G Protein-Coupled Receptor Kinase 2 Selectively Enhances -Arrestin Recruitment to the D2 Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation
BIOMOLECULES(2023)
Abstract
The D2 dopamine receptor (D2R) signals through both G proteins and beta-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. beta-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 beta-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for beta-arrestin recruitment to the D2R, and the role of GRKs in D2R-beta-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which beta-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated beta-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 beta-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-beta-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance beta-arrestin recruitment and activation independently of receptor phosphorylation.
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Key words
GRK, D-2 receptor, phosphorylation, beta-arrestin
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