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

摘要-蛋白质酪氨酸磷酸化和去磷酸化，通过中和来平衡 蛋白酪氨酸激酶(PTKs)和蛋白酪氨酸磷酸酶(PTPs)是分子生物学中不可缺少的一类。 信号转导中的通信级联。受体样蛋白(RPTPs)是一类细胞表面蛋白 含有通常较大的胞外区、单次通过的跨膜区和单一的 或串联胞浆磷酸酶结构域。RPTP的功能归因于这两种催化活性 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 与RPTP活性调节的结构和结构相比，对RPTP活性调节的理解还远远不完整 功能特征良好的RTK。专注于磷酸酶结构域的结构研究未能定义 具有概括性和结论性的机制。我们一直致力于一个结构和功能分析 RPTP家族的重要成员，即CD148/PTPRJ，是血小板和组织中含量最丰富的RPTP 巨核细胞在血小板聚集中发挥积极作用，这是止血的基本过程。 和血栓形成。我们对CD148的初步生化和结构研究产生了许多有趣的结果 开始定义CD148活性调节的结构基础的观察，支持我们的假设 RPTPs的二聚化很大程度上归因于控制二聚体的胞外和跨膜结构域 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 一种新的从诱导多能干细胞(IPS)细胞来源的人巨核祖细胞系，我们将 研究CD148活性是如何通过二聚化和配体结合来调节的，以及这些调节如何 影响人类巨核细胞和血小板的功能。利用最近发展起来的邻近度依赖 标记法，我们将在静息和激活的人中进行CD148底物的蛋白质组学分析 巨核细胞和血小板。使用结晶学、电子显微镜、核磁学 共振，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 胞外结构域大小和胞外二聚化对CD148活性的调节 和跨膜结构域，以及配体结合的结构基础。单个区域的影响，N- 连接的糖基化，特定的疾病相关的多态性，以及结构上的配体结合， 二聚作用和CD148的活性将被检测。这些互补的具体目标将推动我们的 了解CD148 PTP活性调节的分子基础将促进CD148 PTP活性的发展 通过选择性靶向二聚化或配体结合来调节CD148功能的新策略 血栓形成和癌症等疾病的治疗。在这项研究中建立的方法将很容易 适用于RPTP大家庭的其他成员，这将提高我们对RPTP家庭成员的了解 RPTP活性调控的分子机制。

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.