EBI2 regulates CXCL13-mediated responses by heterodimerization with CXCR5.
B-cell movement into lymphoid follicles depends on the expression of the chemokine receptor CXCR5 and the recently reported Epstein-Barr virus-induced receptor 2 (EBI2). In cooperation with CXCR5, EBI2 helps to position activated B cells in the follicle, although the mechanism is poorly understood. Using human HEK293T cells and fluorescence resonance energy transfer (FRET) techniques, we demonstrate that CXCR5 and EBI2 form homo- and heterodimers. EBI2 expression modulated CXCR5 homodimeric complexes, as indicated by the FRET(50) value (CXCR5 homodimer, 0.9851±0.0784; CXCR5 homodimer+EBI2, 1.7320±0.4905; P<0.05). HEK293T cells expressing CXCR5/EBI2 and primary activated murine B cells both down-modulated CXCR5-mediated responses, such as Ca(2+) flux, cell migration, and MAPK activation; this modulation did not occur when primary B cells were obtained from EBI2(-/-) mice. The mechanism involves a reduction in binding affinity of the ligand (CXCL13) for CXCR5 (K(D): 5.05×10(-8) M for CXCR5 alone vs. 1.49×10(-7) M for CXCR5/EBI2) and in the efficacy (E(max)) of G-protein activation in CXCR5/EBI2-coexpressing cells (42.33±4.3%; P<0.05). These findings identify CXCR5/EBI2 heterodimers as functional units that contribute to the plasticity of CXCL13-mediated B-cell responses.
Department of Immunology and Oncology, Centro Nacional de Biotecnología/Consejo Superior de Investigaciones Cientificas (CSIC), Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain.
Rubén
R
Barroso
Laura
L
Martínez Muñoz
Sergio
S
Barrondo
Beatriz
B
Vega
Borja L
BL
Holgado
Pilar
P
Lucas
Amparo
A
Baíllo
Joan
J
Sallés
José M
JM
Rodríguez-Frade
Mario
M
Mellado
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D001667:Binding, Competitive; D015153:Blotting, Western; D002465:Cell Movement; D002478:Cells, Cultured; D054382:Chemokine CXCL13 / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D055503:Protein Multimerization; D011869:Radioligand Assay; D054380:Receptors, CXCR5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
C095085:CXCR5 protein, human; D054382:Chemokine CXCL13; C080105:GPR183 protein, human; D008164:Luminescent Proteins; D054380:Receptors, CXCR5; D043562:Receptors, G-Protein-Coupled; D016244:Guanosine 5'-O-(3-Thiotriphosphate)