RECONSTITUTION OF GROWTH FACTOR RECEPTOR/TYROSINE KINASE

细胞增殖依赖于大分子成分， 称为生长因子，其存在于血清中。 的结合 这些生长因子的特异性细胞表面受体似乎 刺激各种生化和生理反应 最终导致有丝分裂信号。 的理解 这些受体偶联的信号系统 由于信号中的损伤， 转移事件可能对为什么 细胞会癌变。 现在人们怀疑生长因子 可以通过三种成分（受体， 传感器、效应器）系统，类似于在 腺苷酸环化酶活性的激素调节，或 脊椎动物的视觉 我们打算测试这个假设，以及 探讨生长因子分子基础的其他方面 行动，使用最近开发的重组方法。以及 确定的磷脂囊泡系统含有纯化的 表皮生长因子（EGF）受体，或纯化的胰岛素 受体，将形成比较的机制， 这两种生长因子受体/酪氨酸激酶的作用 在脂质环境中。 这里提出的研究分为 三个具体目标：1）EGF的结构-功能研究 受体和胰岛素受体/酪氨酸激酶，两者都在 洗涤剂溶液和脂质囊泡中，使用 重建，稳态动力学（磷酸化）和 水动力学方法，2）生长的表征 因子诱导的构象变化（和/或受体-受体 EGF受体与胰岛素受体的相互作用 荧光光谱技术，和3）检查 不同异源三聚体GTP结合的能力 转导蛋白和ras致癌蛋白，作为 在生长因子作用中的转换器。 在具体的 这些研究将探讨的问题包括：a. 生长因子诱导的构象的本质是什么？ 这些受体/酪氨酸激酶的变化，它们是否可以 从生长因子穿过膜双层 结合结构域与酪氨酸激酶结构域通过 分子内机制，B.）受体-受体之间 相互作用在诱导生长因子依赖性 酪氨酸激酶活性和脂质环境是否影响这些 相互作用，以及c.）EGF或胰岛素受体 直接调节GTP的激活-失活循环 结合蛋白？ 重构磷脂的构建 含有这些纯化受体的囊泡系统应 这是向描绘重要的 蛋白质-蛋白质（蛋白质-转换器）相互作用 生长因子偶联信号转导

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.