Regulation of Dopamine Receptor Subtypes by G Protein-Coupled Receptor Kinase Isoforms

Abstract ID 98801 Poster Board 159 Dopamine receptors (DARs) are G-protein coupled receptors (GPCRs) that regulate diverse physiological functions including cognition, mood, movement, and reward-related behaviors. They are classified as either D1-like (D1R and D5R) or D2-like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. The D1-like DARs couple to Gs and Golf to increase cAMP levels, while the D2-like DARs couple to Gi/o to decrease cAMP levels. All DARs also recruit β-arrestin which activates distinct signaling cascades and can also initiate receptor desensitization and internalization. There are seven GRK isoforms (GRKs1-7), with GRK2, GRK3, GRK5, and GRK6 being widely expressed. Using systematic mutational analyses, our lab has previously mapped the GRK-mediated phosphorylation sites on both the D1R and D2R, with phosphorylation occurring exclusively on ICL3 for the D2R, and both ICL3 and the C-terminus for the D1R. GRK-mediated phosphorylation of the D1R was found to be required for subsequent β-arrestin recruitment. In contrast, while GRKs play a role in β-arrestin recruitment to the D2R, GRK-mediated D2R phosphorylation is dispensable for this process. We have now sought to determine which GRK isoform(s) are involved in regulating β-arrestin recruitment to these receptors using cell lines in which the expression of specific GRK isoforms were selectively eliminated via CRISPR, as well as by utilizing isoform-selective GRK inhibitors. β-arrestin recruitment to both the D1R and D2R was severely impaired in cells lacking all GRKs (total KO), whereas selective expression of GRK isoforms 2, 3, 5 and 6 in the total KO cells was able to rescue β-arrestin recruitment. The kinase activity of each GRK was required for this recue for the D1R. Interestingly, the GRK2/3 and GRK5/6 isoforms differed in their requirement of kinase activity for the rescue of β-arrestin recruitment to the D2R. Individual and double GRK KO cells revealed that the GRK5/6 subfamily is more important for β-arrestin recruitment to the D1R, while the GRK2/3 subfamily is more important for the D2R. Treatment with GRK2/3-selective inhibitors recapitulated these findings with the GRK KO cells in that they impaired β-arrestin recruitment to the D2R, but had limited effect on this process for the D1R. Intriguingly, in cells with endogenous levels of GRK expression, β-arrestin recruitment to both the D1R and D2R were unaffected by inhibitors of GRK5/6. However, these inhibitors could block the rescue of β-arrestin recruitment to the D2R observed with GRK5 or GRK6 overexpression but had little effect on the rescue of β-arrestin recruitment to the D1R or the Gs-coupled β2 adrenergic receptor. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that DAR regulation, and GPCR regulation in general, by specific GRK isoforms can add layers of regulatory fine-tuning through differentially directing signaling or trafficking outcomes.