THE MOLECULAR BASIS OF LIGAND BINDING TO ALPHAIIB BETA3

已显示α IIb和α v整联蛋白在多种疾病过程中发挥重要作用。α IIb β 3是血小板特异性纤维蛋白原受体，对血栓形成和止血至关重要。确定α IIb β 3的配体识别基础对于血小板功能的调节至关重要。本提案的目的是确定与α IIb β 3结合的配体的分子基础。我们假设α IIb的离散区域调节α IIb β 3的配体识别特异性。我们进一步假设，需要多个配体结合点的高亲和力结合α IIb β 3。为了检验这些假设，具体目标是：1）利用基于分子建模的定向方法鉴定α IIb内决定α IIb β 3配体识别特异性的区域和特定氨基酸; 2）利用无偏遗传筛选鉴定α IIb和β 3上配体结合功能所需的其他氨基酸残基。为了接近目标1，我们将用α IIb的相应区域替换α v的预测环区域，并测试配体特异性的阳性表型转变。为了实现目标2，我们将随机诱变表达α IIb或β 3亚基的细胞，并检测配体结合的丧失。一旦发现突变，我们将把这些突变导入野生型alphaIIB β 3，并验证配体结合被破坏。

The alphaIIb and alphav integrins have been shown to play a significant role in a variety of diseases processes. AlphaIIbbeta3 is a platelet-specific fibrinogen receptor that is critical for thrombosis and hemostasis. Determination of the basis of ligand recognition by alphaIIbbeta3 is critical for the modulation of platelet function. The objective of this proposal is to define the molecular basis of ligand binding to alphaIIbbeta3. We hypothesize that discrete regions of alphaIIb regulate the ligand recognition specificity of alphaIIbbeta3. We further hypothesize that multiple ligand binding points are required for high affinity binding to alphaIIbbeta3. To test these hypotheses, the specific aims are 1) to identify regions and specific amino acids within alphaIIb that determine ligand recognition specificity of alphaIIbbeta3 utilizing a directed approach based upon molecular modeling and 2) identify additional amino acid residues on alphaIIb and beta3 that are required for ligand binding function utilizing an unbiased genetic screen. To approach aim 1, we will replace the predicted loop regions of alphav with the corresponding region of alphaIIb and test for a positive phenotypic shift in ligand specificity. To accomplish aim 2, we will randomly mutagenize cells expressing either the alphaIIb or the beta3 subunits and test for loss of ligand binding. Once mutations are identified, we will introduce these mutations into wild type alphaIIBbeta3 and verify that ligand binding is destroyed.