Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

Abstract

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.

GRK2 Protein-mediated Transphosphorylation Contributes to Loss of Function of μ-Opioid Receptors Induced by Neuropeptide FF (NPFF2) Receptors

“…All GPCRs possess several phosphorylation sites (serine, threonine, and tyrosine) in their intracellular domain (51)(52)(53). Differential phosphorylation, tissue-, cell-, or agonist-dependent, has recently been postulated to create a "phosphorylation bar code" that offers a way to regulate signaling and trafficking of GPCRs (52).…”

Reelin and CXCL12 regulate distinct migratory behaviors during the development of the dopaminergic system

“…S7A-C and Table S1). To confirm that the antagonist abolishes CXCR4 signaling, we assessed the phosphorylation of CXCR4, which serves an indicator for CXCR4 activation (Busillo et al, 2010). We could show that phosphorylated CXCR4 is present in untreated, but not in antagonist-treated slice cultures, demonstrating that the antagonist inhibits CXCL12/CXCR4 signaling in our experimental setup (supplementary material Fig.…”

“…Primary antibodies used for immunostaining were: rabbit or mouse anti-TH (1:500, AB152 or MAB318, Millipore), rat anti-GFP (1:1500, 04404-26, Nacalai Tesque), rabbit anti-LMX1A (1:3000, AB10533, Millipore), rabbit anti-NURR1 (1:200, Sc-990, Santa Cruz Biotechnology), mouse anti-POU4F1 (1:50, Sc-8429, Santa Cruz Biotechnology), rabbit anti-calbindin (1:2500, CB38, Swant), rabbit anti-GIRK2 (1:500, APC-006, Alomone Labs), biotinylated rabbit anti-CXCR4 (1:500, AB097, BD Biosciences), anti-phosphorylated CXCR4 [1:250, kindly provided by Dr Benovic (Busillo et al, 2010)]. Secondary antibodies were: donkey anti-rabbit IgG-Alexa 488 (1:500; Life Technologies), donkey anti-mouse or -rabbit Cy3 (1:200, Jackson ImmunoResearch), donkey anti-rat IgG-DyLight 488 or 647 (1:100, Jackson ImmunoResearch).…”

Rapid Uptake and Degradation of CXCL12 Depend on CXCR7 Carboxyl-terminal Serine/Threonine Residues

“…CXCR4 represents a G protein-coupled chemoattractant receptor showing rapid phosphorylation at the C-terminal domain and ␤-arrestin-dependent endocytosis in response to CXCL12 stimulation (19,20). Sustained stimulation causes CXCR4 degradation (21), a mechanism expected to desensitize the CXCL12/CXCR4 pathway once migratory cells reach CXCL12-rich environments.…”