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

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 (LPA₁) is implicated in Cancer cell proliferation and migration. In our preliminary study, we identified LPA₁ as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA₁ 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 LPA₁. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA₁ 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 LPA₁ in recombinant HEK293A cells and the human breast Cancer cell line MDA-MB-231. Coexpression of LPA₁ 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 LPA₁-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 LPA₁. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA₁-selective antagonists enhanced CXCL12-induced Gα_i/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA₁-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA₁ antagonists.

Conclusions: The presence and impact of CXCR4-LPA₁ 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 LPA₁ inhibitors for Cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA₁ antagonists alone for such conditions. Video Abstract.

Cancer; Chemokine receptor 4; Chemotaxis; G protein-coupled receptor; GPCR heteromer; GPCR signaling; Inflammatory disease; Lysophosphatidic acid receptor 1.