STRUCTURE AND FUNCTION OF THE INTEGRIN ALPHA 1 BETA 1

我们的研究集中在整合素的结构和功能， 是许多细胞外的跨膜异二聚体受体 基质分子 这些受体由α和β亚基组成。 最初，我们使用人整合素α 1 β 1作为模型系统， 为了回答结构-功能关系的主要问题， α和β结构域。 我们最近发现， 这些受体的结构域可以控制细胞内的整合素 功能 本提案的目标是确定关系 整合素受体的结构和它们的功能之间的联系。 我们有 这个项目的两个主要目标。 第一，我们将描述 整合素中发生后配体结合事件的机制，以及 分离出调节这些事件的基因。 第二，我们将识别配体 结合结构域，然后分离参与调节的基因， 配体结合的亲和力。 我们最近的结构-功能实验 表明α胞质结构域在细胞内的 整联蛋白β胞质功能的调节。 它通过 这两个领域之间的联系，我们正在研究的网站- 两个结构域的定向诱变。 我们会用一个有力的主导音 阴性构建体作为鉴定β细胞质 结构域结合蛋白，通过一种策略，涉及拯救的 显性阴性表型 我们的实验鉴定了配体结合 alpha 1中的基序继续，我们将精确定位必要的区域， 胶原IV结合。 最后，我们将研究整合素的活化， 血小板整合素IIb/IIIa，然后在CHO细胞中表达。 使用新开发的透化方案，我们将能够 确定由内而外激活所需的物质。 这些实验 将帮助我们理解粘附的调节，这是 动脉粥样硬化中的迁移 此外，这些实验也将有助于 我们对整合素通过由内而外激活机制的理解 信号传导，其在迁移和血小板粘附中是关键的。

Our research is focused on the structure and function of integrins, which are transmembrane heterodimeric receptors for a number of extracellular matrix molecules. These receptors are composed of alpha and beta subunits. Initially, we have used the human integrin alpha1beta1 as a model system in order to answer major questions of the structure-function relationships in alpha and beta domains. We have recently shown that both cytoplasmic domains of these receptors may be controlling integrin intracellular function. The goal of this proposal is to determine the relationship between the structure of integrin receptors and their functions. We have two primary broad goals in this project. One, we will characterize the mechanism by which post-ligand binding events in integrins occur, and isolate genes which mediate those events. Two, we will identify ligand binding domains, and then isolate genes involved in the modulation of the affinity of ligand binding. Our recent structure-function experiments have shown that the alpha cytoplasmic domain plays a regulatory role in the modulation of integrin beta cytoplasmic function. It accomplishes this via an association between these two domains, which we are studying by site- directed mutagenesis of both domains. We will use a potent dominant negative construct as a probe for the identification of beta cytoplasmic domain binding proteins, through a strategy which involves rescue of the dominant negative phenotype. Our experiments identifying ligand binding motifs within alpha1 continue, and we will pinpoint regions necessary for collagen IV binding. Lastly, we will study activation of integrins using the platelet integrin IIb/IIIa, which is then expressed in CHO cells. Using a newly developed permeabilization scheme, we will be able to identify substances required for inside out activation. These experiments will help us understand regulation of adhesion, which is central to migration in atherosclerosis. Furthermore, these experiments will also aid our understanding of the mechanism of integrin activation via inside-out signalling, which is critical in migration and in platelet adhesion.