Functional Studies of Laminin-5

描述（由申请人提供）：层粘连蛋白-5 （Laminin-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的功能依赖于其与两个受体（整合素alpha3beta1和alpha6beta4）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、迁移和半粒体的形成。揭示这些分子机制的关键一步是确定Ln-5上的整合素结合位点。为了说明这一点，我们最近在Ln-5 alpha3链的LG3和LG4结构域上发现了整合素alpha3beta1的两个结合位点，指出了在配体水平上调节alpha3beta1亲和力/亲和力的新分子机制。在本提案中，我们将验证这些机制的核心是LG3和LG4之间的蛋白水解，导致细胞迁移下调的假设。我们提出的研究的另一个重要焦点是alpha6beta4的Ln-5结合位点，该位点尚未表征。破坏alpha6beta4 /Ln- 5结合，例如在遗传性水疱疾病中，完全损害上皮的完整性，因为这种相互作用是半染色体组装的唯一原因。我们打算定义alpha6beta4 LG结合位点，并验证当上皮细胞从迁移转向基于半脂小体的静态粘附时，它参与指导alpha3beta1和alpha6beta4整合素的重新分配的假设。我们的建议分为3个目标。在Aim 1中，我们将通过制造LG重组结构域和Ln-5嵌合分子，并通过粘附、迁移、半小体形成和直接结合试验对Ln-5上的alpha3beta1和alpha6beta4结合位点进行全面分析：在目标2中，我们将通过以下方法来验证LG4的缺失启动了从迁移到静态粘附的转换的假设：i)测量a3（1与LG3和LG4相互作用的Kd，并测试它们是否为迁移建立了亲和/亲和阈值；ii）确定LG4的移除是否会停止迁移并开始半染色体组分的募集；iii)测试特定的LG结构域是否指导了alpha3beta1和alpha6beta4在足小体和半半小体之间的运输。在Aim 3中，我们将研究整合素与两种依赖Ln-5的器官型系统中特定LG结构域相互作用的生物学后果，即乳腺腺泡和肾管的形成。在基础研究层面，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细胞尺度的粘附和迁移机制，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伤口愈合、组织再生和癌症侵袭等生理和病理过程的能力。

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.