{"doi":"10.1016/j.jbc.2025.111114","title":"GRK phosphorylation drives β-arrestin–independent internalization of chemokine receptor CXCR5","abstract":"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 Ser 367 Thr 368 and Ser 370 Ser 371 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.","journal":"Journal of Biological Chemistry","year":2025,"id":587276,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.947,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":498922,"name":"Ya Zhuo","orcid":"0000-0002-3068-3792","position":1,"is_corresponding":false},{"id":1266385,"name":"Aaren R Manz","orcid":null,"position":2,"is_corresponding":false},{"id":1047653,"name":"Julia Drube","orcid":"0000-0001-6188-2279","position":3,"is_corresponding":false},{"id":443582,"name":"Stefan Schulz","orcid":"0000-0002-5997-8885","position":4,"is_corresponding":false},{"id":578790,"name":"Carsten Hoffmann","orcid":"0000-0003-0884-9300","position":5,"is_corresponding":false},{"id":370582,"name":"Adriano Marchese","orcid":null,"position":6,"is_corresponding":false},{"id":905914,"name":"Joseph M. Crecelius","orcid":"0000-0003-2370-9154","position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:59:36.020030Z","pmid":"41475547","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}