{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T18:27:25Z","timestamp":1785868045372,"version":"3.56.0"},"reference-count":37,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100014188","name":"MSIT","doi-asserted-by":"crossref","award":["2022R1F1A1074216"],"award-info":[{"award-number":["2022R1F1A1074216"]}],"id":[{"id":"10.13039\/501100014188","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003710","name":"Korea Health Industry Development Institute","doi-asserted-by":"publisher","award":["RS-2022-KH129263"],"award-info":[{"award-number":["RS-2022-KH129263"]}],"id":[{"id":"10.13039\/501100003710","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>C-C chemokine receptor 7 (CCR7) directs immune cell homing to secondary lymphoid organs and has been implicated in cancer metastasis through its ligands CCL19 and CCL21. Human CCR7 pre-mRNA undergoes alternative splicing to generate five transcripts that encode three protein isoforms with distinct N-termini, termed CCR7A, CCR7B, and CCR7C, but their comparative properties and cross-regulation are not well defined. Here, we cloned these three isoforms and systematically characterized their expression, localization, signaling, and mutual interactions in mammalian cells under both strong (CMV) and weaker (ubiquitin C, UbiC) promoter control to reduce overexpression-related artifacts. Variant-specific RT-PCR revealed that transcripts encoding CCR7A (V1) and CCR7B (V2) predominate in diverse human cell lines, whereas CCR7C-encoding variants (V3\u2013V5) are weakly expressed. EGFP imaging and HiBiT-based assays showed efficient plasma-membrane targeting of CCR7A, partial membrane localization and prominent perinuclear accumulation of CCR7C, and largely cytosolic retention of CCR7B. Under UbiC-driven expression, CCR7A mediated robust CCL19- and CCL21-induced Gi\/o and Gq-like activation, intracellular Ca\u00b2\u2009\u207a\u2009mobilization, ERK phosphorylation, GRK3-dependent G\u03b21 recruitment, and \u03b2-arrestin1 binding, whereas CCR7C displayed weaker and mainly CCL19-biased signaling. CCR7B did not respond to either chemokine in any signaling readout and thus behaved as a non-signaling isoform. NanoBiT-based assays and co-immunoprecipitation demonstrated that all three isoforms form homo- and heterodimers, with particularly strong association between CCR7A and CCR7B. Co-expression of CCR7B reduced CCR7A surface expression and markedly attenuated chemokine-induced Ca\u00b2\u2009\u207a\u2009responses, mini-Gi interaction, and \u03b2-arrestin1 recruitment, while confocal microscopy revealed redistribution of CCR7A-EGFP from the plasma membrane to intracellular compartments. Moreover, MDA-MB-231 breast cancer cells, which express CCR7A and CCR7B transcripts, did not migrate toward CCL19 or CCL21 despite preserved motility toward low-serum medium. These findings identify CCR7A as the dominant functional isoform, CCR7C as a weak CCL19-biased receptor with inefficient membrane targeting, and CCR7B as a non-signaling dominant-negative isoform that dampens CCR7A-mediated responses, suggesting that CCR7 splicing fine-tunes chemokine responsiveness in immune and cancer cells.<\/jats:p>","DOI":"10.1371\/journal.pone.0354819","type":"journal-article","created":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T17:25:57Z","timestamp":1785864357000},"page":"e0354819","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional CCR7A-mediated cellular responses are negatively modulated by the splice variant CCR7B"],"prefix":"10.1371","volume":"21","author":[{"given":"Minyeong","family":"Cho","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hee-Kyung","family":"Park","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lan Phuong","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4908-1185","authenticated-orcid":true,"given":"Thai Uy","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Duc Trung","family":"Nguyen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soyeon","family":"In","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunghoon","family":"Hurh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Beom Jin","family":"Park","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jae Young","family":"Seong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0729-1782","authenticated-orcid":true,"given":"Jong-Ik","family":"Hwang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,8,4]]},"reference":[{"issue":"1","key":"pone.0354819.ref001","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1111\/imr.12752","article-title":"Concepts of GPCR-controlled navigation in the immune system","volume":"289","author":"T L\u00e4mmermann","year":"2019","journal-title":"Immunol Rev"},{"key":"pone.0354819.ref002","doi-asserted-by":"crossref","unstructured":"Griffith JW, Sokol CL, Luster ADJAroi. Chemokines and chemokine receptors: Positioning cells for host defense and immunity. 