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

描述(申请人提供)：作为血小板表面表达第二多的膜受体复合体，糖蛋白(GP)Ib-IX-V复合体是血小板生理所必需的。它主要被认为是一些重要的止血蛋白的受体，包括von Willebrand因子和凝血酶。它还与血小板的起源和清除有关。这种多亚单位受体复合体的功能障碍可导致严重的出血素质，并导致许多心血管疾病。然而，这一综合体如何发挥其多方面的功能尚不清楚，主要是因为对其结构和组织缺乏了解。GPIB-IX-V复合体由4种不同的I型跨膜蛋白组成，其中GPIB-IX复合体具有配体结合活性和信号转导能力。我们假设亚基间的相互作用对GPIB-IX复合体的功能和调节很重要。在这个项目中，我们旨在阐明其组织原理，并探索亚基间相互作用的功能作用。在特定的目标1中，为了表征GPIb1和Gpix亚基胞外结构域之间的弱相互作用，我们将定义这两个结构域的界面区域和残基，并确定它们在受体复合体的组装和由内向外调节中的作用。具体目标2是阐明胞外结构域间相互作用的结构基础。重组GPIb2胞外区的结构将通过X射线结晶学确定。将生产具有改进的稳定性的工程Gpix变体，这些变体保留了与GPIb2和GPIb1的天然结合区域，用于结构确定和功能分析。具体目标3是通过异核三重共振核磁共振波谱阐明GPIB-IX复合体中跨膜螺旋相互作用的结构基础。总之，仔细分析分离蛋白结构域、转基因哺乳动物细胞和人类血小板中的个体相互作用，将有助于阐明GPIB-IX复合体响应细胞内调节信号和配体结合而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机会开发工具和试剂，以专门干扰感兴趣的亚基之间的相互作用，这可能导致新的治疗策略。 公共卫生相关性： 血小板中的糖蛋白Ib-IX-V复合体有助于血液正常凝结。这一复合体的故障会导致严重出血，并可能导致许多心血管疾病。这个项目试图确定这个综合体的结构，并了解它是如何运作的，以及它是如何受到监管的。

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.