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.

Design, synthesis, and biological evaluation of lipophilically modified bisphenol Z derivatives.

In the present study, a small library of bisphenol Z (BPZ) derivatives was synthesized and investigated for anti-proliferative effects in cultured breast and glioblastoma cell lines. Synthesized BPZ derivatives varied in molecular size, polarity, and lipophilicity. Of the 8 derivatives tested, compounds 4 and 6, both of which displayed the highest degree of lipophilicity, were most active at inducing cell death as determined by the XTT assay. Cell membranes were interrogated using trypan blue staining and were shown to remain intact during treatments with 4 and 6. Activation of caspase enzymes (3 and/or 7) was noted to occur following treatment with compound 4. Polar BPZ derivatives, those with a substituted amine or alcohol, were devoid of any inhibitory or proliferative effects. The remaining derivatives seem to lack sufficient lipophilicity to execute an overt toxic effect. Our results suggest that increasing the lipophilic character of BPZ enhances the cytotoxic effects.