HETERODIMERIZATION IN ERBB RECEPTORS

描述(由申请人提供)： EGF受体是一种经典的受体酪氨酸激酶，具有细胞外配体结合域和细胞内酪氨酸激酶域。EGF结合可诱导EGF受体与自身或与ErbB受体酪氨酸激酶家族其他成员的二聚化，ErbB2是首选的二聚化伙伴。EGF受体的二聚化通过形成不对称的激酶二聚体来刺激其细胞内酪氨酸激酶的活性。在这个不对称二聚体中，一个激活域充当另一个激活域的激活剂。作为这一机制的结果，受体二聚体中一次只能激活一个激活域。根据目前的模型，受体的磷酸化然后发生在反式，并且只发生在受体二聚体的背景下。虽然这个模型看起来很简单，但它不能解释EGFR同源二聚体和EGFR/ErbB2异源二聚体功能的所有方面。EGF受体有八种不同的配体，它们能够在同一细胞系中诱导不同的反应。这其中的分子基础尚不清楚，但已假设配体诱导不同构象的EGF受体。由于缺乏适当的工具，这一假说尚未在实验中得到解决。利用荧光素酶片段互补成像，我们记录了EGF受体对其不同配体的反应的明显差异，表明该方法对EGF受体构象的配体特异性变化很敏感。我们还使用放射性配基结合研究来证明受体构象的更细微的差异，反映在配体结合的变构性质的差异上。靶向ErbB受体的治疗策略取决于充分了解这些受体如何相互作用，如何与它们的配体相互作用，以及它们是如何受到调控的。这项工作的目标是建立一个与所有现有数据一致的EGFR/ErbB2激活模型，并了解EGF受体介导的信号转导中配体特异性差异的分子基础。为此，这笔赠款的具体目的是：1)建立EGFR/ErbB2异二聚体激活和磷酸化的准确模型；2)检验不同EGF受体配体诱导EGF受体不同构象的假设；3)量化EGF受体最早信号反应中不同配体特异性的差异。我们将使用放射性配基结合以及分子成像和传统的激酶分析来验证我们的假设。这些研究将为ErbB靶向疗法的开发和利用提供更准确和更全面的基础，以此作为研究战略和临床选择的基础。

DESCRIPTION (provided by applicant): The EGF receptor is a classical receptor tyrosine kinase with an extracellular ligand binding domain and an intracellular tyrosine kinase domain. Binding of EGF induces dimerization of the EGF receptor with itself or with other members of the ErbB family of receptor tyrosine kinases with ErbB2 being the preferred dimerization partner. Dimerization of the EGF receptor leads to the stimulation of its intracellular tyrosine kinase activity via the formation of an asymmetric kinase dimer. In this asymmetric dimer, one kinase domain serves as the activator of the other kinase domain. As a result of this mechanism, only one kinase domain can be activated at a time in the receptor dimer. According to the current model, phosphorylation of the receptor then occurs in trans and only takes place in the context of receptor dimers. While appealingly simple, this model cannot explain all aspects of EGFR homodimer and the EGFR/ErbB2 heterodimer function. The EGF receptor has eight different ligands and they are capable of inducing different responses in the same cell line. The molecular basis for this is unknown but it has been hypothesized that the ligands induce different conformations of the EGF receptor. This hypothesis has not been experimentally addressed due to the lack of appropriate tools. Using luciferase fragment complementation imaging, we have documented distinct differences in the response of the EGF receptor to its different ligands, indicating that this method is sensitive to ligand-specific changes in EGF receptor conformation. We have also used radioligand binding studies to document more subtle differences in receptor conformation reflected in differences in the allosteric properties of ligand binding. Therapeutic strategies for targeting ErbB receptors hinge on having a full understanding of how these receptors interact with each other and with their ligands and how they are regulated. The goal of the proposed work is to develop a model of EGFR/ErbB2 activation that is consistent with all extant data and to understand the molecular basis for ligand-specific differences in EGF receptor-mediated signaling. To this end, the specific aims of this grant are to: 1) Develop an accurate model for the activation and phosphorylation in the EGFR/ErbB2 heterodimer; 2) Test the hypothesis that different EGF receptor ligands induce different conformations of the EGF receptor; 3) Quantify ligand-specific differences in the earliest signaling response of the EGF receptor. We will use radioligand binding as well as molecular imaging and traditional kinase assays to test our hypotheses. These studies will provide a more accurate and comprehensive foundation upon which to base research strategies and clinical choices for the development and utilization of ErbB-targeted therapeutics.