Structure and Function of Platelet Glycoprotein Ib-IX-V Complex

描述（由申请人提供）：糖蛋白（GP） Ib-IX-V复合体是血小板表面表达量第二高的膜受体复合体，对血小板生理至关重要。它主要被认为是一些止血重要蛋白的受体，包括血管性血友病因子和凝血酶。它还与血小板的产生和清除有关。这种多亚单位受体复合物的功能障碍可导致严重的出血性素质，并有助于许多心血管疾病。然而，由于缺乏对其结构和组织的了解，这个综合体是如何实现其多方面功能的尚不清楚。GPIb-IX- v复合物由4种不同的I型跨膜蛋白组成，GPIb-IX复合物具有配体结合活性和信号传导能力。我们假设亚基间的相互作用对GPIb-IX复合物的功能和调控很重要。在本项目中，我们旨在阐明其组织原理，并探讨亚单位间相互作用的功能角色。在Specific Aim 1中，为了描述GPIb1和GPIX亚基的外结构域之间假定的弱相互作用，我们将定义两个结构域的界面区域和残基，并确定它们在受体复合物的组装和内向外调节中的作用。具体目的2是阐明外域相互作用的结构基础。重组GPIb2外畴的结构将通过x射线晶体学来确定。将产生稳定性更好的工程GPIX变体，保留GPIb2和GPIb1的天然结合区域，用于结构测定和功能分析。具体目的3是利用异核三共振核磁共振波谱技术阐明GPIb-IX配合物跨膜螺旋相互作用的结构基础。总的来说，对分离蛋白结构域、转染的哺乳动物细胞和人血小板中的个体相互作用进行细致的生化和结构解剖，将有助于阐明GPIb-IX复合物在响应细胞内调节信号和配体结合时可能发生的结构变化，并为GPIb-IX相关疾病的分子基础提供见解。该项目还将为我们提供开发工具和试剂的机会，以专门干扰感兴趣的亚基间相互作用，这可能会导致新的治疗策略。

DESCRIPTION (provided by applicant): As the second most expressed membrane receptor complex on the platelet surface, the glycoprotein (GP) Ib-IX-V complex is essential to platelet physiology. It is identified primarily as the receptor for a number of hemostatically important proteins including von Willebrand factor and thrombin. It has also been implicated in the genesis and clearance of platelets. Malfunction of this multi-subunit receptor complex can lead to severe bleeding diathesis and contribute to many cardiovascular diseases. However, how this complex carries out its multi-faceted functions is not clear, primarily due to the lack of understanding of its structure and organization. The GPIb-IX-V complex comprises of 4 different type I transmembrane proteins, with the GPIb-IX complex possessing the ligand-binding activity and signaling capability. We hypothesize that inter-subunit interactions are important to the functions and regulation of the GPIb-IX complex. In this project, we aim to elucidate its organizing principle and to explore functional roles of inter-subunit interactions. In Specific Aim 1, to characterize the postulated weak interaction between the ectodomains of GPIb1 and GPIX subunits, we will define the interfacial region and residues in both domains and determine their roles in the assembly and inside-out regulation of the receptor complex. Specific Aim 2 is to elucidate the structural basis for the inter-ectodomain interactions. Structure of the recombinant GPIb2 ectodomain will be determined by X-ray crystallography. Engineered GPIX variants with improved stability that retain the native binding region to GPIb2 and GPIb1 will be produced for structural determination and functional analysis. Specific Aim 3 is to elucidate the structural basis for the interaction among transmembrane helices in the GPIb-IX complex by heteronuclear triple resonance NMR spectroscopy. Overall, careful biochemical and structural dissection of individual interactions in isolated protein domains, in transfected mammalian cells and in human platelets will help to elucidate the likely structural changes in the GPIb-IX complex in response to intracellular regulatory signals and ligand binding, and to provide insights on the molecular basis for GPIb-IX-related diseases. This project will also afford us the opportunity to develop tools and reagents to specifically perturb the inter-subunit interaction of interest, which may lead to novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: The glycoprotein Ib-IX-V complex in the platelet helps the blood to clot properly. Malfunction of this complex leads to severe bleeding and can contribute to many cardiovascular diseases. This project seeks to determine the structure of this complex and to learn how it functions and how it is regulated.