G protein‐coupled receptor kinase 2 can enhance beta‐arrestin recruitment to the D2 dopamine receptor in the absence of receptor phosphorylation

다국어 초록 (Multilingual Abstract)

Dopamine (DA) receptors (DARs) regulate diverse physiological functions and are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. DARs can signal through two main pathways: activation of G proteins and recruitment of β‐arrestin, a multivalent scaffolding protein that coordinates downstream signaling events. Upon agonist binding to the receptor, an active state is formed that activates G proteins and facilitates receptor phosphorylation by one or more members of the G protein‐coupled receptor kinase (GRK) family. GRK‐mediated receptor phosphorylation is widely believed to enhance GPCR‐β‐arrestin interactions, but the precise 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. When F189 is mutated to alanine, the mutant D2R (D2R‐F1895.38A) is capable of activating G proteins, but is unable to recruit β‐arrestin, i.e., D2R‐F1895.38A is a G protein‐biased mutant. 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. GRK2 has been shown to phosphorylate the D2R‐WT in response to agonist stimulation and its overexpression can enhance the ability of DA to promote β‐arrestin recruitment to the receptor. Importantly, we found that the G protein‐biased D2R‐F1895.38A mutant 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), a catalytically inactive GRK2 mutant (GRK2‐K220R), and compound 101, a GRK2/3 inhibitor. Strikingly, the phosphorylation‐defective D2R mutant was not impaired in its ability to recruit either GRK2 or β‐arrestin. In addition, overexpression of GRK2‐K220R, or treatment with compound 101, resulted in a decrease, but not elimination of DA‐stimulated β‐arrestin recruitment to the 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 using a cell line in which GRK2 was knocked out and found that, while β‐arrestin recruitment to the D2R was diminished, it was not eliminated. Taken together, these data suggest that while GRK2 plays 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.
NINDS Intramural Research Program