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

SH2 和 SH3 结构域介导的接头蛋白 Grb2 上的协同相互作用

• 批准号: 8795270
• 负责人: Jon C.D. Houtman
• 金额: $ 5.47万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795270
• 关键词: Adaptor Signaling Protein; Address; Affinity; Asthma; Autoimmune Diseases; Binding; Biochemical; Biochemistry; Biological Models; Biophysics; Calorimetry; Cardiovascular Diseases; Complex; Computer Simulation; Consensus; Conserved Sequence; Data; Diabetes Mellitus; Disease; Exhibits; Future; Goals; Graft Rejection; Growth; Health; Human; Hypersensitivity; Individual; Iowa; Knowledge; Lead; Ligand Binding; Ligands; Link; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Multiprotein Complexes; Organ Transplantation; Physiological Processes; Production; Proline; Protein Biochemistry; Proteins; Publishing; Regimen; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; System; T-Lymphocyte; TCR Activation; Techniques; Testing; Therapeutic; Therapeutic Agents; Titrations; Tyrosine; Universities; Work; analytical ultracentrifugation; base; cell type; clinically relevant; design; driving force; examination questions; human disease; in vivo; inhibitor/antagonist; innovation; insight; novel therapeutics; programs; protein activation; protein protein interaction; receptor; research study; sedimentation velocity; src Homology Domains; src Homology Region 2 Domain; stoichiometry; therapy development

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.

描述（由申请人提供）：普遍表达的衔接蛋白 Grb2 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。除了其在传播激活受体和衔接蛋白下游信号通路中的作用之外，Grb2介导的复合物形成失调与人类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。 Grb2 由一个 Src 同源 2 (SH2) 结构域组成，该结构域结合特定的磷酸化酪氨酸，两侧是两个 Src 同源 3 (SH3) 结构域，与富含脯氨酸的序列相关。 Grb2 与 SH2 和 SH3 结构域配体的相互作用已被广泛研究。因此，Grb2 是我们了解含有 SH 和 SH3 结构域的蛋白质的激活和功能的主要模型系统。尽管这些研究深入了解了潜在的配体以及 Grb2 和其他重要信号蛋白的结合机制，但它们尚未明确解决几个关键问题。如果我们要充分了解 Grb2 和其他相关蛋白如何调节临床相关复合物的形成和功能，对这些问题的研究非常重要。我们的长期目标是表征包含 SH2 和 SH3 结构域的蛋白质如何控制临床相关信号复合物的形成和功能，以促进与这些复合物形成失调相关的疾病的治疗方案的开发。该具体提案的目的是全面检查在 SH2 结构域配体（接头蛋白 LAT）上形成的 Grb2 介导的复合物。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相互作用，控制这些关键多蛋白信号复合物的形成和功能。为了检验这一假设，我们将 1) 从分子角度表征 Grb2 与 SH3 结构域配体 Sos1、HPK1 和 c-Cbl 的相互作用，2) 确定驱动特定 Grb2 SH3 结构域配体与 LAT 结合的力，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，我们将采用多种创新、最先进的生物物理和生化技术，包括用于等温滴定量热法和沉降速度分析超速离心的新的、更强大的分析方法。从这些研究中获得的信息将使我们能够显着增进对 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。这不仅将为SH2和SH3结构域介导的信号复合物的形成提供新的见解，而且还将促进用于治疗使人衰弱的人类疾病的新型治疗剂的生产和/或使用。

项目成果

Activated PLC-γ1 is catalytically induced at LAT but activated PLC-γ1 is localized at both LAT- and TCR-containing complexes.

• DOI: 10.1016/j.cellsig.2013.12.022
• 发表时间: 2014-04
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: Cruz-Orcutt N;Vacaflores A;Connolly SF;Bunnell SC;Houtman JC
• 通讯作者: Houtman JC

The adaptor protein LAT serves as an integration node for signaling pathways that drive T cell activation.

• DOI: 10.1002/wsbm.1194
• 发表时间: 2013-01
• 期刊: WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE
• 影响因子: 7.9
• 作者: Bartelt, Rebekah R.;Houtman, Jon C. D.
• 通讯作者: Houtman, Jon C. D.

TCR-mediated functions are enhanced in activated peripheral blood T cells isolated from leucocyte reduction systems.

从白细胞减少系统中分离出的活化外周血 T 细胞的 TCR 介导功能得到增强。

• DOI: 10.1016/j.jim.2014.11.009
• 发表时间: 2015
• 期刊: Journal of immunological methods
• 影响因子: 2.2
• 作者: Tremblay,MikaelaM;Houtman,JonCD
• 通讯作者: Houtman,JonCD