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

描述（由申请人提供）：血小板糖蛋白（GP） Ib-IX-V复合物介导血小板对受损血管壁的初始粘附，并传导信号诱导血小板活化和聚集。这种多亚单位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。尽管经过数十年的深入研究，复合物功能的调控尚不清楚，血小板激活信号由复合物介导的过程也不清楚。我们假设亚基间的相互作用对复合体的功能和调节很重要。由于复合物的三维组织未知，亚基间相互作用在很大程度上未被探索，作为测试我们假设的必要前奏，本项目旨在识别和表征GP Ib-IX-V复合物中的亚基间相互作用。在Specific Aim 1中，我们提出验证跨膜结构域在亚基间结合中发挥重要作用的假设，因此对GP Ib-IX-V复合物的表达和组装很重要。这些研究是基于我们的观察，用不相关的序列取代GP β或β亚基的跨膜结构域可显著降低复杂的表达水平。将定义跨膜结构域中特定残基的结构和功能角色，包括极性残基和膜近端二硫形成半胱氨酸残基。GP Ib-IXV复合物亚基化学计量学的分子基础也将被阐明。在Specific Aim 2中，我们将定义复合体胞外结构域之间的界面区域和残基，并探索它们对复合体组装和功能的贡献。这些研究是基于我们的观察，首先，GP Ibbeta和IX亚基的胞外结构域相互结合并稳定，其次，GP Ibbeta的可溶性胞外结构域糖钙素直接与重组GP IX的胞外结构域结合。总的来说，仔细解剖分离蛋白、转染的哺乳动物细胞和血小板中的个体相互作用，将使我们能够开发出专门干扰感兴趣的亚基间相互作用的工具和试剂，并将为研究GP Ib-IX-V复合物响应配体结合或细胞内调节信号的可能结构变化铺平道路。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跳板，并可能导致对抗各种复合物相关血液学疾病和疾病的新治疗策略。与公共卫生相关：血小板中的糖蛋白Ib-IX-V复合物有助于血液正常凝块。这种复合物的功能障碍会导致严重的出血，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