G Protein-Coupled Receptor Kinase 2 Selectively Enhances ß-Arrestin Recruitment to the D2 Dopamine Receptor through Mechanisms That Are Independent of Receptor Phosphorylation.

The D2 dopamine receptor (D2R) signals through both G proteins and ß-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. ß-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 ß-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for ß-arrestin recruitment to the D2R, and the role of GRKs in D2R-ß-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which ß-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated ß-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 ß-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R-ß-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance ß-arrestin recruitment and activation independently of receptor phosphorylation.

Delineation of G Protein-Coupled Receptor Kinase Phosphorylation Sites within the D1 Dopamine Receptor and Their Roles in Modulating ß-Arrestin Binding and Activation.

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein ß-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting ß-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third intracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion, where phosphorylation of the C-terminus precedes that of the ICL3. Using ß-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive ß-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for ß-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in ß-arrestin binding and activation.

Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1.

GPR37 and GPR37L1 are glia-enriched G protein-coupled receptors that have been implicated in several neurological and neurodegenerative diseases. To gain insight into the potential molecular mechanisms by which GPR37 and GPR37L1 regulate cellular physiology, proteomic analyses of whole mouse brain tissue from wild-type (WT) versus GPR37/GPR37L1 double knockout (DKO) mice were performed in order to identify proteins regulated by the absence versus presence of these receptors (data are available via ProteomeXchange with identifier PXD015202). These analyses revealed a number of proteins that were significantly increased or decreased by the absence of GPR37 and GPR37L1. One of the most decreased proteins in the DKO versus WT brain tissue was S100A5, a calcium-binding protein, and the reduction of S100A5 expression in KO brain tissue was validated via Western blot. Coexpression of S100A5 with either GPR37 or GPR37L1 in HEK293T cells did not result in any change in S100A5 expression but did robustly increase secretion of S100A5. To dissect the mechanism by which S100A5 secretion was enhanced, cells coexpressing S100A5 with the receptors were treated with different pharmacological reagents. These studies revealed that calcium is essential for the secretion of S100A5 downstream of GPR37 and GPR37L1 signaling, as treatment with BAPTA-AM, an intracellular Ca2+ chelator, reduced S100A5 secretion from transfected HEK293T cells. Collectively, these findings provide a panoramic view of proteomic changes resulting from loss of GPR37 and GPR37L1 and also impart mechanistic insight into the regulation of S100A5 by these receptors, thereby shedding light on the functions of GPR37 and GPR37L1 in brain tissue.