Work

Regulation of CXCR4 function by S1P1 through heteromerization

Year: 2025

Type: article

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 (S1P 1 ). S1P 1 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-S1P 1 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-S1P 1 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. S1P 1 -overexpressing Jurkat T cells were generated via lentiviral transduction, while S1P 1 -deficient KARPAS299 cells and β-arrestin1/2-deficient HEK293A cells were constructed using the CRISPR/Cas9 system. Results CXCR4-S1P 1 heteromerization was observed in HEK293A cells overexpressing both receptors. The S1P/S1P 1 axis interfered with CXCR4-mediated signaling, while CXCR4 did not affect S1P 1 -mediated signaling, indicating a unidirectional modulation of CXCR4 by S1P 1 . CXCL12 binding to CXCR4 remained unchanged in the presence of S1P 1 , and interference of CXCL12-induced Gα i activation by S1P 1 was observed in β-arrestin1/2-deficient cells. BRET analysis revealed that S1P 1 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 S1P 1 as essential for this modulation. In Jurkat T cells overexpressing S1P 1 , CXCR4-mediated signaling and cell migration were diminished, whereas these functions were enhanced in S1P 1 -deficient KARPAS299 cells. Co-activation of S1P 1 attenuated CXCL12-induced migration, while pretreatment with S1P or FTY720-phosphate increased CXCR4-mediated migration by downregulating surface S1P 1 in KARPAS299 cells. In primary T cells, PLA confirmed CXCR4-S1P 1 heteromerization, and S1P interfered with CXCL12-induced migration. Conclusions This study identifies CXCR4-S1P 1 heteromers and demonstrates a unidirectional modulation of CXCR4 by S1P 1 . S1P 1 affects CXCR4 function by disrupting its G protein pre-association and oligomerization. These findings underscore the regulatory role of the S1P/S1P 1 axis in CXCR4 signaling within the heteromeric context and provide novel insights into the intricate mechanisms governing immune cell trafficking.

Source: Cell Communication and Signaling

DOI: 10.1186/s12964-025-02099-x

Authors: Hyun-Tae Kim, Jae-Yeon Jeong, Won-Ki Huh

Institution: Seoul National University

Language: English

Topic: Chemokine receptors and signaling

Subfield: Oncology

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,390