GRK phosphorylation drives β-arrestin-independent internalization of chemokine receptor CXCR5

G protein-coupled receptor kinases (GRKs) and β-arrestins act in concert to regulate G protein-coupled receptor signaling and trafficking. Previously, we showed that β-arrestins are essential for desensitization, but not internalization, of the chemokine receptor CXCR5. Here, we investigated the role of GRKs on β-arrestin recruitment, phosphorylation, and internalization of CXCR5 using gene-edited HEK293 cells in which the ubiquitously expressed GRKs have been deleted (GRK2/3/5/6; ΔQ-GRK). Using novel phospho-site-specific antibodies, we demonstrate that CXCL13 stimulation promotes rapid and sustained phosphorylation of the carboxyl terminal region (C-tail) of CXCR5 at paired Ser³⁶⁷Thr³⁶⁸ and Ser³⁷⁰Ser³⁷¹ residues, which we have previously shown are essential for β-arrestin recruitment. Using ΔQ-GRK HEK293 cells coupled with individual GRK2 or GRK5 re-expression, we show that phosphorylation of these residues was rescued by either GRK2 or GRK5, while rescue of agonist-stimulated β-arrestin recruitment showed a preference for GRK2 over GRK5, suggesting that additional phospho-sites are likely involved in β-arrestin recruitment. Extension of these studies revealed that agonist-stimulated internalization of CXCR5 was significantly reduced in ΔQ-GRK HEK293 cells and that GRK2 or GRK5 equally rescued the internalization of WT or phospho-site variants, indicating GRK isoform redundancy in CXCR5 internalization. Further, we show that siRNA-mediated knockdown of clathrin significantly reduced CXCR5 internalization, suggesting that internalization of CXCR5 is via clathrin-mediated endocytosis. Taken together, these results reveal that GRKs regulate CXCR5 desensitization and internalization and that internalization occurs through an atypical mode that is β-arrestin-independent and requires GRK phosphorylation of the C-tail.