Spectroscopic Studies of EGF-Receptor Interactions

描述(申请人提供)：该项目结合了两个现有的膜蛋白生物物理和生物化学实验室的资源，研究表皮生长因子(EGF)受体与其配体以及与ErbB受体激酶家族的其他受体之间的分子间相互作用的细节。ErbB受体家族，特别是EGF受体，是研究最广泛的多肽激素受体之一，因为它们在正常发育、伤口愈合以及当突变或过度表达解除调控时与肿瘤有关。然而，关于EGF配体家族与ErbB受体家族结合所触发的信号转导机制的基本问题仍未得到解决。这个项目的长期目标是深入了解当EGF家族配体与其各自的受体结合时，信号转导途径中发生的最早事件。本预算期间的具体目标将利用具有良好特性的荧光配体、已被证明能够承受湍流停流快速混合并在没有内源性ErbB受体的情况下表达特定ErbB家族受体的新细胞系，以及专门为这些研究设计和优化的定制停流荧光仪。研究将涉及以下假设：a)ErbB家族其他成员的表达影响处于高亲和力和低亲和力状态的EGF受体的比例；b)当受体存在于同一细胞中时，ErbB3或ErbB4识别的配体可以调节EGF与其受体的结合；c)受体结合的动态开启和关闭速率是决定多肽激素有丝分裂活性的重要参数；以及d)糖基化的异质性可以影响受体对EGF的亲和力。研究还将检验关于EGF受体和ErbB2之间相互作用的相互竞争的假说：当EGF与受体结合时，EGF受体-ErbB2二聚体直接形成，或者EGF受体在结合EGF时二聚，随后招募ErbB2。

DESCRIPTION (provided by applicant): This project combines resources of two established laboratories in membrane protein biophysics and biochemistry to study details of the intermolecular interactions of the epidermal growth factor (EGF) receptor with its ligands and with other receptors of the ErbB family of receptor kinases. The ErbB family of receptors, and, in particular, the EGF receptor, are among the most widely studied polypeptide hormone receptors because of their importance in normal development, wound healing, and, when deregulated by mutation or over-expression, neoplasia. Nonetheless, fundamental questions concerning the mechanisms of signal transduction triggered by the binding of the EGF family of ligands to the ErbB family of receptors remain unresolved. The long term goal of this project is to develop an in-depth understanding of the earliest events in the signal transduction pathway that occur when EGF family ligands bind to their respective receptors. The specific aims for this budget period will exploit well characterized fluorescent ligands, new cell lines that have been shown to withstand turbulent stoppedflow rapid mixing and that express selected ErbB family receptors in the absence of endogenous ErbB receptors, as well as a custom stopped-flow fluorometer specifically designed and optimized for these studies. Studies will address hypotheses a) that expression of other ErbB family members affects the proportion of EGF receptors in high and low affinity states, b) that ligands recognized by ErbB3 or ErbB4 can modulate the binding of EGF to its receptor when the receptors are present in the same cell, c) that the dynamic on and off rates for receptor binding are important parameters for determining the mitogenic activity ofpeptide hormones, and d) that heterogeniety in glycosylation can affect receptor affinity for EGF. Studies will also test competing hypotheses concerning the interactions between the EGF receptor and ErbB2: that EGF receptor-ErbB2 dimers form directly when EGF binds to the receptor, or that EGF receptors dimerize on binding EGF and subsequently recruit ErbB2.