PHYSIOLOGICAL CHEMISTRY OF INTEGRIN FUNCTION

整合素是一类发挥作用的细胞黏附/信号分子家族 在发育、伤口修复、血管生成、免疫、 和组织的完整性。对特定整合素的调节是一种潜在的 针对多种疾病的强大治疗靶点，包括癌症和 自身免疫力。整合素的一个显著特征是 在不改变蛋白质的情况下可以实现的细胞调控 表达级别。例如，整合素LFA-1最初是 对静止的淋巴细胞不活跃，但黏附活性很快 因接触趋化因子或抗原而增加。LFA-1的变化 对配体的亲和力不是调节的主要机制。在……里面 相比之下，越来越多的证据表明整合素与 细胞骨架很重要。我们的假设是整合素活性 由以下主要步骤组成的多级级联进行调节：1) 整合素最初附着在细胞骨架上，以防止 与配体的扩散限制反应，2)活化释放 来自细胞骨架的整合素约束以增加配体结合，3) 配体结合诱导整合素的构象变化，以及4) 连接的整合素结合调节局部的细胞质因子 增强整合素二维亲和力的机械性能- 配基相互作用。我们将通过检查 使用一种新型的整合素相互作用(键)的生理化学 基于荧光的方法。在目标1中，我们将确定不同之处 重要的生物学淋巴细胞激活模式影响LFA-1 在细胞底物接触区域接合。在目标2中，我们将确定 不同的淋巴细胞激活方式对LFA-1侧向的影响 作为从细胞骨架中释放的一种分析方法。在《目标3》中我们 将确定探测整合素的最有效策略 外源胞质表达与细胞骨架的相互作用 域。

Integrins are a family of cell adhesion/signaling molecules that play an important role in development, wound repair, angiogenesis, immunity, and tissue integrity. Regulation of specific integrins is a potentially powerful therapeutic target for diverse diseases including cancer and autoimmunity. A remarkable characteristic of integrins is the degree of cellular regulation that can be achieved without altering protein expression levels. For example, the integrin LFA-1 is initially inactive on resting lymphocytes, but adhesion activity is rapidly increased by exposure to chemokines or antigen. Changes in LFA-1 affinity for ligand are not the primary mechanism for regulation. In contrast, there is mounting evidence that interactions of integrins with the cytoskeleton is important. Our hypothesis is that integrin activity is regulated by a multistep cascade with the following major steps: 1) the integrin is initially attached to the cytoskeleton to prevent diffusion limited reaction with ligands, 2) activation releases the integrin from cytoskeletal constraints to increase ligand binding, 3) ligand binding induces a conformational change in the integrin, and 4) the ligated integrin binds cytoplasmic factors that regulate local mechanical properties to enhance two dimensional affinity of integrin- ligand interaction. We will test these hypotheses by examining the physiological chemistry of integrin interactions (bonds) using a novel fluorescence based approach. In Aim 1 we will determine how different biologically important modes of lymphocyte activation affect LFA-1 engagement in cell substrate contact areas. In Aim 2 we will determine how different modes of lymphocyte activation affect LFA-1 lateral mobility as an assay for release from the cytoskeleton. In Aim 3 we will determine the most effective strategy to probe integrin cytoskeletal interactions through expression of exogenous cytoplasmic domain.