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.

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.

• Authors: Jiansong Luo, John M Busillo, Ralf Stumm, Jeffrey L Benovic
• Journal: Molecular pharmacology (Mol Pharmacol) Vol. 91 Issue 6 Pg. 554-566 (06 2017) ISSN: 1521-0111 [Electronic] United States
• PMID: 28331048 (Publication Type: Journal Article)
• Copyright: Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Chemical References

• CXCR4 protein, human
• Isoenzymes
• Protein Kinase Inhibitors
• Receptors, CXCR4
• beta-Arrestins
• Serine
• Protein Kinase C
• G-Protein-Coupled Receptor Kinase 3
• GRK3 protein, human

Topics

• G-Protein-Coupled Receptor Kinase 3 (metabolism)
• HEK293 Cells
• Humans
• Isoenzymes (antagonists & inhibitors, metabolism)
• Phosphorylation (drug effects, physiology)
• Protein Kinase C (antagonists & inhibitors, metabolism)
• Protein Kinase Inhibitors (pharmacology)
• Receptors, CXCR4 (metabolism)
• Serine (metabolism)
• beta-Arrestins (metabolism)