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 β-Arrestin

  • Mol Pharmacol. 2017 Jun;91(6):554-566. doi: 10.1124/mol.116.106468.
Jiansong Luo  1 ,  John M Busillo  1 ,  Ralf Stumm  1 ,  Jeffrey L Benovic  2
Affiliations
  • 1. 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.).
  • 2. 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.) [email protected].
PMID: 28331048 DOI: 10.1124/mol.116.106468
Abstract

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.