Functional Studies of Laminin-5

描述(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，对上皮形态发生和动态平衡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个受体-整合素α3β1和α6β4的相互作用，但这种相互作用如何机械地转化为细胞黏附、迁移和半桥粒形成尚不清楚。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，我们最近在Ln-5α3链的LG3和LG4结构域上发现了整合素α3beta1的两个结合位点，指出了在配体水平上调节α3beta1亲和力的新的分子机制。在这个提案中，我们将检验这样一个假设，即这些机制的核心是LG3和LG4之间的蛋白质分解，导致细胞迁移的下调。我们建议研究的另一个重要焦点是Alpha6beta4的Ln-5结合部位，该结合部位仍未确定。α6β4/Ln-5结合的破坏，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半染色体组装的唯一原因。我们打算定义α6beta4 LG结合部位，并测试它参与了当上皮细胞从迁移切换到基于半桥粒的静态黏附时α3beta1和alpha6beta4整合素的重新分布的假设。我们的建议分为三个目标。在目标1中，我们将通过产生重组Lg结构域和Ln-5嵌合分子，对Ln-5上的Alpha3beta1和Alpha6beta4结合位点进行全面分析，并在黏附、迁移、半桥体形成和直接结合分析中对它们进行测试：在目标2中，我们将通过以下方式验证以下假设：1)测量A3的Kd(1与LG3和LG4的相互作用，并测试它们是否建立了迁移的亲和力阈值；ii)确定移除LG4是否停止了迁移，并开始招募半染色体组分；iii)测试特定的LG结构域是否指导了Al3beta1和Alpha6beta4在剂量体和血粒之间的贩运。在目标3中，我们将研究整合素与Ln-5依赖的两个器官型系统中特定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。在基础研究水平上，我们的结果将使我们能够将整合素/Ln-5结合的分子尺度量化转化为细胞尺度的黏附和迁移机制，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.