2014;32(1):659\u2013702.","DOI":"10.1146\/annurev-immunol-032713-120145"},{"issue":"4","key":"pone.0354819.ref003","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1038\/cmi.2017.134","article-title":"Chemokines in homeostasis and diseases","volume":"15","author":"K Chen","year":"2018","journal-title":"Cell Mol Immunol"},{"issue":"5","key":"pone.0354819.ref004","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1038\/nri2297","article-title":"CCR7 and its ligands: Balancing immunity and tolerance","volume":"8","author":"R F\u00f6rster","year":"2008","journal-title":"Nat Rev Immunol"},{"key":"pone.0354819.ref005","doi-asserted-by":"crossref","first-page":"568","DOI":"10.3389\/fimmu.2016.00568","article-title":"Differential CCR7 Targeting in Dendritic Cells by Three Naturally Occurring CC-Chemokines","volume":"7","author":"GM Hjort\u00f8","year":"2016","journal-title":"Front Immunol"},{"issue":"3","key":"pone.0354819.ref006","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/j.cytogfr.2013.03.001","article-title":"A myriad of functions and complex regulation of the CCR7\/CCL19\/CCL21 chemokine axis in the adaptive immune system","volume":"24","author":"I Comerford","year":"2013","journal-title":"Cytokine Growth Factor Rev"},{"issue":"1","key":"pone.0354819.ref007","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/j.celrep.2017.09.016","article-title":"CCR7 modulates the generation of thymic regulatory T cells by altering the composition of the thymic dendritic cell compartment","volume":"21","author":"Z Hu","year":"2017","journal-title":"Cell Rep"},{"key":"pone.0354819.ref008","doi-asserted-by":"crossref","first-page":"110431","DOI":"10.1016\/j.intimp.2023.110431","article-title":"CCL21\/CCR7 axis as a therapeutic target for autoimmune diseases","volume":"121","author":"L Han","year":"2023","journal-title":"Int Immunopharmacol"},{"issue":"4","key":"pone.0354819.ref009","doi-asserted-by":"crossref","first-page":"656","DOI":"10.3390\/cells11040656","article-title":"C-C chemokine receptor 7 in cancer","volume":"11","author":"CA Bill","year":"2022","journal-title":"Cells"},{"issue":"2","key":"pone.0354819.ref010","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1016\/j.jns.2006.06.021","article-title":"Expression of CCR7 and its ligands CCL19\/CCL21 in muscles of polymyositis","volume":"249","author":"M Tateyama","year":"2006","journal-title":"J Neurol Sci"},{"issue":"5","key":"pone.0354819.ref011","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1002\/ijc.21123","article-title":"Prediction of lymph node metastasis in colorectal carcinoma by expressionof chemokine receptor CCR7","volume":"116","author":"K G\u00fcnther","year":"2005","journal-title":"Int J Cancer"},{"issue":"6","key":"pone.0354819.ref012","doi-asserted-by":"crossref","first-page":"354","DOI":"10.1593\/tlo.10178","article-title":"Expression of the C-C chemokine receptor 7 mediates metastasis of breast cancer to the lymph nodes in mice","volume":"3","author":"HD Cunningham","year":"2010","journal-title":"Transl Oncol"},{"issue":"10","key":"pone.0354819.ref013","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1038\/s41423-018-0056-5","article-title":"The chemokine receptor CCR7 is a promising target for rheumatoid arthritis therapy","volume":"16","author":"GL Moschovakis","year":"2019","journal-title":"Cell Mol Immunol"},{"issue":"7","key":"pone.0354819.ref014","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1007\/s00018-019-03235-w","article-title":"CCL21\/CCR7 signaling in macrophages promotes joint inflammation and Th17-mediated osteoclast formation in rheumatoid arthritis","volume":"77","author":"K Van Raemdonck","year":"2020","journal-title":"Cell Mol Life Sci"},{"key":"pone.0354819.ref015","doi-asserted-by":"crossref","first-page":"736758","DOI":"10.3389\/fonc.2021.736758","article-title":"CCR7 in blood cancers - Review of Its pathophysiological roles and the potential as a therapeutic target","volume":"11","author":"C Cuesta-Mateos","year":"2021","journal-title":"Front Oncol"},{"issue":"3","key":"pone.0354819.ref016","doi-asserted-by":"crossref","first-page":"886","DOI":"10.1182\/blood-2006-03-013532","article-title":"CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity","volume":"109","author":"UE H\u00f6pken","year":"2007","journal-title":"Blood"},{"key":"pone.0354819.ref017","doi-asserted-by":"crossref","unstructured":"Markovic D, Challiss RJJC, sciences ml. Alternative splicing of G protein-coupled receptors: physiology and pathophysiology. 2009;66(20):3337\u201352.","DOI":"10.1007\/s00018-009-0093-4"},{"issue":"7835","key":"pone.0354819.ref018","first-page":"650","volume":"587","author":"M Marti-Solano","year":"2020","journal-title":"Combinatorial expression of functionally distinct GPCR isoforms can diversify receptor signalling response"},{"issue":"1","key":"pone.0354819.ref019","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1186\/s13578-022-00787-6","article-title":"Analysis of CCR2 splice variant expression patterns and functional properties","volume":"12","author":"H-K Park","year":"2022","journal-title":"Cell Biosci"},{"issue":"5","key":"pone.0354819.ref020","doi-asserted-by":"crossref","first-page":"281","DOI":"10.14348\/molcells.2023.2096","article-title":"Functional analysis of CXCR3 splicing variants and their ligands using NanoBiT-based molecular interaction assays","volume":"46","author":"HT Nguyen","year":"2023","journal-title":"Mol Cells"},{"issue":"12","key":"pone.0354819.ref021","doi-asserted-by":"crossref","first-page":"2914","DOI":"10.1111\/j.1742-4658.2005.04731.x","article-title":"Methods to monitor the quaternary structure of G protein-coupled receptors","volume":"272","author":"G Milligan","year":"2005","journal-title":"FEBS