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

描述（由申请人提供）：普遍表达的衔接蛋白Grb 2对于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。沿着其在活化的受体和衔接蛋白下游传播信号传导途径中的作用，Grb 2介导的复合物的失调形成与人类中的许多病理状况有关，包括癌症、糖尿病、自身免疫性疾病、过敏/哮喘和心血管疾病。Grb 2由Src同源2（SH 2）结构域组成，其结合特异性磷酸化酪氨酸，侧翼为两个Src同源3（SH 3）结构域，其与富含脯氨酸的序列缔合。Grb 2与SH 2和SH 3结构域配体的相互作用已被广泛研究。因此，Grb 2是我们理解SH和SH 3结构域蛋白的激活和功能的主要模型系统。尽管这些研究提供了对Grb 2和其他重要信号蛋白的潜在配体和结合机制的深入了解，但它们尚未明确解决几个关键问题。如果我们要充分了解Grb 2和其他相关蛋白质如何调节临床相关复合物的形成和功能，那么对这些问题的研究是很重要的。我们的长期目标是表征含SH 2和SH 3结构域的蛋白如何控制临床相关信号传导复合物的形成和功能，以促进与这些复合物的失调形成相关的疾病的治疗方案的开发。这个具体的建议的目的是全面检查Grb 2介导的复合物，形成在SH 2结构域配体，衔接蛋白LAT。我们的中心工作假设是Grb 2与LAT和单个SH 3结构域配体形成合作相互作用，这些配体控制这些关键的多蛋白信号复合物的形成和功能。为了验证这一假设，我们将1）从分子上表征Grb 2与SH 3结构域配体Sos 1、HPK 1和c-Cbl的相互作用，2）确定驱动特定Grb 2 SH 3结构域配体与LAT结合的力，3）确定SH 3结构域配体在Grb 2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物化学技术，包括我们的新的，更强大的等温滴定量热法和沉降速度分析超离心分析方法。从这些研究中获得的信息将使我们能够显着推进我们对Grb 2和其他相关蛋白质与SH 2和SH 3结构域配体结合机制的理解。这不仅将提供对SH 2和SH 3结构域介导的信号传导复合物的形成的新见解，而且还将促进用于治疗使人衰弱的疾病的新型治疗剂的生产和/或使用。 公共卫生相关性：这项提案将研究如何合作的蛋白质-蛋白质相互作用控制的特异性和结合能力的SH 2和SH 3结构域的普遍表达的衔接蛋白Grb 2。从本研究中获得的知识将有助于我们了解Grb 2和其他相关临床重要蛋白的激活和功能，预计将为合理设计和/或使用这些蛋白的抑制剂提供关键信息。这些试剂将可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。

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.