THE MOLECULAR BASIS OF LIGAND BINDING TO ALPHAIIB BETA3

AlphaIIb和Alphav整合素已被证明在各种疾病过程中发挥重要作用。AlphaIIbbeta3是一种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AlphaIIbbeta3识别配体的基础是调节血小板功能的关键。这项建议的目的是确定配体与AlphaIIbbeta3结合的分子基础。我们假设AlphaIIb的离散区域调节AlphaIIbbeta3的配体识别特异性。我们进一步假设，与αIIbbeta3的高亲和力结合需要多个配体结合点。为了验证这些假说，具体目标是1)利用基于分子模型的定向方法识别决定AlphaIIb中决定AlphaIIbbeta3配体识别特异性的区域和特定氨基酸，以及2)利用无偏见的遗传筛选识别AlphaIIb和Beta3上的额外氨基酸残基，这些氨基酸残基是配体结合功能所必需的。为了接近目标1，我们将用alphaIIb的相应区域替换预测的alphav环区，并测试配体特异性的正表型转变。为了实现目标2，我们将随机诱变表达AlphaIIb或Beta3亚基的细胞，并测试配体结合的丢失。一旦发现突变，我们将把这些突变引入野生型alphaIIBbeta3，并验证配体结合是否被破坏。

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.