Intrinsic Disorder and Agonist Bias in EGF Receptor Signaling

摘要 EGF 受体 (EGFR) 是一种细胞表面受体酪氨酸激酶，经常发生突变或过度突变。 在多种肿瘤中表达。 EGF 对受体的刺激导致其细胞内的激活 酪氨酸激酶，其自身磷酸化其本质上无序的 C 末端尾上的多个酪氨酸。 这些磷酸酪氨酸作为包含 SH2 和 PTB 结构域的蛋白质的结合位点，介导 EGF 的下游效应。 SH2 和 PTB 结构域通过短线性基序识别其结合位点 其中嵌入了 pTyr。在初步实验中，我们发现这些区域之外的区域 短基序有助于 SH2 和 PTB 结构域蛋白的结合。重要性的进一步证据 SH2 结构域与 EGFR 尾部结合中更远的序列源自我们的初步研究 计算机建模研究表明，当 Grb2 SH2 结构域与 Tyr-1068 结合时，序列 Tyr-1148 周围是 EGFR 自身磷酸化的主要位点，与二级结合相互作用 SH2 结构域上的表面。这种相互作用限制了 Tyr-1148 与其伙伴蛋白结合的可用性。 因此，我们假设远离 pTyr 基序的尾部区域参与了 SH2 的结合 结构域蛋白，这些扩展的相互作用改变了尾部的构象整体并改变 它以整体变构的形式与其他伙伴互动的能力。 EGFR 可以被七种不同配体中的任何一种激活，从而引发不同的长期效应。 应用于同一细胞时的生物效应。用两种不同的配体刺激同一细胞可以 产生两种完全不同的生物学结果。我们已经证明了能力上的差异 不同的配体诱导受体同源和异源二聚化并结合下游信号蛋白。 在初步研究中，我们还记录了它们刺激糖酵解通量的能力的差异，这是一个关键 快速生长细胞的代谢特征。已知不同的肿瘤优先被转化 结构上不同形式的 EGFR 并表达不同的 EGFR 配体。在许多情况下，临床 结果已被证明与配体的表达相关。我们假设结构上 不同的致癌 EGFR 在功能上有所不同，并且 EGFR 变体和配体的某些组合 比其他人更具变革性。我实验室的目标是阐明基本原理 通过受体酪氨酸激酶进行潜在信号传导，并应用此来了解突变型 EGFR 如何诱导 肿瘤。该提案的具体目标是： 1. 确定酪氨酸间序列如何调节 SH2 结构域蛋白与 EGFR C 末端尾部的结合； 2) 识别功能差异 致癌 EGFR 及其对 EGFR 激动剂的反应与转化如何相关 建立并维持。

Abstract The EGF receptor (EGFR) is a cell surface receptor tyrosine kinase that is frequently mutated or over- expressed in a variety of tumors. Stimulation of the receptor by EGF leads to the activation of its intracellular tyrosine kinase which autophosphorylates multiple tyrosines on its intrinsically-disordered C-terminal tail. These phosphotyrosines serve as binding sites for the SH2 and PTB domain-containing proteins that mediate the downstream effects of EGF. SH2 and PTB domains recognize their binding sites via a short linear motif in which the pTyr is embedded. In preliminary experiments, we have found that regions well outside of these short motifs contribute to the binding of SH2 and PTB domain proteins. Further evidence for the importance of more distant sequences in the binding of SH2 domains to the EGFR tail was derived from our preliminary computer modeling studies that show that when the Grb2 SH2 domain is bound to Tyr-1068, the sequence surrounding Tyr-1148, the major site of autophosphorylation of the EGFR, interacts with a secondary binding surface on the SH2 domain. This interaction limits the availability of Tyr-1148 to bind to its partner proteins. We therefore hypothesize that regions of the tail distant from the pTyr motif participate in the binding of SH2 domain proteins and that these extended interactions modify the conformational ensemble of the tail and alter its ability to interact with other partners in a form of ensemble allostery. The EGFR can be activated by any one of seven different ligands, which can elicit different long-term biological effects when applied to the same cell. Stimulation of the same cell with two different ligands can give rise to two completely different biological outcomes. We have demonstrated differences in the ability of different ligands to induce receptor homo- and hetero-dimerization and to bind downstream signaling proteins. In preliminary studies, we have also documented differences in their ability to stimulate glycolytic flux, a key metabolic feature of rapidly growing cells. Different tumors are known to be preferentially transformed by structurally different forms of the EGFR and to express different EGFR ligands. In many instances, clinical outcome has been shown to correlate with which ligand is expressed. We hypothesize that the structurally diverse oncogenic EGFRs are functionally different and that certain combinations of EGFR variant and ligand are more transforming than others. The goal of my laboratory is to elucidate the fundamental principles underlying signaling via receptor tyrosine kinases and to apply this to understand how mutant EGFRs induce tumors. The specific aims of this proposal are to: 1. Determine how inter-tyrosine sequences modulate the binding of SH2 domain proteins to the C-terminal tail of the EGFR; and, 2) Identify functional differences among oncogenic EGFRs and their response to EGFR agonists that are relevant to how transformation is established and maintained.