ACTIVATION OF EPIDERMAL GROWTH FACTOR RECEPTOR

该项目的长期目标是阐明所有步骤 在表皮生长因子(EGF)的分子机制中 激活表皮生长因子受体(EGF)的酪氨酸激酶 受体)。细胞内各种蛋白质的磷酸化 这种酪氨酸激酶启动一系列事件，导致细胞 分裂和增长。已有研究表明，含有某些物质的细胞 EGF受体的突变形式在转化状态下无法控制地生长 某些病毒癌基因是这种细胞的改变的病毒形式 受体。这些观察表明这种蛋白质可能是一个点 异常致癌生长的启动。 EGF受体的激活机制包括与EGF的结合， EGF受体的二聚化，酪氨酸激酶的激活，以及 受体的自我磷酸化。这些步骤中的每一个都将 仔细检查了一下。表皮生长因子单体和二聚体的亲和力 将测定EGF受体以确定是否 单体EGF受体的二聚化和酪氨酸的激活 激酶与这一结合相连接。的二阶速率常数 将测量胞外结构域的二聚化，以便它可以 可以直接与完整蛋白质的二聚化进行比较。我们 将在完整的细胞质结构域中寻找构象变化 由结合EGF启动但先于EGF的EGF受体 二聚化。EGF受体的游离胞质结构域将是 与二价免疫球蛋白二聚化，看看二聚化是否 足以激活酪氨酸激酶。激活一个 缺乏所有自我磷酸化位点的截短EGF受体将 被研究以验证在过程中的二聚化要求 即使在没有自我磷酸化的情况下也能激活。的能力 EGF受体的启动子在激活的二聚体中自身磷酸化 将会被评估。自我磷酸化的进展将是 与酪氨酸二聚化和激活的时间相关 激活剂。表皮生长因子受体二聚化的可逆性与血管内皮生长因子的关系 我们将检查酪氨酸激酶的激活情况。结果来自于 这些实验将增加我们对各个步骤的理解 在这种生长因子受体的激活机制中。

The long term objective of this project is to elucidate all of the steps in the molecular mechanism by which epidermal growth factor (EGF) activates the tyrosine kinase of epidermal growth factor receptor (EGF receptor). The phosphorylation of various proteins within the cell by this tyrosine kinase initiates a cascade of events leading to cell division and growth. It has been shown that cells containing certain mutant forms of EGF receptor grow uncontrollably in a transformed state and certain viral oncogenes are altered viral forms of this cellular receptor. These observations suggest that this protein could be a point of initiation for abnormal oncogenic growth. The mechanism of activation of EGF receptor involves binding of EGF, dimerization of the EGF receptor, activation of the tyrosine kinase, and self-phosphorylation of the receptor. Each of these steps will be examined in detail. the affinity of monomeric and dimeric forms of EGF receptor for EGF will be measured to determine whether or not dimerization of monomeric EGF receptor and activation of the tyrosine kinase is linked to this binding. The second-order rate constant for the dimerization of the extracellular domain will be measured so that it can be compared directly to that for dimerization of the intact protein. We will look for conformational changes in the cytoplasmic domain of intact EGF receptor that are initiated by binding EGF but that precede dimerization. The free cytoplasmic domain of EGF receptor will be dimerized with bivalent immunoglobulins to see if dimerization is sufficient to activate the tyrosine kinase. the activation of a truncated EGF receptor lacking all of the self-phosphorylation sites will be studied to verify the requirement for dimerization in the process of activation even in the absence of self-phosphorylation. The ability of a promoter of EGF receptor to phosphorylate itself in the activated dimer will be assessed. The progress of self-phosphorylation will be correlated temporally with dimerization and activation of the tyrosine kinase. The reversibility of the dimerization of EGF receptor and the activation of the tyrosine kinase will be examined. The results from these experiments will increase our understanding of the various steps in the mechanism of activation of this growth factor receptor.