CONTROL OF IGF-1 ACTIVITY BY INTERACTION WITH CELL ASSOCIATED BINDING PROTEINS

胰岛素样生长因子(IGF-I和IGF-II)在血管紧张性疾病中的作用 局部生长过程和细胞和组织分化现在是 久负盛名。严格控制表达和表达的机制 IGF-I的活性必须存在，因为多种细胞类型产生和 对IGF-I做出回应。这两种IGF的许多作用都是由IGF-I介导的 受体，一种异四聚体酪氨酸特异性蛋白激酶 在结构上与胰岛素受体相关。我的长期目标是 实验室已经了解了功能之间的相互作用 IGF-I及其细胞表面受体和结合蛋白。我们以前的 研究阐明了IGF-I受体亚基之间的相互作用 对于高亲和力的IGF-I结合和IGF-I- 刺激的自磷酸化。在此应用程序中，有两种机制 可能控制IGF-I的活性，特别是IGF-I与 细胞相关胰岛素样生长因子结合蛋白与胰岛素样生长因子-I的产生 可能需要通过有限的蛋白分解来激活的原蛋白 E域，将对其进行调查。提出了三个具体目标。 第一，IGF结合蛋白与细胞表面的相互作用 和/或人成纤维细胞中的细胞外基质。我们 之前曾描述过一名身材矮小儿童的成纤维细胞 (CSC)产生丰富的细胞相关IGF结合蛋白。这个 CSC成纤维细胞中相互作用的细胞分子将被定位并 纯化后，将对其识别位点进行表征。第二， 胰岛素样生长因子结合的主要分子IGFBP-3寡糖单元的结构 将对血清和成纤维细胞中的蛋白质进行分析。功能界别 糖基化对IGFBP-3转位的重要性 细胞，用于其细胞关联，和/或其稳定性 将检测蛋白质的降解情况。第三，生物性 将确定人IGF-IA和IGF-IB的性质和激活。 在与Peter Rotwein博士(项目1)的合作下，我们已经表达了 IGF-IA在杆状病毒系统中的表达。将同时表达IGF-IA和IGF-IB 提纯的数量将使我们能够比较它们的生物活性 与成熟的70个残基IGF-I结合，并阐明它们的翻译后 正在处理。预计这些研究将导致更好的 对正常人IGF-I活性调控机制的认识 成长。

The importance of the insulin-like growth factors (IGF-I and IGF-II) on local growth processes and in cell and tissue differentiation is now well-establish. Mechanisms that tightly control the expression and activity of IGF-I must exist because numerous cell types produce and respond to IGF-I. Many actions of both IGFs are mediated by the IGF-I receptor, a heterotetrameric tyrosine-specific protein kinase structurally related to the insulin receptor. The long-term goal of my laboratory has been to understand the functional interactions between IGF-I and its cell surface receptor and binding proteins. Our previous studies have elucidated the interactions between IGF-I receptor subunits that are necessary for high-affinity IGF-I binding and for IGF-I- stimulated autophosphorylation. In this application, two mechanisms that may control IGF-I activity, specifically, the interaction of IGF-I with cell-associated IGF binding proteins and the production of IGF-I as two proproteins which may require activation by limited proteolysis of their E domains, will be investigated. Three specific aims are proposed. First, the interaction of IGF binding proteins with the cell surface and/or extracellular matrix in human fibroblasts will be defined. We have previously characterized fibroblasts from a child with short stature (CSC) that produce abundant cell-associated IGF binding proteins. The interacting cellular molecules in CSC fibroblasts will be localized and purified, and their recognition sites will be characterized. Second, the structures of the oligosaccharide units of IGFBP-3, the major IGF binding protein in serum and in fibroblasts, will be analyzed. The functional importance of glycosylation for the translocation of IGFBP-3 through the cell, for its cellular association, and/or for its stability to proteolytic degradation will be examined. Third, the biological properties and activation of human IGF-IA and IGF-IB will be determined. In collaboration with Dr. Peter Rotwein (Project #1), we have expressed IGF-IA in a baculovirus system. Both IGF-IA and IGF-IB will be expressed and purified in amounts that will allow us to compare their bioactivity with mature 70 residue IGF-I and to elucidate their post-translational processing. It is anticipated that these studies will lead to a better understanding of the mechanisms that control IGF-I activity in normal growth.