背景:G蛋白偶联受体异聚化被认为对信号转导产生动态调节影响。CXC趋化因子受体4(CXCR4)及其配体CXCL12在许多癌症中都过表达,在转移中起关键作用。同样,溶血磷脂酸受体1(LPA1)与癌细胞增殖和迁移有关。在我们的初步研究中,我们确定LPA1为前瞻性CXCR4相互作用物。在本研究中,我们详细研究了CXCR4-LPA1异聚体的形成,并表征了这种异聚体的独特分子特征和功能。
方法:我们采用双分子荧光互补,生物发光共振能量转移,和邻近连接测定以证明CXCR4和LPA1之间的异聚化。为了阐明CXCR4-LPA1异聚体的独特分子特征和功能含义,我们进行了各种检测,包括cAMP,BRET用于G蛋白激活,β-抑制素招募,配体结合,和transwell迁移测定。
结果:我们观察到CXCR4在重组HEK293A细胞和人乳腺癌细胞系MDA-MB-231中与LPA1形成异聚体。LPA1与CXCR4的共表达减少了CXCL12介导的cAMP抑制,ERK激活,Gαi/o激活,和β-抑制素招募,而CXCL12与CXCR4的结合未受影响。相比之下,CXCR4对LPA1介导的信号没有影响。溶血磷脂酸(LPA)的添加进一步阻碍了CXCL12诱导的Gαi/o募集至CXCR4。LPA或烷基-OMPT在内源性表达CXCR4和LPA1的各种癌细胞中抑制CXCL12诱导的迁移。相反,CXCL12诱导的钙信号和迁移在LPAR1敲除细胞中增加,和LPA1选择性拮抗剂在亲本MDA-MB-231细胞中增强CXCL12诱导的Gαi/o信号传导和细胞迁移,但在LPA1缺陷细胞中不增强。最终,只有在CXCR4和LPA1拮抗剂同时存在的情况下,才能完全抑制细胞向CXCL12和烷基-OMPT的迁移.
结论:已经在各种细胞系中证明了CXCR4-LPA1异聚体对CXCL12诱导的信号传导和细胞迁移的存在和影响。这一发现为CXCR4通过异聚化的有价值的调节机制提供了重要的见解。此外,我们的发现提出了联合CXCR4和LPA1抑制剂治疗与这些受体相关的癌症和炎性疾病的治疗潜力,同时引起人们对单独使用LPA1拮抗剂治疗此类疾病的担忧。视频摘要。
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.
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.
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, Gαi/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 Gαi/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 Gαi/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.
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.