J"},{"issue":"11","key":"pone.0354819.ref022","doi-asserted-by":"crossref","first-page":"909","DOI":"10.14348\/molcells.2020.0144","article-title":"Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by optimizing calmodulin-binding motif and protein expression levels","volume":"43","author":"LP Nguyen","year":"2020","journal-title":"Mol Cells"},{"issue":"2","key":"pone.0354819.ref023","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1002\/JLB.2VMA1217-492R","article-title":"A unique signal sequence of the chemokine receptor CCR7 promotes package into COPII vesicles for efficient receptor trafficking","volume":"104","author":"E Uetz-von Allmen","year":"2018","journal-title":"J Leukoc Biol"},{"issue":"5","key":"pone.0354819.ref024","volume":"18","author":"H-K Park","year":"2023","journal-title":"The N-terminus of CXCR4 splice variants determines expression and functional properties"},{"issue":"1","key":"pone.0354819.ref025","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1186\/s13578-020-00497-x","article-title":"CXCR7: a \u03b2-arrestin-biased receptor that potentiates cell migration and recruits \u03b2-arrestin2 exclusively through G\u03b2\u03b3 subunits and GRK2","volume":"10","author":"HT Nguyen","year":"2020","journal-title":"Cell Biosci"},{"issue":"4","key":"pone.0354819.ref026","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0175642","article-title":"Mini-G proteins: Novel tools for studying GPCRs in their active conformation","volume":"12","author":"R Nehm\u00e9","year":"2017","journal-title":"PLoS One"},{"issue":"19","key":"pone.0354819.ref027","doi-asserted-by":"crossref","first-page":"7466","DOI":"10.1074\/jbc.RA118.001975","article-title":"Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells","volume":"293","author":"Q Wan","year":"2018","journal-title":"J Biol Chem"},{"issue":"1","key":"pone.0354819.ref028","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S0092-8674(00)80059-8","article-title":"CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs","volume":"99","author":"R F\u00f6rster","year":"1999","journal-title":"Cell"},{"issue":"12","key":"pone.0354819.ref029","doi-asserted-by":"crossref","first-page":"7118","DOI":"10.1074\/jbc.273.12.7118","article-title":"Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7","volume":"273","author":"R Yoshida","year":"1998","journal-title":"J Biol Chem"},{"issue":"1","key":"pone.0354819.ref030","doi-asserted-by":"crossref","first-page":"4151","DOI":"10.1038\/s41467-021-24438-5","article-title":"Structural basis for chemokine recognition and receptor activation of chemokine receptor CCR5","volume":"12","author":"H Zhang","year":"2021","journal-title":"Nat Commun"},{"key":"pone.0354819.ref031","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/S0076-6879(09)05407-X","article-title":"Chapter 7. Tyrosine sulfation of HIV-1 coreceptors and other chemokine receptors","volume":"461","author":"H Choe","year":"2009","journal-title":"Methods Enzymol"},{"issue":"2","key":"pone.0354819.ref032","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1124\/mol.117.108522","article-title":"C-X-C Motif Chemokine Receptor 3 Splice Variants Differentially Activate Beta-Arrestins to Regulate Downstream Signaling Pathways","volume":"92","author":"JS Smith","year":"2017","journal-title":"Mol Pharmacol"},{"issue":"1","key":"pone.0354819.ref033","first-page":"1","volume":"7","year":"2012","journal-title":"Wise HJJoms. The roles played by highly truncated splice variants of G protein-coupled receptors"},{"issue":"4","key":"pone.0354819.ref034","doi-asserted-by":"crossref","first-page":"1884","DOI":"10.1210\/en.2005-1488","article-title":"A dominant-negative human growth hormone-releasing hormone (GHRH) receptor splice variant inhibits GHRH binding","volume":"147","author":"AT McElvaine","year":"2006","journal-title":"Endocrinology"},{"issue":"5","key":"pone.0354819.ref035","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1002\/cam4.1039","article-title":"CCR7 mediates human breast cancer cell invasion, migration by inducing epithelial-mesenchymal transition and suppressing apoptosis through AKT pathway","volume":"6","author":"B Xu","year":"2017","journal-title":"Cancer Med"},{"issue":"1","key":"pone.0354819.ref036","article-title":"MicroRNA let-7a suppresses breast cancer cell migration and invasion through downregulation of C-C chemokine receptor type 7","volume":"14","author":"S-J Kim","year":"2012","journal-title":"Breast Cancer Res"},{"issue":"9","key":"pone.0354819.ref037","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1007\/s12032-014-0180-8","article-title":"CCL21\/CCR7 axis activating chemotaxis accompanied with epithelial-mesenchymal transition in human breast carcinoma","volume":"31","author":"F Li","year":"2014","journal-title":"Med Oncol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0354819","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T17:26:05Z","timestamp":1785864365000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0354819"}},"subtitle":[],"editor":[{"given":"Joseph J","family":"Mattapallil","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,4]]},"references-count":37,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8,4]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0354819","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,4]]}}}