Structural Mechanisms Underlying the Activity Regulation of the Receptor-like Protein Tyrosine Phosphatase, CD148/PTPRJ

受体样蛋白酪氨酸磷酸酶 CD148/PTPRJ 活性调节的结构机制

• 批准号: 10391480
• 负责人: Jieqing Zhu
• 金额: $ 33万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10391480
• 关键词: Affect; Antiplatelet Drugs; Biochemical; Biology; Biotinylation; Blood Platelets; CRISPR/Cas technology; Cell Line; Cell Surface Proteins; Cell model; Cell physiology; Cell surface; Cells; Clinical; Communication; Crystallization; Crystallography; Data; Development; Differentiation and Growth; Dimerization; Disease; Electron Microscopy; Enzymes; Event; Excision; Extracellular Domain; Family; Family member; Genes; Genetic Polymorphism; Hemostatic function; Human; Immune; In Situ; Individual; Inflammation; Investigation; Investigational Therapies; Label; Ligand Binding; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Megakaryocytes; Methods; Molecular; Nuclear Magnetic Resonance; PTPRJ gene; Patients; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Platelet aggregation; Process; Protein Dephosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Proteomics; Reagent; Receptor Protein-Tyrosine Kinases; Regulation; Research; Resolution; Rest; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Structure; Technology; Therapeutic; Thrombosis; Transmembrane Domain; Tyrosine; Tyrosine Phosphorylation; angiogenesis; base; biophysical techniques; cancer cell; cell growth; chemical bond; comparative; crosslink; design; design and construction; dimer; disulfide bond; extracellular; glycosylation; improved; induced pluripotent stem cell; innovation; member; monomer; mouse model; new therapeutic target; novel; novel strategies; receptor; receptor function; stem cells; therapeutic target

SUMMARY - Protein tyrosine phosphorylation and dephosphorylation, which are balanced by counteracting protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are essential for molecular communications in signal transduction cascades. The receptor-like PTPs (RPTPs) are a family of cell surface PTPs containing a usually large extracellular domain, a single-pass transmembrane domain, and either a single or tandem cytoplasmic phosphatase domain. The functions of RPTPs are attributable to both catalytic activities and extracellular interactions, resembling that of receptor tyrosine kinases (RTKs). However, our molecular understanding of RPTP activity regulation is far from complete compared with that of the structurally and functionally well-characterized RTKs. Structural studies focused on phosphatase domains failed to define generalizable and conclusive mechanisms. We have been engaged in structural and functional analysis of an important RPTP family member, namely CD148/PTPRJ, which is the most abundant RPTP in platelets and megakaryocytes and has an established positive role in platelet aggregation, an essential process for hemostasis and thrombosis. Our preliminary biochemical and structural studies of CD148 have yielded many intriguing observations that begin to define the structural basis of CD148 activity regulation, supporting our hypothesis that dimerization of RPTPs is largely attributable to extracellular and transmembrane domains that govern dimer formation of the phosphatase domain and regulation of catalytic activity. Using innovative construct designs and a novel human megakaryocyte progenitor cell line derived from induced pluripotent stem (iPS) cells, we will examine how the CD148 activity is regulated by dimerization and ligand binding, and how these regulations affect the function of human megakaryocytes and platelets. Using the recently developed proximity-dependent labeling method, we will perform proteomic profiling of CD148 substrates in both resting and activated human megakaryocytes and platelets. Using a combination of crystallographic, electron microscopy, nuclear magnetic resonance, and other multifaceted biochemical and biophysical approaches, we will define the structural basis of CD148 activity regulation through the size of ectodomain and the dimerization mediated by the extracellular and transmembrane domains, and the structural basis of ligand binding. The effect of individual domains, N- linked glycosylation, specific disease-associated polymorphisms, and ligand binding on the structure, dimerization, and activity of CD148 will be examined. These complementary Specific Aims will advance our understanding of the molecular basis for the regulation of CD148 PTP activity and will facilitate the development of novel strategies for modulating CD148 function by selectively targeting either dimerization or ligand binding in the treatment of diseases such as thrombosis and cancer. Approaches established in this study will be readily applicable to other members of the RPTP family, which will improve our family-wide understanding of the molecular mechanisms of RPTP activity regulation.

摘要-蛋白质酪氨酸磷酸化和去磷酸化，这是平衡的抵消 蛋白酪氨酸激酶（PTK）和蛋白酪氨酸磷酸酶（PTPs），是分子生物学中必需的。 信号转导级联中的通信。受体样PTP（receptor-like PTPs，RPTPs）是细胞表面的一个家族， PTPs通常含有一个大的胞外结构域，一个单通道跨膜结构域，或一个单通道跨膜结构域。 或串联胞质磷酸酶结构域。RPTP的功能可归因于两种催化活性 和细胞外相互作用，类似于受体酪氨酸激酶（RTK）。然而，我们的分子 对RPTP活性调节的理解与对结构和 功能良好的RTK。结构研究集中在磷酸酶域未能定义 可概括的和结论性的机制。我们一直在从事结构和功能分析， 重要的RPTP家族成员，即CD 148/PTPRJ，其是血小板中最丰富的RPTP， 巨核细胞，并在血小板聚集（止血的基本过程）中发挥积极作用 和血栓形成。我们对CD 148的初步生化和结构研究已经产生了许多有趣的结果。 这些观察开始定义了CD 148活性调节的结构基础，支持我们的假设， RPTP的二聚化主要归因于控制二聚体的细胞外和跨膜结构域 磷酸酶结构域的形成和催化活性的调节。使用创新的结构设计， 一种新的人巨核细胞祖细胞系来源于诱导多能干细胞（iPS），我们将 研究CD 148活性是如何通过二聚化和配体结合来调节的，以及这些调节是如何进行的。 影响人巨核细胞和血小板的功能。使用最近开发的邻近依赖 标记的方法，我们将进行蛋白质组学分析的CD 148底物在静息和活化的人 巨核细胞和血小板。结合晶体学，电子显微镜，核磁共振 共振和其他多方面的生物化学和生物物理方法，我们将定义结构基础 CD 148活性通过胞外域的大小和由细胞外膜介导的二聚化调节 和跨膜结构域，以及配体结合的结构基础。单个域的影响，N- 连接的糖基化，特定疾病相关的多态性，和结构上的配体结合， 将检查CD 148的二聚化和活性。这些互补的具体目标将促进我们的 了解CD 148 PTP活性调节的分子基础，将有助于开发 通过选择性靶向二聚化或配体结合来调节CD 148功能的新策略， 治疗血栓和癌症等疾病。本研究中建立的方法将很容易 适用于RPTP家族的其他成员，这将提高我们整个家族对 RPTP活性调节的分子机制。