Structure-Function Studies on Intestinal Epithelial JAM

描述（由申请方提供）：上皮屏障功能受到调节 在很大程度上是由最顶端的细胞间连接，称为 紧密连接（TJ）。TJ不仅将内腔室与 组织空间，而且还调节溶质穿过细胞旁的运动。 空间在不同的生理和病理状态。上皮缺陷 已经在粘膜标本中观察到TJs的屏障和变化， 患有炎症性肠病的个体在功能性和结构性 水平我们最近克隆了一种新的TJ相关膜的人类同源物 连接粘附分子（JAM）。我们的初步数据显示 该蛋白在调节细胞旁通透性中的重要作用， 肠上皮中的TJ组织。因此，本报告的总体目标 建议在结构和职能层面上界定机制， 其中JAM调节TJ组装和上皮屏障功能。在这 我们将测试两个假设：第一个假设是， JAM的胞外结构域对于蛋白质功能至关重要，并介导蛋白质的功能。 细胞间连接的形成。这一假设将由以下人员进行检验： 检查一组JAM细胞外结构域特异性mAb对 屏障功能和TJ重组，然后绘制这些表位 单克隆抗体此外，还研究了重组JAM和突变型JAM构建体对细胞凋亡的影响。 将检查细胞-细胞相互作用。我们的第二个假设是 JAM的胞质尾区是与TJ蛋白特异性相互作用所必需的 家族成员和信号转导蛋白，调节细胞旁 磁导率我们将通过扩展对以下方面的初步研究来检验这一假设： TJ相关蛋白结合到JAM的胞质尾区， 通过分析突变型JAM构建体形成的细胞-细胞连接。信息 从这些研究应该提供机制的见解， TJ功能和潜在的新的治疗途径，旨在调节粘膜 磁导率

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.