Receptor determinants for ß-arrestin functional specificity at C-X-C chemokine receptor 5 (CXCR5).

Abstract

ß-arrestins are multi-faceted adaptor proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and signaling. It is emerging that receptor-specific determinants specify these divergent functions at GPCRs, yet this remains poorly understood. Here, we set out to identify the receptor determinants responsible for ß-arrestin-mediated regulation of the chemokine receptor CXCR5. Using bioluminescence resonance energy transfer (BRET) we show that ß-arrestin1 and ß-arrestin2 are dose-dependently recruited to CXCR5 by its cognate ligand CXCL13. The carboxy-terminal tail of CXCR5 contains several Ser/Thr residues that can be divided into 3 discrete phospho-site clusters based on their position relative to transmembrane domain 7. Mutagenesis experiments revealed that the distal and medial phospho-site clusters, but not the proximal, are required for agonist-stimulated ß-arrestin1 or ß-arrestin2 recruitment to CXCR5. Consistent with this, we provide evidence that the distal and medial, but not proximal, phospho-site clusters are required for receptor desensitization. Surprisingly, the individual phospho-site clusters are not required for agonist-stimulated internalization of CXCR5. Further, we show that CXCL13-stimulated CXCR5 internalization and ERK1/2 phosphorylation, but not desensitization, remain intact in HEK293 cells lacking ß-arrestin1 and ß-arrestin2. Our study provides evidence that b-arrestins are recruited to CXCR5 and are required for desensitization but are dispensable for internalization or signaling, suggesting that discrete receptor determinants specify the divergent functions of ß-arrestins. Significance Statement CXCL13 and CXCR5 are important in the immune system and are linked to diseases, yet regulation of CXCR5 signaling remains poorly understood. We provide evidence that a phospho-site cluster located at the extreme distal carboxyl-terminal tail of the receptor is responsible for ß-arrestin recruitment and receptor desensitization. ß-arrestins are not required for CXCL13-stimulated internalization or signaling, indicating that ß-arrestins perform only one of their functions at CXCR5 and that discrete receptor determinants specify the divergent functions of ß-arrestins.