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

Abstract

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 β-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 β-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 β-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive β-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for β-arrestin activation, leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in β-arrestin binding and activation.

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

“…While some DAR subtypes, such as the D1R [14,15], follow the canonical model of GPCR regulation as described above, the D2R appears to be an exception to this rule. Using site-directed mutagenesis and in situ phosphorylation assays, we previously identified all of the GRK-mediated phosphorylation sites on D2R and, using phosphorylation-deficient receptor mutants, demonstrated that phosphorylation does not regulate D2R interactions with β-arrestin [16,17].…”

“…Typically, GRK phosphorylation of GPCRs is thought to mediate β-arrestin recruitment, receptor internalization, and downstream signaling pathways [4][5][6]14,37]. Our lab has previously mapped out all the GRK-mediated phosphorylation sites within the D2R [16,17] as well as those mediated by PKCs [38], which together comprise all the sites of known phosphorylation within the D2R when expressed in HEK293 cells.…”

Arrestin-3-assisted activation of JNK3 mediates dopaminergic behavioral sensitization

“…All five DA receptors are GPCRs and interact with arrestins, although the mode of interaction and its functional consequences are unique for each subtype. 17 , 18 , 19 , 20 , 21 , 22 Long-term use of dopaminergic drugs causes persistent changes in DA-dependent behaviors. In rodents with unilateral ablation of the dopaminergic input to the striatum, stimulation with dopaminergic agonists or the DA precursor L-DOPA causes persistent behavioral and molecular sensitization (reviewed in the study by Bastide et al.…”