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

通过与细胞相关结合蛋白的相互作用来控制 IGF-1 活性

• 批准号: 6240980
• 负责人: SHERIDA E TOLLEFSEN
• 金额: $ 19.27万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240980
• 关键词: Baculoviridae; SDS polyacrylamide gel electrophoresis; affinity chromatography; binding proteins; carbohydrate structure; chemical association; chemical kinetics; extracellular matrix proteins; fibroblasts; glycosylation; growth /development; growth factor receptors; human tissue; insulinlike growth factor; intermolecular interaction; molecular site; oligosaccharides; postnatal growth disorder; posttranslational modifications; protein biosynthesis; protein purification; protein structure function; proteolysis; receptor binding; serum

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.

胰岛素样生长因子（IGF-I和IGF-II）的重要性 局部生长过程以及细胞和组织分化现在 良好的。 紧紧控制表达的机制和 IGF-I的活性必须存在，因为许多细胞类型会产生，并且 回应IGF-I。 这两个IGF的许多动作均由IGF-I介导 受体，一种杂酸酪氨酸特异性蛋白激酶 与胰岛素受体结构相关。 我的长期目标 实验室已经了解 IGF-I及其细胞表面受体和结合蛋白。 我们的先前 研究阐明了IGF-I受体亚基之间的相互作用 对于高亲和力IGF-1结合和IGF-i的必要条件是 刺激自磷酸化。 在此应用中，有两种机制 可能控制IGF-I活性，具体来说是IGF-I与 与细胞相关的IGF结合蛋白和IGF-I的产生为两个 可能需要通过有限的蛋白水解激活其原则 E域将被调查。 提出了三个具体目标。 首先，IGF结合蛋白与细胞表面的相互作用 将定义人成纤维细胞中的细胞外基质。 我们 以前已经表征了身材矮小的孩子的成纤维细胞 （CSC）产生丰富的细胞相关IGF结合蛋白。 这 CSC成纤维细胞中相互作用的细胞分子将被定位，并且 纯化，他们的识别站点将被描述。 第二， IGFBP-3的寡糖单位的结构，主要IGF结合 将分析血清和成纤维细胞中的蛋白质。 功能 糖基化对于IGFBP-3易位的重要性 细胞，其细胞关联和/或稳定性 将检查蛋白水解降解。 第三，生物学 将确定人IGF-IA和IGF-IB的特性和激活。 与Peter Rotwein博士（项目＃1）合作，我们已经表达了 杆状病毒系统中的IGF-IA。 IGF-IA和IGF-IB都将被表达 并纯化，可以使我们比较他们的生物活性 与成熟的70残留IGF-I一起，以阐明其翻译后 加工。 预计这些研究将导致更好 了解控制正常IGF-I活性的机制 生长。