Phosphorylation of the D 1 Dopamine Receptor by G Protein‐Coupled Receptor Kinases: phosphorylation site identification and linkage to functional effects

Abstract

Dopamine (DA) receptors (DARs) are G protein-coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward-related behaviors, as well as cardiovascular and renal physiology. Multiple diseases are linked to dysregulated dopaminergic functioning including Parkinson's disease, schizophrenia, substance use disorder, and hypertension. DARs 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 (D1R and D5R) increase cAMP, while the D2-like DARs (D2R, D3R, D4R) decrease cAMP. All DARs also recruit β-arrestin which activates separate signaling cascades and also initiates receptor desensitization and internalization. Generally, agonist activation of GPCRs, including DARs, rapidly leads to receptor desensitization and a return to basal levels of signaling. This occurs even in the continued presence of agonist, ensuring homeostasis. This desensitization process is intimately linked with receptor phosphorylation. The D1R is highly phosphorylated, with 32 intracellular serine and threonine residues, and is known to be phosphorylated by several kinases including protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs). Previous studies by our lab indicate that the D1R is phosphorylated on its third intracellular loop (ICL3) and C-terminus in a hierarchical fashion, in that phosphorylation must first occur on the C-terminus before the ICL3 can be phosphorylated. Using systematic mutational analyses, we previously identified the PKC-mediated D1R phosphorylation sites. We have now extended these studies to completely identify the DA-induced, GRK-mediated phosphorylation sites on the D1R. We found that GRK-mediated phosphorylation involves several serine and threonine residues on the C-terminus and ICL3. Mutation of these residues to alanine or valine, respectively, abolishes DA-induced D1R phosphorylation and severely impairs β-arrestin recruitment, but causes little effect on G protein-mediated signaling. Our results indicate that a large fraction of DA-induced D1R phosphorylation occurs on residues T360 and S362 in the proximal C-terminus, and that these residues are also responsible for the majority of DA-induced β-arrestin recruitment to the D1R. Using HEK cells that have had their endogenous GRKs knocked out via CRISPR, we found that DA-induced β-arrestin recruitment to the D1R is severely impaired. However, β-arrestin recruitment can be restored by expressing exogenous GRKs in these cells, further suggesting that DA-induced β-arrestin recruitment to the D1R is highly dependent on GRK phosphorylation. As GRK distribution varies by tissue and brain region, it is intriguing to postulate that D1R phosphorylation by different GRKs can add layers of regulatory fine-tuning through differential effects on D1R signaling or trafficking outcomes.