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

描述（由申请方提供）：血小板糖蛋白（GP）Ib-IX-V复合物介导初始血小板粘附至受损血管壁，并转导信号以诱导血小板活化和聚集。这种多亚基受体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。尽管进行了数十年的深入研究，但复合物功能的调节尚不清楚，血小板活化信号由复合物介导的过程也不清楚。我们推测亚基间的相互作用对复合物的功能和调节很重要。由于复杂的3维组织是未知的，亚基间的相互作用在很大程度上是未开发的，作为一个必要的前奏，以测试我们的假设，这个项目旨在确定和表征GP Ib-IX-V复杂的亚基间的相互作用。在具体目标1中，我们提出测试跨膜结构域在亚基间关联中起重要作用的假设，因此对于GP Ib-IX-V复合物的表达和组装是重要的。这些研究是基于我们的观察，即用不相关的序列替换GP Ib α或Ib β亚基的跨膜结构域显著降低了复合物的表达水平。将定义跨膜结构域中特定残基的结构和功能作用，包括极性残基和近膜二硫键形成半胱氨酸残基。GP Ib-IXV复合物中亚基化学计量的分子基础也将被阐明。在具体目标2中，我们将定义复合物胞外结构域之间的界面区域和残基，并探索它们对复合物组装和功能的贡献。这些研究是基于我们的观察，第一，GP Ibbeta和IX亚基的细胞外结构域相互关联和稳定，第二，glycocalicin，GP Ibalpha的可溶性细胞外结构域，直接结合到GP IX的重组细胞外结构域。总体而言，在分离的蛋白质，在转染的哺乳动物细胞和血小板中的个体相互作用的仔细解剖将使我们能够开发工具和试剂，特别是干扰感兴趣的亚基间的相互作用，并将铺平道路的GP Ib-IX-V复合物中的可能的结构变化的研究，在响应配体结合或细胞内调节信号。该项目还将为研究GP Ib-IX-V复合物与其他受体的相互作用提供垫脚石，并可能导致新的治疗策略，以对抗各种与复合物相关的血液学疾病和疾病。与公共卫生的相关性：血小板中的糖蛋白Ib-IX-V复合物有助于血液适当凝结。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。该项目旨在了解Ib-IX-V复合体的结构并了解其功能

DESCRIPTION (provided by applicant): Platelet glycoprotein (GP) Ib-IX-V complex mediates initial platelet adhesion to the injured vessel wall and transduces signals to induce platelet activation and aggregation. Malfunction of this multi-subunit receptor complex leads to severe bleeding and can contribute to many cardiovascular diseases. Despite decades of intensive study, regulation of the complex function is not clear, nor is the process by which the platelet activating signal is mediated by the complex. We hypothesize that inter-subunit interactions are important to the functions and regulation of the complex. Since the 3-dimensional organization of the complex is unknown and inter-subunit interactions are largely unexplored, as a necessary prelude to test our hypothesis this project seeks to identify and characterize the inter-subunit interactions in the GP Ib-IX-V complex. In Specific Aim 1, we propose to test the hypothesis that the transmembrane domains play vital roles in inter-subunit associations and thus are important for expression and assembly of the GP Ib-IX-V complex. The studies are based on our observation that replacing the transmembrane domain of GP Ibalpha or lbbeta subunit with an unrelated sequence decreases significantly the complex expression level. Structural and functional roles of specific residues in the transmembrane domains, including polar residues and membrane-proximal disulfide forming cysteine residues, will be defined. The molecular basis for the subunit stoichiometry in the GP Ib-IXV complex will also be elucidated. In Specific Aim 2, we will define the interfacial regions and residues among the extracellular domains of the complex and explore their contribution to complex assembly and function. The studies are based on our observations that first, the extracellular domains of GP Ibbeta and IX subunits associate and stabilize each other, and second, glycocalicin, the soluble extracellular domain of GP Ibalpha, binds directly to the recombinant extracellular domain of GP IX. Overall, careful dissection of individual interactions in the isolated proteins, in the transfected mammalian cells and in the platelets will enable us to develop tools and reagents that specifically perturb the inter-subunit interaction of interest and will pave the way for studies of the likely structural changes in the GP Ib-IX-V complex in response to ligand binding or intracellular regulatory signals. This project will also provide a stepping stone for studies on the interaction of the GP Ib-IX-V complex with other receptors and may lead to novel therapeutic strategies to combat various complex-related hematological disorders and diseases. Relevance to public health: 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 understand the structure of the Ib-IX-V complex and to learn how it functions