Physiological Chemistry of Integrin Function

描述（由申请人提供）：整合素是一类细胞粘附/信号分子，在发育、伤口修复、血管生成、免疫和组织完整性中发挥重要作用。特异性整合素的调控是包括癌症和自身免疫在内的多种疾病的潜在强有力的治疗靶点。整合素的一个显著特征是可以在不改变蛋白表达水平的情况下实现细胞调节的程度。例如，整合素淋巴细胞功能相关-1 （LFA-1）最初对静止淋巴细胞无活性，但通过暴露于趋化因子或抗原，粘附活性迅速增加。LFA-1对配体亲和力的改变是重要的，但不是调控的唯一机制；越来越多的证据表明，整合素与细胞骨架的相互作用同样重要。我们的假设是，整合素活性是由以下几个主要步骤的多步骤级联调节的：1)中间亲和整合素最初附着在细胞骨架上，以防止与配体的相互作用；2)激活将中间亲和整合素从细胞骨架的约束中释放出来，以增加配体的结合；3)配体结合诱导整合素的构象变化到高亲和构象；4)连接的整合素结合细胞质因子，调节局部力学性质，增强整合素与配体相互作用的二维亲和力。我们将通过检测整合素相互作用（键）的生理化学来检验这些假设，利用支撑平面双层中的配体来确定LFA-1在不同条件下的动力学和平衡结合。我们还将在适当的地方使用单分子测量。在Aim 1中，我们将比较分化、趋化因子和抗原受体介导的LFA-1激活。在目标2中，我们将确定不同模式的淋巴细胞活化如何影响不同形式LFA-1的横向迁移，作为细胞骨架释放的测定。在Aim 3中，我们将确定细胞骨架调节剂Wiskott Aldrich综合征蛋白（WASP）和WASP相互作用蛋白（WIP）的缺陷如何影响LFA-1的横向迁移及其介导调节粘附的能力。在Aim 4中，我们将确定包括粘附和脱粒促进适配器蛋白（ADAP）和蛋白激酶C-0 （PKC-0）介导的信号通路对LFA-1迁移和聚集的影响。这些实验将为整合素功能的调控提供新的见解，并确定白细胞整合素调控的潜在治疗靶点。

DESCRIPTION (provided by applicant): 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 Lymphocyte Function Associated-1 (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 important, but are not the exclusive mechanism for regulation; there is mounting evidence that integrin interactions with the cytoskeleton are equally important. Our hypothesis is that integrin activity is regulated by a multistep cascade with the following major steps: 1) intermediate affinity integrin is initially attached to the cytoskeleton to prevent interaction with ligands, 2) activation releases the intermediate affinity integrin from cytoskeletal constraints to increase ligand binding, 3) ligand binding induces a conformational change in the integrin to the high affinity conformation, 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 ligands in supported planar bilayers to determine the kinetic and equilibrium binding of LFA-1 under different conditions. We will also make use of single molecule measurements where appropriate. In Aim 1 we will compare differentiation, chemokine and antigen receptor mediated LFA-1 activation. In Aim 2 we will determine how different modes of lymphocyte activation affect lateral mobility of different forms of LFA-1 as an assay for release from the cytoskeleton. In Aim 3 we will determine how defects in the cytoskeleton regulators Wiskott Aldrich Syndrome protein (WASP) and WASP interacting protein (WIP) influence the lateral mobility of LFA-1 and its ability to mediate regulated adhesion. In Aim 4 we will determine the effect of signaling pathways including those mediated by Adhesion and Degranulation promoting Adapter Protein (ADAP) and Protein Kinase C-0 (PKC-0) on LFA-1 mobility and clustering. These experiments should provide new insight into regulation of integrin function and identify potential therapeutic targets for regulation of leukocyte integrins.