TY - JOUR T1 - G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of <em>β</em>-Arrestin JF - Molecular Pharmacology JO - Mol Pharmacol SP - 554 LP - 566 DO - 10.1124/mol.116.106468 VL - 91 IS - 6 AU - Jiansong Luo AU - John M. Busillo AU - Ralf Stumm AU - Jeffrey L. Benovic Y1 - 2017/06/01 UR - http://molpharm.aspetjournals.org/content/91/6/554.abstract N2 - Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines (Ser) 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a Gαiβγ heterotrimer–biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared with CXCL12. Knockdown of G protein-coupled receptor kinase (GRK) 2, GRK3, or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional protein kinase C (PKC) isoforms, particularly PKCα, reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of β-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζ effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a nonspecific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome. ER -