Signaling Through Membrane Type 1 Matrix Metalloproteinase

描述(申请人提供)：膜型1基质金属蛋白酶(MT1-MMPs)是一种具有胞外蛋白水解区和短胞浆尾巴的跨膜蛋白水解酶。基质金属蛋白酶组织抑制因子-2(TIMP-2)生理性地抑制了MT1-MMPs的蛋白降解活性。TIMP-2与MT1-MMP2结合，该复合体通过胞浆尾部介导的内吞作用被内化。我们大量的前期工作表明，TIMP-2与MT1-MMPs结合可迅速诱导ERK1/2 MAP激酶激活，从而上调细胞的增殖和迁移。这些作用需要MT1-基质金属蛋白酶的胞质尾巴，而不是蛋白分解活性。 因此，我们建议： 1.确定TIMP-2与MT1-MMP结合后激活ERK1/2的生理意义。为此，将使用野生型和MT1-MMP基因敲除的小鼠胚胎成纤维细胞来确定这种信号机制及其产生的生物学效应是否在表达生理数量的MT1-MMP的基质细胞中活跃，并依赖于细胞质尾部，而不是蛋白水解酶的活性。 2.探讨MT1-MMP胞内信号转导的分子机制。我们建议鉴定在TIMP-2加入时与MT1-MMP胞浆尾相互作用的蛋白质，并表征TIMP-2-MT1-MMP络合物的内吞作用在MT1-MMP介导胞内信号转导中的作用。 这项拟议的研究项目的结果将阐明细胞表面蛋白之间的相互作用产生细胞内信号的新机制，该信号在许多细胞功能的调节中具有重要作用。因此，对这些机制的详细了解对于我们理解各种生理和病理过程以及开发旨在控制它们的药理学工具具有重要意义。

DESCRIPTION (provided by applicant): Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane proteinase with an extracellular proteolytic domain and a short cytoplasmic tail. The proteolytic activity of MT1-MMP is inhibited physiologically by tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). TIMP-2 binds to MT1-MMP and the complex is internalized by endocytosis mediated by the cytoplasmic tail of the proteinase. Our extensive preliminary work has shown that binding of TIMP-2 to MT1-MMP rapidly induces ERK1/2 MAP kinase activation, which upregulates cell proliferation and migration. These effects require the cytoplasmic tail but not the proteolytic activity of MT1 -MMP. We therefore propose to: 1. Determine the physiologic significance of ERK1/2 activation generated by TIMP-2 binding to MT1-MMP. For this purpose, wild-type and MT1-MMP knockout mouse embryonic fibroblasts will be used to determine if this signaling mechanism and its resulting biological effects are active in stromal cells expressing physiologic amounts of MT1 -MMP and dependent on the cytoplasmic tail but not the proteolytic activity of the proteinase. 2. Investigate the molecular mechanism of intracellular signaling by MT1-MMP. We propose to identify proteins that interact with the cytoplasmic tail of MT1-MMP upon TIMP-2 addition, and to characterize the role of endocytosis of the TIMP-2-MT1-MMP complex in the control of MT1-MMP-mediated intracellular signaling. The results of the proposed research project will elucidate novel mechanisms through which interactions among cell surface proteins generate intracellular signaling that has important roles in the regulation of numerous cell functions. A detailed understanding of these mechanisms can therefore have relevant implications for our comprehension of a variety of physiological and pathological processes, and for the development of pharmacological tools aimed to control them.