STRUCTURAL AND BIOCHEMICAL STUDIES OF PROTEIN TYROSINE PHOSPHATASE FUNCTION

描述（由申请人提供）： 受体蛋白酪氨酸磷酸酶（RPTPs）在细胞粘附和细胞信号转导中起着重要作用。然而，缺乏信息的细胞外结合的合作伙伴和细胞粘附和细胞内磷酸酶活性之间的功能联系，目前是一个很大的差距，我们了解这些受体如何介导的细胞外结合和酪氨酸去磷酸化的神经组织的发展所必需的事件。在RPTP中，PTPRZ（RPTP 2/PTP 6）主要在神经胶质细胞上表达，并与细胞外基质中的生腱蛋白-C和神经元上的接触蛋白1等多种结合伴侣相关。PTPRZ和contactin 1之间的相互作用诱导神经突生长，并降低表达PTPRZ的细胞中的磷酸酪氨酸水平。有趣的是，PTPRZ和接触蛋白1之间的相互作用在生腱蛋白-C的存在下受损，揭示了这些蛋白质之间复杂的相互作用以及神经元和神经胶质细胞之间的相互作用。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粘附和细胞信号传导的机制，这些机制是神经网络构建的基础。本提案的目的是提供一个结构基础的结合PTPRZ的结合伙伴，这是一个先决条件的第一步，以确定涉及PTPRZ的细胞粘附复合物的功能。这里提出的研究的基本原理是，涉及PTPRZ的受体-配体对的结构表征将代表理解RPTP介导的细胞粘附的重大进展。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，以满足对RPTP介导的细胞粘附的结构方面的详细理解的需要：（1）确定PTPRZ和contactin 1之间复合物的晶体结构，为这两种蛋白介导的粘附相互作用提供结构基础，（2）分析contactin 1结合对PTPRZ寡聚状态及其细胞内磷酸酶活性的影响;（3）通过测定PTPRZ-tenascin-C和contactin 1-tenascin-C复合物的晶体结构，建立tenascin-C干扰PTPRZ与CNTN 1结合的机制。我们的贡献是显着的，因为我们将能够可视化涉及RPTP的细胞粘附复合物中的蛋白质界面，产生宝贵的信息配体-受体特异性以及潜在的构象变化和/或寡聚状态的变化，发生在配体结合。这些结构的见解将奠定基础，RPTP功能的生化研究将建立。总体而言，这些研究将与NIH的使命相关，因为它们将提供支持神经系统发育和维持的细胞粘附事件的更清晰的分子图像，并最终阐明细胞粘附和RPTP介导的细胞信号传导之间的关系。 公共卫生相关性： 酪氨酸激酶和酪氨酸磷酸酶之间持续不断的拔河调节胚胎发生和成年期的关键信号事件。在这个应用中，我们建议确定受体蛋白酪氨酸磷酸酶之间的相互作用的结构基础称为PTPRZ和它的两个细胞外结合伙伴。这些研究的完成将构成一个重要的一步，在确定的机制，调节磷酸酪氨酸信号的水平在神经网络的建设。这项工作与公共卫生有关，因为它为从分子水平理解控制神经系统发育的粘合剂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.