Premium
G protein‐coupled receptor kinase 2 can enhance β‐arrestin recruitment to the D 2 dopamine receptor in the absence of receptor phosphorylation
Author(s) -
Sanchez Soto Marta,
Dasaro Christopher,
Schardien Kayla,
Inbody Laura,
Free R.,
Drube Julia,
Haider Raphael,
Hoffmann Carsten,
Sibley David
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02799
Subject(s) - arrestin , dopamine receptor d2 , g protein coupled receptor kinase , g protein coupled receptor , 5 ht5a receptor , phosphorylation , enzyme linked receptor , receptor , microbiology and biotechnology , agonist , biology , beta adrenergic receptor kinase , chemistry , signal transduction , biochemistry
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‐F189 5.38 A mutant is capable of activating G proteins, but is unable to recruit β‐arrestin, i.e., D2R‐F189 5.38 A is a G protein‐biased receptor. To investigate the mechanisms underlying the biased signaling of D2R‐F189 5.38 A, its interactions with GRK2 were explored using a variety of bioluminescence resonance energy transfer (BRET) assays. Importantly, we found that D2R‐F189 5.38 A does not recruit GRK2 upon agonist stimulation. This finding suggests that the absence of D2R‐F189 5.38 A ‐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.