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

描述（由申请方提供）：作为血小板表面上表达第二多的膜受体复合物，糖蛋白（GP）Ib-IX-V复合物对血小板生理学至关重要。它主要被鉴定为许多止血重要蛋白质的受体，包括血管性血友病因子和凝血酶。它也与血小板的生成和清除有关。这种多亚基受体复合物的功能障碍可导致严重的出血素质，并有助于许多心血管疾病。然而，这个综合体如何履行其多方面的职能尚不清楚，主要是由于对其结构和组织缺乏了解。GPIb-IX-V复合物由4种不同的I型跨膜蛋白组成，其中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复合物的功能和调节很重要。在这个项目中，我们的目标是阐明其组织原理，并探讨亚基间相互作用的功能作用。在具体目标1中，为了表征GPIb 1和GPIX亚基的胞外域之间假定的弱相互作用，我们将定义两个域中的界面区域和残基，并确定它们在受体复合物的组装和由内而外调节中的作用。具体目标2是阐明胞外域间相互作用的结构基础。重组GPIb 2胞外域的结构将通过X射线晶体学确定。将产生具有改进的稳定性的工程化GPIX变体，其保留GPIb 2和GPIb 1的天然结合区，用于结构测定和功能分析。具体目标3是阐明的结构基础之间的相互作用的跨膜螺旋在GPIb-IX复杂的异三重共振NMR光谱。总体而言，在分离的蛋白质结构域中，在转染的哺乳动物细胞和人血小板中的个体相互作用的仔细的生物化学和结构解剖将有助于阐明GPIb-IX复合物响应于细胞内调节信号和配体结合的可能的结构变化，并提供GPIb-IX相关疾病的分子基础上的见解。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。 公共卫生相关性： 血小板中的糖蛋白Ib-IX-V复合物有助于血液正确凝结。这种复合物的功能障碍会导致严重出血，并可能导致许多心血管疾病。该项目旨在确定这一综合体的结构，并了解它如何运作以及如何受到监管。

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.