CONVERGENCE OF INTEGRIN AND GROWTH-FACTOR RECEPTOR SIGNALING PATHWAYS WITHIN THE FOCAL ADHESION COMPLEX
CONVERGENCE OF INTEGRIN AND GROWTH-FACTOR RECEPTOR SIGNALING PATHWAYS WITHIN THE FOCAL ADHESION COMPLEX
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DOI：
10.1091/mbc.6.10.1349
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发表时间：
1995-10-01
影响因子：
3.3
通讯作者：
INGBER, DE
中科院分区：
文献类型：
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作者：
PLOPPER, GE;MCNAMEE, HP;INGBER, DE
细胞外基质通过结合整合素受体从而激活与生长因子诱导的信号机制快速整合的化学信号应答来控制毛细血管内皮细胞对可溶性有丝分裂原的敏感性。在这里，我们报告说，除了整联蛋白，生长因子受体和多种分子，这两种类型的受体传递的信号被固定在细胞骨架（CSK）和空间整合内的粘着斑复合物（FAG）在网站的整联蛋白结合。在圆形细胞中快速诱导FACs，并在洗涤剂提取后使用包被有纤连蛋白或合成的含RGD肽的磁性微珠从剩余的CSK中物理分离。免疫荧光显微镜显示，多种信号分子（例如，pp 60（c-src）、pp 125（FAK）磷脂酰肌醇-3-激酶、磷脂酶C-γ和Na-/H-反向转运蛋白）在结合至包被有整联蛋白配体的珠后15分钟内与FAC的CSK框架结合。酪氨酸激酶向FAC的募集也伴随着酪氨酸磷酸化的局部增加，如整合素结合位点处磷酸酪氨酸染色增强所示。相比之下，既没有招聘的信号分子，也没有增加磷酸酪氨酸染色时，观察到细胞结合到珠包被与对照配体（乙酰化低密度脂蛋白），连接跨膜清道夫受体，但不诱导FAC的形成。Western印迹分析证实，与保留脂质双层的完整CSK或基底细胞表面制剂相比，使用RGD-珠分离的FACs富含pp 60（c-src）、pp 125（FAK）、磷脂酶C-γ和Na+/H+反向转运蛋白。分离的FACs也大大富集了高亲和力成纤维细胞生长因子受体Jig。最重要的是，分离的FACs在体外继续表现出多种化学信号传导活性，包括蛋白酪氨酸激酶活性（pp 60（c-src）和pp 125（FAK））以及在肌醇脂质合成级联中经历多个连续步骤的能力。这些数据表明，毛细血管细胞中由整合素和生长因子受体诱导的许多化学信号传导事件可以在FAC内的不溶性CSK支架上以“固态”有效地起作用，并且FAC可能代表这两种调节途径之间信号整合的主要位点。这种方法可能有助于进一步研究信号转导的生物化学和生物物理基础，从而分离出保留CSK框架以及多种相关化学信号传导活性的FACs。
Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAG) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60(c-src), pp125(FAK) phosphatidylinositol-3-kinase, phospholipase C-gamma, and Na-/H- antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp60(c-src), pp125(FAK), phospholipase C-gamma, and the Na+/H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor Jig. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60(c-src) and pp125(FAK)) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a ''solid-state'' on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.