Functional Studies of Laminin-5

描述（由申请人提供）：层粘连蛋白-5（Ln-5）是一种基底膜细胞外基质大分子，对上皮形态发生和稳态的基本过程至关重要。在很大程度上，Ln-5的功能依赖于与其两种受体整合素α 3 β 1和α 6 β 4的相互作用，但尚不清楚这种相互作用如何机械地转化为细胞粘附，迁移和半桥粒形成。揭示这些分子机制的关键一步是定义Ln-5上的整合素结合位点。为了说明这一点，我们最近发现的两个结合位点的整合素α 3 β 1的LG 3和LG 4域的Ln-5 α 3链点的新的分子机制，在配体水平上，调节α 3 β 1的亲和力/亲合力。在这项提案中，我们将测试的假设，这些机制的核心是LG 3和LG 4之间的蛋白水解，导致细胞迁移的下调。我们提出的研究的另一个重要焦点是α 6 β 4的Ln-5结合位点，这仍然是未知的。破坏α 6 β 4/Ln- 5结合，例如，在遗传性起泡疾病中，完全损害上皮完整性，因为这种相互作用是唯一负责半桥粒组装的。我们打算定义的α 6 β 4 LG结合位点和测试的假设，它是参与指导重新分配的α 3 β 1和α 6 β 4整合素上皮细胞从迁移切换到半桥粒为基础的静态粘附。我们的建议分为三个目标。在目标1中，我们将通过产生LG重组结构域和Ln-5嵌合分子，并在粘附、迁移、半桥粒形成和直接结合测定中测试它们，对Ln-5上的α 3 β 1和α 6 β 4结合位点进行全面分析：在目的2中，我们将通过i）测量a3的Kd来检验LG 4的损失引发从迁移到静态粘附的转变的假设（1）与LG 3和LG 4的相互作用，并测试它们是否建立了迁移的亲和力/亲合力阈值; ii）确定LG 4去除是否停止迁移并开始募集半桥粒组分; iii）测试特定LG结构域是否指导α 3 β 1和α 6 β 4在足粒和半桥粒之间的运输。在目标3中，我们将研究整合素与两种Ln-5依赖性器官型系统中特定LG结构域相互作用的生物学后果，即乳腺腺泡和肾小管的形成。在基础研究水平上，我们的研究结果将使我们能够将整合素/Ln-5结合的分子尺度定量转化为粘附和迁移的细胞尺度机制，然后观察这些机制如何在上皮形态发生的尺度上运作。我们的发现应该与人类健康相关，因为它们将增强我们操纵生理和病理过程的能力，如伤口愈合，组织再生和癌症侵袭。

DESCRIPTION (provided by applicant): Laminin-5 (Ln-5) is a basement membrane extracellular matrix macromolecule crucial to fundamental processes of epithelial morphogenesis and homeostasis. In large part Ln-5 functions rely on interactions with its two receptors, integrins alpha3beta1 and alpha6beta4, but it is not clear how such interactions translate mechanistically into cell adhesion, migration and hemidesmosome formation. A key step towards unveiling these molecular mechanisms is the definition of integrin binding sites on Ln-5. To illustrate this point, our recent uncovering of two binding sites for integrin alpha3beta1 on the LG3 and LG4 domains of the Ln-5 alpha3 chain points to novel molecular mechanisms, at the ligand level, for modulating alpha3beta1 affinity/avidity. In this proposal, we will test the hypothesis that the core of these mechanisms is proteolysis between LG3 and LG4, resulting in downregulation of cell migration. Another important focus of our proposed studies is the Ln-5 binding site for alpha6beta4, which is still uncharacterized. Disruption of alpha6beta4 /Ln- 5 binding, e.g., in inherited blistering diseases, totally compromises epithelial integrity, since this interaction is uniquely responsible for hemidesmosome assembly. We intend to define the alpha6beta4 LG binding site and test the hypothesis that it is involved in directing redistribution of alpha3beta1 and alpha6beta4 integrins as epithelial cells switch from migration to hemidesmosome based static adhesion. Our proposal is divided into 3 Aims. In Aim 1, we will carry out a comprehensive analysis of alpha3beta1 and alpha6beta4 binding sites on Ln-5 by producing LG recombinant domains and Ln-5 chimeric molecules, and testing them in adhesion, migration, hemidesmosome formation and direct binding assays: In Aim 2 we will test the hypothesis that loss of LG4 initiates a switch from migration to static adhesion by i) measuring the Kd of a3(1 interactions with LG3 and LG4 and testing if they establish an affinity/avidity threshold for migration; ii) determining whether LG4 removal halts migration and starts recruitment of hemidesmosomal components; iii) testing whether specific LG domains direct the trafficking of alpha3beta1 and alpha6beta4 between podosomes and hemidesmosomes. In Aim 3 we will study the biological consequences of integrin interactions with specific LG domains in two Ln-5 dependent organotypic systems, formation of mammary gland acini and of kidney tubes. At a basic research level, our results will enable us to translate molecular scale quantitation of integrin/Ln-5 binding into cellular scale mechanisms of adhesion and migration, and then observe how these mechanisms operate at the scale of epithelial morphogenesis. Our findings should be relevant to human health as they will enhance our ability to manipulate physiological and pathological processes like wound healing, tissue regeneration and cancer invasion.