LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration

Abstract

Abstract Background G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA 1 ) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA 1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA 1 heteromer and characterized the unique molecular features and function of this heteromer. Methods We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA 1 . To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA 1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, β-arrestin recruitment, ligand binding, and transwell migration assays. Results We observed that CXCR4 forms heteromers with LPA 1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA 1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gα i/o activation, and β-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA 1 -mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gα i/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA 1 . Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA 1 -selective antagonists enhanced CXCL12-induced Gα i/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA 1 -deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA 1 antagonists. Conclusions The presence and impact of CXCR4-LPA 1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA 1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA 1 antagonists alone for such conditions.

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