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，通过抵消来平衡 蛋白酪氨酸激酶（PTK）和蛋白质酪氨酸磷酸酶（PTPS）对于分子至关重要 信号转导级联中的通信。受体样PTP（RPTP）是细胞表面的家族 PTP包含一个通常大的细胞外域，单个跨跨膜结构域和一个单一的PTP 或串联细胞质磷酸酶结构域。 RPTP的功能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）。但是，我们的分子 与结构上的RPTP活动调节的理解远非完整 功能良好的RTK。集中于磷酸酶结构域的结构研究未能定义 可概括和结论性的机制。我们一直从事结构和功能分析 重要的RPTP家庭成员，即CD148/PTPRJ，这是血小板中最丰富的RPTP 巨核细胞，在血小板聚集中具有既定的积极作用，这是止血的基本过程 和血栓形成。我们的CD148的初步生化和结构研究产生了许多有趣的 开始定义CD148活性调节的结构基础的观察结果，支持我们的假设 RPTP的二聚化在很大程度上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。使用创新的构造设计和 一种新型的人类巨核细胞祖细胞细胞系，衍生自诱导的多能茎（IPS）细胞，我们将 检查CD148活性如何通过二聚化和配体结合以及这些法规如何调节 影响人类巨核细胞和血小板的功能。使用最近开发的接近度依赖性 标记方法，我们将在静止和激活的人类中对CD148底物进行蛋白质组学分析 巨核细胞和血小板。结合晶体学，电子显微镜，核磁 共鸣以及其他多方面的生化和生物物理方法，我们将定义结构基础 CD148的活性调节，通过胞外域的大小和细胞外介导的二聚化调节 和跨膜结构域，以及配体结合的结构基础。单个领域的影响，n- 连接的糖基化，特定疾病相关的多态性和配体与结构的结合， 将检查二聚化和CD148的活性。这些互补的特定目标将推动我们的 了解CD148 PTP活性调节的分子基础，并将促进发展 通过选择性靶向二聚化或配体结合，以调节CD148功能的新型策略 血栓形成和癌症等疾病的治疗。本研究中建立的方法很容易 适用于RPTP家族的其他成员，这将提高我们对家庭的理解 RPTP活性调节的分子机制。