Research Article
G protein–coupled receptor kinase phosphorylation of distal C-tail sites specifies βarrestin1-mediated signaling by chemokine receptor CXCR4

https://doi.org/10.1016/j.jbc.2022.102351Get rights and content
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G protein–coupled receptor (GPCR) kinases (GRKs) and arrestins mediate GPCR desensitization, internalization, and signaling. The spatial pattern of GPCR phosphorylation is predicted to trigger these discrete GRK and arrestin-mediated functions. Here, we provide evidence that distal carboxyl-terminal tail (C-tail), but not proximal, phosphorylation of the chemokine receptor CXCR4 specifies βarrestin1 (βarr1)-dependent signaling. We demonstrate by pharmacologic inhibition of GRK2/3-mediated phosphorylation of the chemokine receptor CXCR4 coupled with site-directed mutagenesis and bioluminescence resonance energy transfer approaches that distal, not proximal, C-tail phosphorylation sites are required for recruitment of the adaptor protein STAM1 (signal-transducing adaptor molecule) to βarr1 and focal adhesion kinase phosphorylation but not extracellular signal–regulated kinase 1/2 phosphorylation. In addition, we show that GPCRs that have similarly positioned C-tail phosphoresidues are also able to recruit STAM1 to βarr1. However, although necessary for some GPCRs, we found that distal C-tail sites might not be sufficient to specify recruitment of STAM1 to βarr1 for other GPCRs. In conclusion, this study provides evidence that distal C-tail phosphorylation sites specify GRK–βarrestin-mediated signaling by CXCR4 and other GPCRs.

Keywords

GPCR
phosphorylation
arrestin
GRK
BRET

Abbreviations

95% CI
95% confidence interval
β2AR
β2-adrenergic receptor
βarr1
βarrestin1
AT1a
angiotensin II type 1A receptor
BRET
bioluminescence resonance energy transfer
BSA
bovine serum albumin
C-tail
carboxyl-terminal tail
CXCR
chemokine receptor
DMEM
Dulbecco's modified Eagle's medium
ERK1/2
extracellular signal–regulated kinase 1/2
FAK
focal adhesion kinase
FBS
fetal bovine serum
GPCR
G protein–coupled receptor
GRK
G protein–coupled receptor kinase
HA
hemagglutinin
HEK293
human embryonic kidney 293 cell line
MAP
mitogen-activated protein
NTS1
neurotensin receptor 1
PEI
polyethylenimine
Rluc
Renilla luciferase
Ser
serine
STAM
signal-transducing adaptor molecule
TBS
Tris-buffered saline
TBS-T
Tris-buffered saline with Tween-20
Thr
threonine

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