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.

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 Gα_i activation by S1P₁ was observed in β-arrestin1/2-deficient cells. BRET analysis revealed that S1P₁ interfered with CXCR4-Gα_i 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.

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.