{"doi":"10.1101/2023.03.25.534231","title":"Multiple mechanisms of self-association of chemokine receptors CXCR4 and CCR5 demonstrated by deep mutagenesis","abstract":"Chemokine receptors are members of the rhodopsin-like class A GPCRs whose signaling through G proteins drives the directional movement of cells in response to a chemokine gradient. Chemokine receptors CXCR4 and CCR5 have been extensively studied due to their roles in white blood cell development and inflammation and their status as coreceptors for HIV-1 infection, among other functions. Both receptors form dimers or oligomers but the function/s of self-associations are unclear. While CXCR4 has been crystallized in a dimeric arrangement, available atomic resolution structures of CCR5 are monomeric. To investigate the dimerization interfaces of these chemokine receptors, we used a bimolecular fluorescence complementation (BiFC)-based screen and deep mutational scanning to find mutations that modify receptor self-association. Many disruptive mutations promoted self-associations nonspecifically, suggesting they aggregated in the membrane. A mutationally intolerant region was found on CXCR4 that matched the crystallographic dimer interface, supporting this dimeric arrangement in living cells. A mutationally intolerant region was also observed on the surface of CCR5 by transmembrane helices 3 and 4. Mutations from the deep mutational scan that reduce BiFC were validated and were localized in the transmembrane domains as well as the C-terminal cytoplasmic tails where they reduced lipid microdomain localization. The reduced self-association mutants of CXCR4 had increased binding to the ligand CXCL12 but diminished calcium signaling. There was no change in syncytia formation with cells expressing HIV-1 Env. The data highlight that multiple mechanisms are involved in self-association of chemokine receptor chains.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":396085,"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":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9516,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":414607,"name":"Kritika Mehta","orcid":"0000-0002-4312-9950","position":1,"is_corresponding":false},{"id":413314,"name":"Jeremiah D. Heredia","orcid":"0000-0002-0109-3937","position":2,"is_corresponding":false},{"id":510048,"name":"Vishnu V. Krishnamurthy","orcid":"0000-0001-9905-5965","position":3,"is_corresponding":false},{"id":321413,"name":"Kai Zhang","orcid":"0000-0002-6687-4558","position":4,"is_corresponding":false},{"id":343873,"name":"Erik Procko","orcid":"0000-0002-0028-490X","position":5,"is_corresponding":false},{"id":413312,"name":"Kevin S. Gill","orcid":"0000-0002-9456-3693","position":0,"is_corresponding":true}],"reference_count":46,"raw_metadata":null,"created_at":"2026-07-19T01:19:27.044553Z","pmid":"36993221","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":[]}