| Summary: | Abstract Background The trafficking of immune cells between lymphoid organs and circulation depends on gradients of CXCL12 and sphingosine-1-phosphate (S1P), mediated through their cognate receptors C-X-C chemokine receptor type 4 (CXCR4) and S1P receptor type 1 (S1P1). S1P1 facilitates the egress of hematopoietic stem cells and lymphocytes by counteracting CXCR4-mediated retention signals. However, the molecular mechanisms underlying this interplay remain poorly understood. In this study, we uncover CXCR4-S1P1 heteromerization and explore their functional interactions. Methods Bimolecular fluorescence complementation (BiFC) assay, proximity ligation assay (PLA), and quantitative bioluminescence resonance energy transfer (BRET) assay were employed to detect CXCR4-S1P1 heteromerization. Functional properties of the heteromers were assessed using cAMP assay, G protein activation, β-arrestin recruitment, ligand binding, calcium mobilization, and transwell migration assays. S1P1-overexpressing Jurkat T cells were generated via lentiviral transduction, while S1P1-deficient KARPAS299 cells and β-arrestin1/2-deficient HEK293A cells were constructed using the CRISPR/Cas9 system. Results CXCR4-S1P1 heteromerization was observed in HEK293A cells overexpressing both receptors. The S1P/S1P1 axis interfered with CXCR4-mediated signaling, while CXCR4 did not affect S1P1-mediated signaling, indicating a unidirectional modulation of CXCR4 by S1P1. CXCL12 binding to CXCR4 remained unchanged in the presence of S1P1, and interference of CXCL12-induced Gαi activation by S1P1 was observed in β-arrestin1/2-deficient cells. BRET analysis revealed that S1P1 interfered with CXCR4-Gαi pre-association and CXCR4 oligomerization, both of which are critical for CXCR4 function. Domain-swapping experiments identified transmembrane domain 3 of S1P1 as essential for this modulation. In Jurkat T cells overexpressing S1P1, CXCR4-mediated signaling and cell migration were diminished, whereas these functions were enhanced in S1P1-deficient KARPAS299 cells. Co-activation of S1P1 attenuated CXCL12-induced migration, while pretreatment with S1P or FTY720-phosphate increased CXCR4-mediated migration by downregulating surface S1P1 in KARPAS299 cells. In primary T cells, PLA confirmed CXCR4-S1P1 heteromerization, and S1P interfered with CXCL12-induced migration. Conclusions This study identifies CXCR4-S1P1 heteromers and demonstrates a unidirectional modulation of CXCR4 by S1P1. S1P1 affects CXCR4 function by disrupting its G protein pre-association and oligomerization. These findings underscore the regulatory role of the S1P/S1P1 axis in CXCR4 signaling within the heteromeric context and provide novel insights into the intricate mechanisms governing immune cell trafficking.
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