Hamatake Makiko, Aoki Toru, Futahashi Yuko, Urano Emiko, Yamamoto Naoki, Komano Jun
AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Cancer Sci. 2009 Jan;100(1):95-102. doi: 10.1111/j.1349-7006.2008.00997.x. Epub 2008 Oct 30.
CXCR4, a G-protein-coupled receptor of CXCL12/stromal cell-derived factor-1alpha, mediates a wide range of physiological and pathological processes, including the targeted metastasis of cancer cells. CXCR4 has been shown to homo-oligomerize in several experimental systems. However, it remains unclear with which domains CXCR4 interacts homotypically, and whether it dimerizes or forms a higher-order complex. To address these issues, we used bioluminescent resonance energy transfer and bimolecular fluorescence complementation analyses to measure the homotypic interactions of CXCR4 in living cells. Both assays indicated that CXCR4 interacts homotypically, which is consistent with previous studies. By studying CXCR4 mutants lacking various domains, we found that multiple transmembrane domains probably serve as potential molecular interaction surfaces for oligomerization. The relative contribution of the amino- or carboxy-termini to oligomerization was small. To differentiate between a dimer and a multimer consisting of more than two molecules, bioluminescent resonance energy transfer-bimolecular fluorescence complementation analysis was conducted. It revealed that CXCR4 engages in higher-order oligomerization in a ligand-independent fashion. This is the first report providing direct experimental evidence for the higher-order multimerization of CXCR4 in vivo. We hypothesize that CXCR4 distributes to the cell surface as a multimer, in order to effectively sense, with increased avidity, the chemotaxis-inducing ligand in the microenvironment. Studying the structure and function of the oligomeric state of CXCR4 may lead us to develop novel CXCR4 inhibitors that disassemble the molecular cluster of CXCR4.
CXCR4是CXCL12/基质细胞衍生因子-1α的G蛋白偶联受体，介导多种生理和病理过程，包括癌细胞的靶向转移。在多个实验系统中，CXCR4已被证明可发生同源寡聚化。然而，CXCR4与哪些结构域进行同源相互作用，以及它是形成二聚体还是更高阶复合物仍不清楚。为了解决这些问题，我们使用生物发光共振能量转移和双分子荧光互补分析来测量活细胞中CXCR4的同源相互作用。两种分析均表明CXCR4发生同源相互作用，这与先前的研究一致。通过研究缺失不同结构域的CXCR4突变体，我们发现多个跨膜结构域可能作为寡聚化的潜在分子相互作用表面。氨基或羧基末端对寡聚化的相对贡献较小。为了区分由两个以上分子组成的二聚体和多聚体，进行了生物发光共振能量转移-双分子荧光互补分析。结果表明CXCR4以不依赖配体的方式进行高阶寡聚化。这是第一份提供CXCR4在体内发生高阶多聚化的直接实验证据的报告。我们推测CXCR4以多聚体形式分布于细胞表面，以便以更高的亲和力有效感知微环境中诱导趋化性的配体。研究CXCR4寡聚体状态的结构和功能可能会引导我们开发出能够拆解CXCR4分子簇的新型CXCR4抑制剂。
