STRUCTURAL AND BIOCHEMICAL STUDIES OF PROTEIN TYROSINE PHOSPHATASE FUNCTION

描述(由申请人提供)： 长期以来，受体蛋白酪氨酸磷酸酶(RPTPs)在细胞黏附和细胞信号转导中起着关键作用。然而，关于它们的胞外结合伙伴以及细胞黏附和细胞内磷酸酶活性之间的功能联系的信息缺乏，目前我们对这些受体如何介导神经组织发育所需的胞外结合和酪氨酸去磷酸化事件的理解存在很大差距。在RPTPs中，PTPRZ(RPTP2/PTP6)主要在胶质细胞上表达，并与多种结合伙伴有关，如细胞外基质中的tenascin-C和神经元上的Contactin1。PTPRZ和Conactin1之间的相互作用诱导表达PTPRZ的细胞中的突起生长并降低磷酸酪氨酸水平。有趣的是，PTPRZ和Contactin1之间的相互作用在tenascin-C的存在下受到损害，这揭示了这些蛋白之间以及神经元和神经胶质细胞之间存在复杂的相互作用。然而，这些相互作用的结构基础仍然不清楚。我们的长期目标是剖析细胞黏附和细胞信号的机制，这是构建神经网络的基础。这项建议的目的是为PTPRZ与其结合伙伴的结合提供结构基础，这是确定涉及PTPRZ的细胞黏附复合体功能的先决条件第一步。这项研究的理论基础是，涉及PTPRZ的受体-配体对的结构特征将代表着在理解RPTP介导的细胞黏附方面取得的重大进展。我们的初步数据证明了我们的结构方法的可行性，我们提出了以下具体目标来满足详细了解RPTP介导的细胞黏附结构方面的需要：(1)确定PTPRZ与Conactin1之间复合体的晶体结构，为这两种蛋白质介导的黏附相互作用提供结构基础；(2)分析Conactin1结合对PTPRZ的寡聚态及其细胞内磷酸酶活性的影响；(3)通过确定PTPRZ-tenascin-C和Conactin1-tenascin-C复合体的晶体结构，建立tenascin-C干扰PTPRZ和CNTN1之间结合的机制。我们的贡献是重大的，因为我们将能够可视化涉及RPTP的细胞黏附复合体中的蛋白质界面，产生关于配体-受体特异性的宝贵信息，以及在配体结合时发生的潜在构象变化和/或寡聚体状态的变化。这些结构性的见解将为RPTP功能的生化研究奠定基础。总体而言，这些研究将与NIH的任务相关，因为它们将提供支持神经系统发育和维护的细胞黏附事件的更清晰的分子图像，并最终阐明细胞黏附与RPTPs介导的细胞信号之间的关系。 公共卫生相关性： 酪氨酸激酶和酪氨酸磷酸酶之间的持续拉锯战调节着胚胎发育和成年期的关键信号事件。在这个应用中，我们建议确定一个名为PTPRZ的受体蛋白酪氨酸磷酸酶与其两个细胞外结合伙伴之间相互作用的结构基础。这些研究的完成将是确定神经网络构建过程中调节磷酸酪氨酸信号水平的机制的重要一步。这项工作与公众健康相关，因为它为从分子上理解控制神经系统发育的粘连和信号事件奠定了基础。

DESCRIPTION (provided by applicant): It has long been appreciated that receptor protein tyrosine phosphatases (RPTPs) play key roles in cell adhesion and cell signaling. However, the lack of information on their extracellular binding partners and on the functional link between cell adhesion and intracellular phosphatase activity is currently a substantial gap in our understanding of how these receptors mediate the extracellular binding and tyrosine dephosphorylation events necessary for the development of neural tissues. Among RPTPs, PTPRZ (RPTP2/PTP6) is expressed predominantly on glial cells and has been associated with multiple binding partners such as tenascin-C in the extracellular matrix and contactin1 on neurons. Interactions between PTPRZ and contactin1 induce neurite outgrowth and reduce phosphotyrosine levels in cells expressing PTPRZ. Interestingly, the interactions between PTPRZ and contactin1 are impaired in the presence of tenascin-C, revealing the presence of an intricate interplay between these proteins and the interactions between neurons and glial cells. However, the structural basis for these interactions remains unclear. Our long-term goal of is to dissect the mechanisms of cell adhesion and cell signaling that underlie the construction of neural networks. The objective of this proposal is to provide a structural basis for the binding of PTPRZ to its binding partners, which is a prerequisite first step towards defining the function of the cell adhesion complexes involving PTPRZ. The rationale for the research proposed here is that structural characterizations of receptor-ligand pairs involving PTPRZ would represent a significant progress towards understanding RPTP-mediated cell adhesion. Our preliminary data demonstrate the feasibility of our structural approach and we propose the following specific aims to fulfill the need for a detailed understanding of the structural aspects of RPTP-mediated cell adhesion: (1) to determine the crystal structure of the complex between PTPRZ and contactin1 to provide a structural basis for the adhesive interactions mediated by these two proteins, (2) to analyze the effect of contactin1 binding on the oligomeric state of PTPRZ and its intracellular phosphatase activity and (3) to establish the mechanism by which tenascin-C interferes with the binding between PTPRZ and CNTN1 by determining the crystal structures of PTPRZ-tenascin-C and contactin1-tenascin-C complexes. Our contribution is significant because we will be able to visualize protein interfaces in cell adhesion complexes involving RPTPs, yielding precious information about ligand-receptor specificity as well as potential conformational changes and/or changes in oligomeric states that occur upon ligand binding. These structural insights will lay the foundations upon which biochemical studies of RPTP function will be built. Overall, these studies will be relevant to the mission of NIH because they will provide a clearer molecular picture of the cell adhesion events that underpin the development and maintenance of the nervous system and ultimately illuminate the relationship between cell adhesion and cell signaling mediated by RPTPs. PUBLIC HEALTH RELEVANCE: The incessant tug of war between tyrosine kinases and tyrosine phosphatases regulates critical signaling events in embryogenesis and adulthood. In this application, we propose to determine the structural basis for the interactions between a receptor protein tyrosine phosphatase called PTPRZ and two of its extracellular binding partners. Completion of these studies will constitute an important step in defining the mechanisms that regulate the levels of phosphotyrosine signaling during the construction of neural networks. This work is relevant to public health because it lays the foundation for a molecular understanding of the adhesive and signaling events that govern the development of the nervous system.