published in: Biomolecules (Basel, Switzerland) Vol. 13; no. 10
• Main Authors: Sánchez-Soto, Marta, Boldizsar, Noelia M, Schardien, Kayla A, Madaras, Nora S, Willette, Blair K A, Inbody, Laura R, Dasaro, Christopher, Moritz, Amy E, Drube, Julia, Haider, Raphael S, Free, R Benjamin, Hoffman, Carsten, Sibley, David R
• Format: Journal Article
• Language: English
• Published: Switzerland 20.10.2023
• Subjects: Arrestins - metabolism; beta-Arrestins - metabolism; G-Protein-Coupled Receptor Kinases - metabolism; HEK293 Cells; Humans; Phosphorylation; Protein Isoforms - metabolism; Receptors, Dopamine - metabolism; Receptors, G-Protein-Coupled - metabolism; GRK; β-arrestin; D2 receptor; phosphorylation
• ISSN: 2218-273X

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.