THROMBOSPONDIN 4

血小板反应蛋白是一个细胞外钙结合蛋白家族， 具有粘附和反粘附活性的蛋白质。血小板反应蛋白-1 是家族中最具特征的成员。 已经 显示调节细胞粘附、迁移和生长。的目标 该建议是为了表征两个新的血小板反应蛋白成员， 基因家族失神经支配后肌肉中凝血酶敏感蛋白-4的表达上调 并支持神经元附着和神经突生长。 软骨 寡聚基质蛋白（COMP或血小板反应蛋白-5）已被证明 含有骨发育不良的突变 该研究旨在 为了将从蛋白质中获得的体外和体内数据相关联， 生物化学、细胞生物学和遗传学方法。具体重点将 主要涉及以下几个方面：（1）功能特性 血小板反应蛋白-4和COMP。所提出的研究的一个具体目的是 特异性地定位糖胺聚糖结合位点， 结合，以及通过产生这些蛋白质内的细胞结合 （1）蛋白水解片段和融合蛋白， 活性，（2）阻断功能的多克隆和单克隆抗体， (3)定点诱变，和（4）模拟或 抑制完整蛋白质的活性。这些方法还将 用于绘制支持神经突的血小板反应蛋白-4内的活性位点 结果 血小板反应蛋白-4的正常和突变形式及其与 将测定结构域纠正异常的能力， 血小板反应蛋白-4缺陷小鼠和细胞系。(2)删除 凝血酶敏感蛋白4基因。 拟议研究的一个目标是删除 通过同源重组从小鼠基因组中获得血小板反应蛋白-4基因 在胚胎干细胞中。 选择的胚胎干细胞将用于 产生嵌合体小鼠， 血小板反应蛋白-4缺乏症的杂合或纯合， 两种遗传背景。 如果纯合子不能存活， 确定胚胎致死的发育阶段和原因。 如果纯合子动物存活，则主要器官的形态 系统将被分析，特别关注大脑，心脏和 骨骼肌 其他成员赔偿的可能性 将通过定量mRNA来研究血小板反应蛋白基因家族 水平，并通过杂交两种小鼠，这是缺乏其他 血小板反应蛋白(3)软骨发育不良中的COMP突变。 特定 这项研究的目的是确定这些突变和其他 引起PSACH和多发性骨骺发育不良（MED）。 我们假设 这些软骨发育不良是由于 软骨细胞活力和过早停止生长或缺陷， 软骨细胞与细胞外基质的相互作用。 到 为了确定COMP突变在PRACH和MED中的作用，我们计划 检查突变对结构、生物合成和 突变蛋白质将通过定点突变制备。 诱变和杆状病毒表达。 突变蛋白质将 就其（1）钙依赖性折叠而言， (2)与分子伴侣的相互作用，（3）结合糖胺聚糖的能力， 和（4）它们与细胞表面的相互作用。

The thrombospondins are a family of extracellular, calcium-binding proteins with adhesive and counter-adhesive activity. Thrombospondin-1 is the most extensively characterized member of the family. It has been shown to modulate cell adhesion, migration and growth. The objective of this proposal is to characterize two new members of the thrombospondin gene family. Thrombospondin-4 is upregulated in muscle after denervation and supports neuronal attachment and neurite outgrowth. Cartilage oligomeric matrix protein (COMP or thrombospondin-5) has been shown to contain mutations in bone dysplasias. The proposed study is designed to correlate in vitro and in vivo data that is obtained from protein biochemical, cell biological and genetic approaches. Specific focus will be directed toward the following areas:(1) Functional properties of thrombospondin-4 and COMP. A specific aim of the proposed study is to specifically map the sites for glycoasminoglycan binding, calcium binding, and cell binding within these proteins through the production of (1) proteolytic fragments and fusion proteins that retain functional activity, (2) polyclonal and monoclonal antibodies that block function, (3) site-directed mutagenesis, and (4) synthetic peptides that mimic or inhibit the activity of the intact proteins. These approaches will also be used to map active sites within thrombospondin-4 that support neurite outgrowth. The normal and mutant forms of thrombospondin-4 and its domains will be assayed for the ability to correct abnormalities in thrombospondin-4-deficient mice and cell lines. (2) Deletion of the thrombospondin-4 gene. A goal of the proposed study is to delete the thrombospondin-4 gene from the mouse genome by homologous recombination in embryonic stem cells. Selected embryonic stem cells will be used to produce chimeric mice that can be bred to establish a strain of mice that are heterozygous or homozygous for thrombospondin-4 deficiency in two genetic backgrounds. If the homozygotes are not viable, we will determine the developmental stage and cause of the embryonic lethality. If the homozygous animals are viable, the morphology of the major organ systems will be analyzed, with particular focus on brain, heart and skeletal muscle. The possibility of compensation by other members of the thrombospondin gene family will be investigated by quantitating mRNA levels and by crossing two strains of mice that are deficient in other thrombospondins. (3) COMP mutations in chondrodysplasias. A specific aim of the proposed study is to determine how these mutations and others cause PSACH and multiple epiphyseal dysplasia (MED). We hypothesize that these chondrodysplasias are a result of either decreased chondrocyte viability and premature cessation of growth or a defect in the interaction of chondrocytes with the extracellular matrix. To determine the role of COMP mutations in PSACH and MED, we plan to examine the effect of the mutations on the structure, biosynthesis and function of COMP. Mutant proteins will be made by site directed mutagenesis and baculovirus expression. The mutant proteins will be compared to normal COMP in terms of their (1) calcium-dependent folding, (2) interaction with chaperones, (3) ability to bind glycosaminoglycans, and (4) their interaction with cell surfaces.