G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment ofβ-Arrestin.
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 Gαiβγheterotrimer-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 ofβ-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζeffectively 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.
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.L., J.M.B., J.L.B.); and Institute of Pharmacology and Toxicology, University Hospital, Friedrich-Schiller University, Jena, Germany (R.S.).
Jiansong
J
Luo
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.L., J.M.B., J.L.B.); and Institute of Pharmacology and Toxicology, University Hospital, Friedrich-Schiller University, Jena, Germany (R.S.).
John M
JM
Busillo
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.L., J.M.B., J.L.B.); and Institute of Pharmacology and Toxicology, University Hospital, Friedrich-Schiller University, Jena, Germany (R.S.).
Ralf
R
Stumm
Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania (J.L., J.M.B., J.L.B.); and Institute of Pharmacology and Toxicology, University Hospital, Friedrich-Schiller University, Jena, Germany (R.S.) jeffrey.benovic@jefferson.edu.
Jeffrey L
JL
Benovic
D054770:G-Protein-Coupled Receptor Kinase 3 / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019718:Receptors, CXCR4 / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D000071557:beta-Arrestins / Q000378:metabolism*
C527276:CXCR4 protein, human; D007527:Isoenzymes; D047428:Protein Kinase Inhibitors; D019718:Receptors, CXCR4; D000071557:beta-Arrestins; D012694:Serine; D011493:Protein Kinase C; D054770:G-Protein-Coupled Receptor Kinase 3; C517848:GRK3 protein, human