MOLECULAR BIOLOGY OF PLATELET ALPHAIIB-BETA3 RECEPTOR

血小板是一种循环的、高度分化的有核细胞。 来自巨核细胞的碎片。其中一个组织特有的特征是 这些细胞中有它们的纤维蛋白原受体AlphaII/Beta3。这种整合素 受体密集地堆积在血小板表面，起着关键作用 在血小板聚集和血栓形成中的作用。此应用程序是一个 计划项目的一部分，将研究这一现象的生物学 关键受体，关注AlphaIIb的调节表达 基因与Glanzmann的分子基础分析 血栓减少症。这个应用程序有三个具体目标。我。 确定调控AlphaIIb表达的近端启动子元件。 拟议的研究将集中于以下三个方面的进一步分析 区域：第一个是以Sp1为中心的负面监管区域 -转录起始点上游135个碱基对。我们计划定义其 局部相互作用和生物学作用。我们还计划研究 潜在的PU1结合位点在-510个碱基对上的生物学相关性 潜在的GATA-1相互作用区在-450到-350碱基对之间。二、 AlphaIIb基因远端区域的特征。两种方法将是 紧随其后的是寻找这样的远端调控元件：第一个意愿 检查组织特异性DNA酶1的alphaIIb基因座 过敏部位(HSS)。我们已经初步确定了两个这样的地点 人类αIIb上游分别为5和6千巴空间(Kb) 吉恩。使用可用的P1 AlphaIIb克隆，我们计划对它们进行亚克隆 区域，对它们进行排序，并在体外和 转基因小鼠模型。在第二种方法中，我们计划创建一个 一系列含有不同长度人类的转基因动物 从lambda、Cosmidd和P1克隆中分离到的alphaIIb基因座将 进行测试，以了解以下各项所需的最小构造大小 持续、高水平的组织特异性人类AlphaIIb基因表达 在老鼠身上。一旦定义了这样的区域，我们将重点关注DNase1 HSS 进一步定位潜在远端巨核细胞特异性的研究 并将这些区域整合到进一步的表达中 构造。IIIb/Beta3受体的功能分析这些 研究将集中在分析具有以下特点的血栓生成突变 已由我们发布或正在分析中。这些 研究将包括进一步分析钙结合结构域。 AlphaIIb和Beta3在细胞内受体加工中的作用 一种新的AlphaIIb变异体(P176~A)及其该区域的作用 在配体结合中，以及在alphaIIb(Q819到 P)和C端AlphaIIb重链在受体中的作用 处理和激活。这些研究和相关研究主要集中在 细胞内加工和配体结合，并应提供重要的 对这种重要的整合素受体生物学的新见解。

Platelets are circulating, highly differentiated, a nuclear cellular fragments derived from megakaryocytes. One of the tissue-specific features of these cells is their fibrinogen receptor, alphaII/beta3. This integrin receptor is densely packed on the surface of the platelet and plays a key role in platelet aggregation and thrombus formation. This application, a part of the proposed Program Project, will study the biology of this critical receptor, focusing on the regulated expression of the alphaIIb gene and on the analysis of the molecular basis of Glanzmann thrombasthenia. There are three specific aims to this application. I. Defining the Proximal Promoter Elements Regulating alphaIIb Expression. Proposed studies will concentrate on the further analysis of three regions: The first is an Sp1-based negative regulatory region centered at -135bp upstream to the transcriptional start site. We plan to define its local interactions and biological role. We also plan to examine the biological relevance of a potential PU.1-binding site at -510 bp and a potential GATA-1 interacting region between -450 to -350 bp. II. Characterizing Distal Regions of the alphaIIb Gene. Two approaches will be followed to search for such distal regulatory elements: The first will examine the alphaIIb gene locus for tissue-specific, DNase 1 hypersensitive sites (HSS). We have preliminarily defined two such sites 5 and 6 kilobasepairs (kb), respectively, upstream of the human alphaIIb gene. Using available P1 alphaIIb clones, we plan to subclone these regions, sequence them, and characterize them in vitro and also in transgenic mice models. In the second approach, we plan on creating a series of transgenic animals containing various lengths of the human alphaIIb gene locus isolated from lambda, cosmid and P1 clones that will be tested to see what is the minimal construct size required for consistent, high levels of tissue-specific human alphaIIb gene expression in mice. Once such a region is defined, we will focus on Dnase 1 HSS studies to further localize potential distal megakaryocyte-specific regulatory elements and incorporate these regions into further expression constructs. III. Functional Analysis of the alphaIIb/beta3 Receptor. These studies will focus on the analysis of thrombasthenic mutations that have already been published by us or that are under present analysis. These studies will include further analysis of the Ca2+-binding domains of alphaIIb and beta3 in intracellular receptor processing, and analysis of a new alphaIIb variant (P176 to A) and the role of this region of alphaIIb in ligand binding, and a mutation in the C-terminus of alphaIIb (Q819 to P) and the role of the C-terminal alphaIIb heavy chain in receptor processing and activation. These and related studies focus on intracellular processing and ligand binding and should provide important new insights into the biology of this important integrin receptor.