Cooperative SH2 and SH3 domain-mediated interactions at the adaptor protein Grb2

描述(申请人提供)：普遍表达的接头蛋白Grb2对控制许多人类生理过程的信号通路的激活和传播至关重要。除了在激活的受体和接头蛋白下游传播信号通路的作用外，Grb2介导的复合体的失控形成与人类的许多病理疾病有关，包括癌症、糖尿病、自身免疫性疾病、过敏/哮喘和心血管疾病。Grb2由一个Src同源2(SH2)结构域组成，它与特定的磷酸化酪氨酸结合，两侧有两个Src同源3(SH3)结构域，它们与富含Pro的序列相关联。Grb2与SH2和SH3结构域配体的相互作用已被广泛研究。正因为如此，Grb2是我们了解含SH和SH3结构域蛋白的激活和功能的主要模型系统。虽然这些研究已经深入了解了Grb2和其他重要信号蛋白的潜在配体和结合机制，但它们还没有清楚地解决几个关键问题。如果我们要充分了解Grb2和其他相关蛋白质如何调节临床相关复合体的形成和功能，对这些问题的研究是重要的。我们的长期目标是研究含有SH2和SH3结构域的蛋白质如何控制临床相关信号复合体的形成和功能，以促进与这些复合体的失控形成相关的疾病的治疗方案的开发。这一特定建议的目标是全面研究在SH2结构域配体(即适配蛋白LAT)处形成的Grb2介导的复合体。我们的中心工作假设是Grb2与LAT和单个SH3结构域配体形成协同作用，控制这些关键多蛋白信号复合体的形成和功能。为了验证这一假设，我们将1)从分子上表征Grb2与SH3结构域配体Sos1、HPK1和c-Cb1的相互作用，2)确定驱动特定Grb2 SH3结构域配体与LAT结合的作用力，以及3)确定SH3结构域配体在Grb2与LAT相互作用中的作用。为了实现这些目标，我们将采用几种创新的、最先进的生物物理和生化技术，包括我们的新的、更强大的分析方法，用于等温滴定量热法和沉积速度分析超速离心法。从这些研究中获得的信息将使我们对Grb2和其他相关蛋白与SH2和SH3结构域配体的结合机制的理解有很大的进步。这不仅为SH2和SH3结构域介导的信号复合体的形成提供了新的视角，也将有助于生产和/或使用新型治疗药物来治疗人类衰弱的疾病。 与公共卫生相关：这项提案将研究蛋白质-蛋白质相互作用如何控制普遍表达的适配蛋白Grb2的SH2和SH3结构域的特异性和结合能力。本研究所获得的知识将有助于我们了解Grb2和其他相关的临床重要蛋白的激活和功能，这有望为这些蛋白的合理设计和/或抑制剂的使用提供关键信息。这些药物将用于治疗多种人类疾病，包括癌症、心血管疾病、自身免疫性疾病、哮喘/过敏和移植器官的排斥反应。

DESCRIPTION (provided by applicant): The ubiquitously expressed adaptor protein Grb2 is crucial for the activation and propagation of signaling pathways that control many human physiological processes. Along with its role in propagating signaling pathways downstream of activated receptors and adaptor proteins, the disregulated formation of Grb2-mediated complexes has been linked to numerous pathological conditions in humans, including cancer, diabetes, autoimmune disorders, allergies/asthma and cardiovascular disease. Grb2 is composed of a Src homology 2 (SH2) domain, which binds specific phosphorylated tyrosines, flanked by two Src homology 3 (SH3) domains, which associate with proline- rich sequences. The interaction of Grb2 with SH2 and SH3 domain ligands has been extensively examined. Because of this, Grb2 is the primary model system for our understanding of the activation and function of SH and SH3 domain-containing proteins. Although these studies have provided insight into potential ligands and the binding mechanism of Grb2 and other important signaling proteins, they have not clearly addressed several critical questions. An examination of these questions is important if we are to fully understand how Grb2 and other related proteins regulate the formation and function of clinically relevant complexes. Our long-term goal is to characterize how SH2 and SH3 domain- containing proteins control the formation and function of clinically relevant signaling complexes in order to facilitate the develop of therapeutic regimens for diseases linked to the disregulated formation of these complexes. The objective of this specific proposal is to comprehensively examine the Grb2- mediated complexes that form at an SH2 domain ligand, the adaptor protein LAT. Our central working hypothesis is that Grb2 forms cooperative interactions with both LAT and individual SH3 domain ligands that control the formation and function of these critical multiprotein signaling complexes. To test this hypothesis, we will 1) molecularly characterize the interaction of Grb2 with the SH3 domain ligands Sos1, HPK1 and c-Cbl, 2) determine the forces that drive the association of specific Grb2 SH3 domain ligands with LAT and 3) determine the role of SH3 domain ligands in the interaction of Grb2 with LAT. To achieve these aims, we will employ several innovative, state-of-the-art biophysical, and biochemical techniques, including our new, more robust analysis methods for isothermal titration calorimetry and sedimentation velocity analytical ultracentrifugation. The information gained from these studies will allow us to significantly advance our understanding of the binding mechanism of Grb2 and other related proteins to both SH2 and SH3 domains ligands. This will not only provide new insight into the formation of SH2 and SH3 domain-mediated signaling complexes, but will also facilitate the production and/or use of novel therapeutic agents for the treatment of debilitating human diseases. PUBLIC HEALTH RELEVANCE: This proposal will examine how cooperative protein-protein interactions control the specificity and binding capacity of the SH2 and SH3 domains of the ubiquitously expressed adaptor protein Grb2. The knowledge gained from this study will help to us to understand the activation and function of Grb2 and other related clinically important proteins, which is expected to provide critical information for the rational design and/or use of inhibitors for these proteins. These agents will be useful for the treatment of multiple human diseases, including cancer, cardiovascular disease, autoimmune diseases, asthma/ allergies and the rejection of transplanted organs.