SIGNAL TRANSDUCTION BY ERBB2/ERBB3 OLIGOMERS

ErbB受体的酪氨酸激酶活性通过激动剂配体的结合而刺激， 通过受体亚基的二聚化。亚基的磷酸化在磷酸化的背景下反式发生。 组装二聚体。ErbB 2/ErbB 3异二聚体是ErbB的最高度转化组合 受体。这是出乎意料的，因为ErbB 2具有激酶活性，但不结合配体，而ErbB 3结合配体。 配体，但激酶受损。最近的研究表明，除了传统的背靠背 二聚体，这对形成异源四聚体，允许一个异源二聚体中的ErbB 2磷酸化ErbB 2 在四聚体结构的另一个异二聚体中。这种二聚体间的磷酸化作用是蛋白质合成所必需的。 通过含SH 2和PTB结构域的蛋白质与C-末端的结合介导的信号激活 ErbB 2的尾部。除了形成更高级低聚物的要求之外，本发明的其它特征也是有利的。 ErbB 2/ErbB 3系统与当前范例中描述的系统不同，表明需要更多的 该模型结合了ErbB受体信号传导的日益复杂性。此外，委员会认为， 虽然很清楚ErbB受体的C-末端尾募集磷酸酪氨酸结合蛋白， 信号传导中这一步骤的结构和功能细节完全未知。的目标 拟议的研究是了解ErbB 2和ErbB 3的不同寡聚状态是如何形成的， 它们如何调节输出信号，并提供对结构的分子理解， C-末端尾部的动力学，因为它被磷酸化并结合蛋白质以启动下游信号传导 事件为此，该补助金的具体目标是：1）开发一个模型，准确描述 并预测ErbB 2和ErbB 3的低聚态之间的动力学，并确定这是如何 这些受体中的致瘤突变改变了动态; 2）！描述ErbB 2/ErbB 3二聚体的作用 和四聚体在信号转导中的作用;以及，3）发展对C-末端尾如何 ErbB 2介导下游信号传导。这些目标将通过结合体内、体内和体外试验来实现。 体外和计算机模拟分析。特别地，我们将使用ErbB 2的C-末端尾部的原子模拟， 产生关于磷酸化和SH 2与含PTB结构域的 蛋白质改变了尾巴的局部和整体结构。这些计算机模拟的预测将使用一种新的 荧光素酶片段互补技术，使我们能够监测特定蛋白质的结合， ErbB受体的C末端尾部它们还将在体外通过尾部的质谱足迹进行测试 在不存在和存在磷酸酪氨酸结合蛋白的情况下磷酸化。SH 2的相互作用 和含有PTB结构域的蛋白质与磷酸酪氨酸残基是一种信号传导机制， 激素和生长因子的宿主。在这些研究中， 将提供普遍适用于许多不同信令系统的信息。 !

The tyrosine kinase activity of ErbB receptors is stimulated by the binding of agonist ligand, followed by dimerization of the receptor subunits. Phosphorylation of the subunits occurs in trans in the context of the assembled dimer. The ErbB2/ErbB3 heterodimer is the most highly transforming combination of ErbB receptors. This is unexpected as ErbB2 is kinase active but does not bind a ligand while ErbB3 binds a ligand but is kinase-impaired. Recent studies have suggested that in addition to the traditional back-to-back dimers, this pair forms heterotetramers that allow the ErbB2 in one heterodimer to phosphorylate the ErbB2 in the other heterodimer across the tetrameric structure. This inter-dimer phosphorylation is required for the activation of signals mediated by the binding of SH2 and PTB domain-containing proteins to the C-terminal tail of ErbB2. Beyond requirement for the formation of higher order oligomers, other features of the ErbB2/ErbB3 system differ from those described in the current paradigm, indicating the need for a more refined model that incorporates the increasing complexity of signaling by ErbB receptors. Furthermore, while it is clear that the C-terminal tails of ErbB receptors recruit phosphotyrosine binding proteins, the structural and functional specifics of this step in signaling are completely unknown. The goal of the proposed research is to understand how the different oligomeric states of ErbB2 and ErbB3 are formed and how they regulate the output signal, and to provide a molecular understanding of the structure and dynamics of the C-terminal tail as it is phosphorylated and binds proteins to initiate downstream signaling events. To this end, the specific aims of this grant are to: 1) Develop a model that accurately describes and predicts the dynamics among the oligomeric states of ErbB2 and ErbB3 and determine how this dynamic is altered by tumorigenic mutations in these receptors; 2)!Delineate the role of ErbB2/ErbB3 dimers and tetramers in signal transduction; and, 3) Develop a molecular understanding of how the C-terminal tail of ErbB2 mediates downstream signaling. These aims will be addressed using a combination of in vivo, in vitro and in silico analyses. In particular, we will use atomistic simulations of the C-terminal tail of ErbB2 to generate hypotheses regarding how phosphorylation and the binding of SH2 and PTB domain-containing proteins alter the local and global structure of the tail. These in silico predictions will be tested using a novel luciferase fragment complementation technique that allows us to monitor the binding of specific proteins to the C-terminal tails of ErbB receptors. They will also be tested in vitro by mass spec footprinting of the tails phosphorylated in the absence and presence of phosphotyrosine binding proteins. The interaction of SH2 and PTB domain-containing proteins with phosphotyrosine residues is a signaling mechanism used by a host of hormones and growth factors. The mechanistic insight into this process developed in these studies will provide information universally applicable to many different signaling systems. !