RECONSTITUTION OF GROWTH FACTOR RECEPTOR/TYROSINE KINASE

生长因子受体/酪氨酸激酶的重建

• 批准号: 3298426
• 负责人: RICHARD A. CERIONE
• 金额: $ 14.78万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298426
• 关键词: binding proteins; cell cell interaction; conformation; enzyme mechanism; epidermal growth factor; fluorescence spectrometry; fluorescent dye /probe; gel electrophoresis; growth factor; guanine nucleotide binding protein; guanosine triphosphate; hormone receptor; hormone regulation /control mechanism; human tissue; insulin receptor; lipids; phospholipids; phosphorylation; protein reconstitution; protein structure function; protein tyrosine kinase; radionuclides; vesicle /vacuole

Cell proliferation is dependent on macromolecular components, termed growth factors, which are present in serum. The binding of these growth factors to specific cell surface receptors appears to stimulate a variety of biochemical and physiological responses which culminate in a mitogenic signal. An understanding of the mechanisms by which these receptor-coupled signaling systems operate is of increasing interest since impairments in the signal transfer events may have important implications regarding why cells become cancerous. It is now suspected that growth factors may trigger mitogenesis through three component (receptor, transducer, effector) systems analogous to those operating in the hormonal regulation of adenylate cyclase activity, or in vertebrate vision. We intend to test this hypothesis as well as address other aspects of the molecular basis of growth factor action, using recently developed reconstitution approaches. Well defined phospholipid vesicle systems containing the purified epidermal growth factor (EGF) receptor, or the purified insulin receptor, will form the basis for comparing the mechanisms of action of these two growth factor receptor/tyrosine kinases within a lipid milieu. The studies proposed here are divided into three specific aims: 1) Structure-function studies of the EGF receptor and the insulin receptor/tyrosine kinases, both in detergent solution and in lipid vesicles, using a combination of reconstitution, steady state kinetic (phosphorylation) and hydrodynamic approaches, 2) The characterization of growth factor-induced conformational changes (and/or receptor-receptor interactions) in the EGF receptor and the insulin receptor by fluorescence spectroscopic techniques, and 3) An examination of the capabilities of different heterotrimeric GTP binding transducer proteins, and the ras oncogenic proteins, to act as transducers in growth factor action. Among the specific questions which will be addressed in these studies include: a.) what is the nature of the growth factor-induced conformational changes in these receptor/tyrosine kinases, and can they be transmitted across a membrane bilayer from the growth factor binding domain to the tyrosine kinase domain via an intramolecular mechanism, b.) what roles do receptor-receptor interactions play in the induction of growth factor-dependent tyrosine kinase activity and does a lipid milieu influence these interactions, and c.) can either the EGF or insulin receptor directly regulate the activation-deactivation cycles of GTP binding proteins? The construction of reconstituted phospholipid vesicle systems containing these purified receptors should constitute an important step toward delineating the important protein-protein (protein-transducer) interactions involved in growth factor-coupled signal transduction.

细胞的增殖依赖于大分子成分， 称为生长因子，存在于血清中。装订 在这些生长因子中，出现了特定的细胞表面受体 刺激各种生化和生理反应 最终形成有丝分裂信号。对这一概念的理解 这些受体偶联信号系统的机制 操作越来越受关注，因为信号中的损伤 转会事件可能会对原因产生重要影响 细胞变得癌变。现在人们怀疑，增长因素 可能通过三种成分(受体、 换能器、效应器)系统类似于在 腺苷环化酶活性的激素调节，或在 脊椎动物的视觉。我们打算测试这一假设以及 探讨生长因子分子基础的其他方面 行动，使用最近制定的重建办法。井 定义的磷脂囊泡系统，包含纯化的 表皮生长因子受体，或纯化的胰岛素 受体，将形成比较机制的基础 这两种生长因子受体/酪氨酸激酶的作用 在脂类环境中。这里提出的研究分为 三个具体目标：1)表皮生长因子的结构-功能研究 受体和胰岛素受体/酪氨酸激酶，两者都在 洗涤剂溶液和脂泡中，使用 重组、稳态动力学(磷酸化)和 流体动力学方法，2)生长的表征 因子诱导的构象变化(和/或受体-受体 相互作用)在EGF受体和胰岛素受体中 荧光光谱技术，以及3)检测 不同异源三聚体GTP结合能力的研究 转导蛋白和ras癌基因蛋白作为 生长因子作用中的转导分子。在具体的 这些研究将涉及的问题包括：a.) 生长因子诱导的构象的性质是什么 这些受体/酪氨酸激酶的变化，它们是否会 从生长因子通过膜双层传输 通过一种途径将结构域与酪氨酸激酶结构域结合 分子内机制，b.)受体-受体的作用是什么 相互作用在诱导生长因子依赖中发挥作用 酪氨酸激酶活性和脂质环境是否会影响这些 相互作用，以及c.)EGF或胰岛素受体能否 直接调节GTP的激活-失活周期 结合蛋白？重组磷脂的构建 含有这些纯化受体的囊泡系统应该 构成了向描述重要的 蛋白质-蛋白质(蛋白质-转导)相互作用参与 生长因子偶联信号转导。