Receptor Determinants for β-Arrestin Functional Specificity at C-X-C Chemokine Receptor 5.

β-arrestins are multifaceted 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 β-arrestin-mediated regulation of the chemokine receptor C-X-C motif chemokine receptor 5 (CXCR5). Using bioluminescence resonance energy transfer, we show that β-arrestin1 and β-arrestin2 are dose-dependently recruited to CXCR5 by its cognate ligand C-X-C motif chemokine ligand 13 (CXCL13). The carboxy-terminal tail of CXCR5 contains several serine/threonine residues that can be divided into three 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 β-arrestin1 or β-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 human embryonic kidney 293 cells lacking β-arrestin1 and β-arrestin2. Our study provides evidence that β-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 β-arrestins. SIGNIFICANCE STATEMENT: C-X-C motif ligand 13 (CXCL13) and C-X-C motif chemokine receptor 5 (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 β-arrestin recruitment and receptor desensitization. β-arrestins are not required for CXCL13-stimulated internalization or signaling, indicating that β-arrestins perform only one of their functions at CXCR5 and that discrete receptor determinants specify the divergent functions of β-arrestins.