G protein‐coupled receptor kinase 2 can enhance β‐arrestin recruitment to the D 2 dopamine receptor in the absence of receptor phosphorylation

Abstract

The D2 dopamine (DA) receptor (D2R) signals through two main pathways: activation of G proteins and recruitment of β-arrestin. Upon agonist binding to the receptor, an active signaling state is formed that leads to receptor phosphorylation by one or more members of the G protein-coupled receptor kinase (GRK) family. GRK-mediated receptor phosphorylation enhances β-arrestin interactions with many GPCRs, but this mechanism remains unclear for the D2R. We recently identified a single amino acid (F189 (5.38 using Ballesteros-Weinstein numbering)) within the ligand binding site of the D2R that acts as a micro-switch for regulating D2R interactions with β-arrestin (Sanchez-Soto et al., Sci. Signal. 13(617): eaaw5885, 2020). When F189 is mutated to alanine, the D2R-F1895.38A mutant is capable of activating G proteins, but is unable to recruit β-arrestin, i.e., D2R-F1895.38A is a G protein-biased receptor. To investigate the mechanisms underlying the biased signaling of D2R-F1895.38A, its interactions with GRK2 were explored using a variety of bioluminescence resonance energy transfer (BRET) assays. Importantly, we found that D2R-F1895.38A does not recruit GRK2 upon agonist stimulation. This finding suggests that the absence of D2R-F1895.38A -GRK2 interactions may underlie the receptor's inability to recruit β-arrestin. To test this hypothesis, we examined DA-stimulated β-arrestin and GRK2 recruitment using a phosphorylation-defective D2R mutant (Namkung et. al., JBC 284:34103, 2009), the GRK2/3 inhibitor Compound 101, and cells in which expression of one or more GRK subtypes were eliminated by CRISPR-Cas9 technology. Strikingly, the phosphorylation-defective D2R mutant was not impaired in its ability to recruit either GRK2 or β-arrestin. In addition, treatment with Compound 101 resulted in a decrease, but not elimination of DA-stimulated β-arrestin recruitment to the WT D2R, suggesting that, while GRK2 kinase activity is required for enhancement of β-arrestin recruitment, phosphorylation of the D2R is not involved. We further examined the role of GRK2 by using a GRK2 KO cell line and found that, while β-arrestin recruitment to the D2R and D2R internalization were diminished, they were not eliminated. In addition, cells completely lacking all GRK subtypes (Total GRK KO) showed an even lower β-arrestin recruitment and D2R internalization suggesting that multiple GRK subtypes might facilitate D2R-β-arrestin interactions. Finally, overexpression of GRKs2, 3, 4, 5, or 6 in the Total GRK KO cells increased β-arrestin recruitment to the D2R. Taken together, these data suggest that while GRK2, and potentially other GRK subtypes, play a facilitatory role in regulating β-arrestin recruitment to the D2R, and thus can potentially mediate receptor signaling bias, receptor phosphorylation itself does not appear to be involved.