EGF RECEPTOR ACTIVATION AND INTERACTION WITH ERBB FAMILY RECEPTORS

EGF 受体是生长因子受体酪氨酸激酶 ErbB 家族的第一个成员， 还包括ErbB2、ErbB3和ErbB4。 EGF与其受体结合诱导EGF受体的形成 同二聚体，导致 EGF 受体自身磷酸化。 EGF受体能够异二聚化 与 ErbB 家族的其他成员一起，其中 ErbB2 是首选的异二聚化伙伴。最近 数据表明 ErbB 受体可能形成异四聚体，并且激活的寡聚体可能解离成 在称为二次二聚体形成的过程中与非活性单体重新二聚的单体。 EGF 与其受体结合时表现出亲和力的异质性。这一观察被归因于 存在两个独立的站点类别。这些是如何由单个 EGF 的表达产生的 受体蛋白从未被解释过。最近，我们发现 EGF 结合可以通过以下方式描述： 聚合系统中涉及负合作性的模型。在此模型中，EGF 对 单体和未占据的二聚体是相似的。然而，与 EGF 受体二聚体的第二个位点结合 是消极合作的。该模型预测负协同性将导致 EGF 诱导的解离 高浓度 EGF 下二聚体的变化，我们已使用荧光证实了这一预测 相关光谱和酶互补检查 EGF 受体寡聚化。 我们的长期目标是了解 ErbB 受体如何被激活并开发一个模型 预测 ErbB 家族成员之间的相互作用。使用我们的新模型作为进一步的起点 调查，该提案的具体目标是： 1）量化 EGF 受体和 ErbB2 通过配体结合研究； 2) 确定是否EGF受体介导ErbB2的激活 通过形成异源四聚体复合物而发生和/或涉及二聚体受体的解离 复合体； 3) 确定不同的 EGF 受体配体是否诱导 EGF 的特征模式 受体寡聚体以及这是否与下游信号传导的差异有关。 EGF 平衡结合研究将用于定量表征 EGF 的相互作用 ErbB2 受体。荧光相关光谱和酶亮度分析 将利用互补来评估 EGFR/ErbB2 异聚复合物的寡聚状态 而化学诱导的二聚体形成将用于探测这些中 ErbB2 的激活机制 复合物。这些方法的组合将用于评估 EGF 受体的寡聚化 由三种不同的激动剂引起。三种激动剂刺激的信号传导将通过测量来评估 下游通路的激活。这些数据将共同描述 EGF 相互作用的特征 受体和 ErbB2 允许开发通过 EGF 激活 ErbB2 的定量模型 受体并深入了解 ErbB 家族寡聚体在细胞信号传导过程中的作用。

The EGF receptor is the first member of the ErbB family of growth factor receptor tyrosine kinases that also includes ErbB2, ErbB3 and ErbB4. Binding of EGF to its receptor induces the formation of EGF receptor homodimers, resulting in autophosphorylation of the EGF receptor. The EGF receptor is able to heterodimerize with other members of the ErbB family, with ErbB2 being the preferred heterodimerization partner. Recent data suggest that ErbB receptors may form heterotetramers and that activated oligomers may dissociate into monomers that re-dimerize with inactive monomers in a process referred to as secondary dimer formation. EGF shows heterogeneity in affinity when binding to its receptor. This observation has been attributed to the existence of two independent classes of sites. How these arise from the expression of a single EGF receptor protein has never been explained. Recently, we have shown that EGF binding can be described by a model that involves negative cooperativity in an aggregating system. In this model, the affinity of EGF for a monomer and an unoccupied dimer is similar. However, binding to the second site on an EGF receptor dimer is negatively cooperative. This model predicts that the negative cooperativity will lead to EGF-induced dissociation of the dimer at high concentrations of EGF, a prediction that we have confirmed using fluorescence correlation spectroscopy and enzyme complementation to examine EGF receptor oligomerization. Our long-term goal is to understand how ErbB receptors are activated and to develop a model for predicting the interactions among ErbB family members. Using our new model as the starting point for further investigations, the specific aims of this proposal are to: 1) Quantify the interaction of the EGF receptor and ErbB2 through ligand binding studies; 2) Determine whether EGF receptor-mediated activation of ErbB2 occurs via the formation of a hetero-tetrameric complex and/or involves dissociation of dimeric receptor complexes; and, 3) Determine whether different EGF receptor ligands induce characteristic patterns of EGF receptor oligomers and whether this can be related to differences in downstream signaling. EGF equilibrium binding studies will be used to quantitatively characterize the interaction of the EGF receptor with ErbB2. Fluorescence correlation spectroscopy and brightness analysis along with enzyme complementation will be employed to assess the oligomerization state of EGFR/ErbB2 heteromeric complexes while chemically-induced dimer formation will be used to probe the activation mechanism of ErbB2 within these complexes. A combination of these methods will be applied to assess oligomerization of the EGF receptor elicited by three different agonists. Signaling stimulated by the three agonists will be assessed by measuring the activation of downstream pathways. Together, these data will characterize the interactions of the EGF receptor and ErbB2 allowing the development of a quantitative model for the activation of ErbB2 via the EGF receptor and provide insight into the role of ErbB family oligomers in the process of cell signaling.