MOLECULAR BIOLOGY OF PLATELET ALPHAIIB-BETA3 RECEPTOR

血小板是循环的，高度分化的，核细胞 来源于巨核细胞的片段。其中一个组织特异性特征 纤维蛋白原受体α II/β 3。这种整联蛋白 受体密集地堆积在血小板表面， 在血小板聚集和血栓形成中的作用。此应用程序，a 作为拟议计划项目的一部分，将研究这种生物学 关键受体，重点关注α IIb的调节表达 Glanzmann基因及其分子基础的分析 血栓无力症本申请有三个具体目标。I. 定义调节alphaIIb表达的近端启动子元件。 拟议的研究将集中在进一步分析三个 区域：第一个是以Sp1为中心的负调控区域， 转录起始位点上游-135bp处。我们计划定义其 局部相互作用和生物学作用。我们亦计划研究 在-510 bp处的潜在PU.1结合位点的生物学相关性和 在-450至-350 bp之间的潜在的加塔-1相互作用区域。二. 表征alphaIIb基因的远端区域。两种方法将是 随后寻找这样的远端调控元件：第一个将 检查alphaIIb基因位点的组织特异性DNA酶1 过敏部位（HSS）。我们已经初步确定了两个这样的网站 人α IIb上游分别为5和6个β-内酰胺酶对（kb） 基因利用现有的P1 α IIb克隆，我们计划亚克隆这些 区域，对它们进行测序，并在体外和体内对其进行表征， 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的人类基因的转基因动物 从λ、粘粒和P1克隆中分离的α IIb基因座， 进行测试，以了解所需的最小结构尺寸 一致的、高水平的组织特异性人α IIb基因表达 对小鼠一旦这样的区域被定义，我们将专注于Dnase 1 HSS 进一步定位潜在的远端巨核细胞特异性 调控元件，并将这些区域纳入进一步的表达 结构。三.α IIb/β 3受体的功能分析。这些 研究将集中在分析血栓形成突变， 我们已经发表或正在进行分析。这些 研究将包括进一步分析的钙离子结合域， α IIb和β 3在细胞内受体加工中的作用， 一种新的alphaIIb变体（P176 to A）和alphaIIb该区域的作用 在配体结合中，以及在α IIb的C-末端中的突变（Q819至 P）和C-末端α IIb重链在受体中的作用 处理和激活。这些研究和相关研究的重点是 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的生物学有了新的认识。

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.