Regulation of CXCR4 function by S1P1 through heteromerization

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.

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.

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.

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.