TI - Site -specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling . AB - 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 .