Structure-Function Studies on Intestinal Epithelial JAM

描述(申请人提供)：上皮屏障功能受调节 在很大程度上由顶端最大的细胞间连接称为 紧密连接(TJ)。TJ不仅将腔室与管腔隔开 组织空间，但也调节溶质在细胞旁的运动 处于不同生理和病理状态的空间。上皮性缺陷 在猪的粘膜标本中观察到TJs的屏障和变化 炎症性肠病患者在功能和结构上都有 水平。我们最近克隆了一种新的TJ相关膜的人类同源物 称为连接黏附分子(JAM)的蛋白质。我们的初步数据显示 该蛋白在调节细胞旁通透性和 肠上皮中的TJ组织。因此，这一行动的总体目标是 建议在结构和功能层面上定义机制(S)，由 它调节TJ的组装和上皮屏障功能。在这 提议，我们将检验两个假设：我们的第一个假设是 JAM的胞外结构域对蛋白质功能至关重要，并介导 细胞间连接的形成。这一假设将通过以下方式检验 检测一组果酱细胞外区特异性单抗对小鼠血管内皮细胞的影响 屏障功能和TJ重组，然后映射这些表位 单抗。此外，重组Jam和突变型Jam构建的影响 我们将研究细胞间的相互作用。我们的第二个假设是 JAM的细胞质尾巴是与TJ蛋白特异相互作用所必需的 调节细胞旁的家族成员和信号转导蛋白 渗透性。我们将通过扩展初步研究来检验这一假设 与Jam和Jam的细胞质尾部区域结合的TJ相关蛋白 通过分析突变JAM结构形成的细胞-细胞连接。信息 从这些研究中应该提供对监管的机械性见解 TJ的功能及其潜在的粘膜调节治疗新途径 渗透性。

DESCRIPTION (provided by applicant): Epithelial barrier function is regulated to a large extent by the apical most intercellular junction referred to as the tight junction (TJ). The TJ not only separates the lumenal compartment from the tissue space, but also regulates movement of solutes across the paracellular space in diverse physiologic and pathologic states. Defects in epithelial barrier and changes in TJs have been observed in mucosal specimens from individuals with inflammatory bowel disease at both a functional and structural level. We recently cloned the human homolog of a novel TJ-associated membrane protein termed junction adhesion molecule (JAM). Our preliminary data suggests an important role of this protein in regulating paracellular permeability and TJ organization in intestinal epithelia. Thus, the overall objective of this proposal is to define on a structural and functional level the mechanism(s) by which JAM regulates TJ assembly and epithelial barrier function. In this proposal, we will test two hypotheses: Our first hypothesis is that the extracellular domain of JAM is critical for protein function and mediates the formation of intercellular junctions. This hypothesis will be tested by examining the effects of a panel of JAM extracellular domain-specific mAbs on barrier function and TJ reassembly followed by mapping the epitopes for these mAbs. In addition, the effect of recombinant JAM and mutant JAM constructs on cell-cell interactions will be examined. Our second hypothesis is that the cytoplasmic tail of JAM is necessary for specific interactions with TJ protein family members and signal transduction proteins that regulate paracellular permeability. We will test this hypothesis by extending preliminary studies on TJ associated proteins that bind to regions of the cytoplasmic tail of JAM and by analysis of cell-cell junctions formed by mutant JAM constructs. Information from these studies should provide mechanistic insights into the regulation of TJ function and potential new therapeutic avenues aimed at modulating mucosal permeability.