CELLULAR MECHANISMS OF MACROMOLECULAR TRANSPORT ACROSS EPITHELIA

粘液上皮细胞作为载有抗原的管腔上皮细胞与 与外部环境相邻的隔室， 粘膜免疫系统的抗原敏感细胞。在森林里 车厢 在粘膜组织中，抗原通过 通过上皮吸收。 同样，产生的抗体 在对特异性抗原的反应中， 转化为粘膜分泌物发挥作用 这 大分子的跨上皮转运是一种高度特化的形式 膜交通，这是本提案的主要目标， 研究这个过程的分子机制。 也许 最好理解的经上皮转运的例子是 多聚免疫球蛋白伊加和IgM，由多聚IG介导 受体（分泌成分）。 初步数据显示， 受体跨上皮细胞的结合显著增强 二聚体伊加，并在很大程度上独立于受体磷酸化。 数据进一步表明，这种差异是配体之间的行为- 被占据和未被占据的受体可能是由于 在配体的存在下，受体的二聚化/寡聚化。 该提议的一个目的是将异源三聚体GTP结合蛋白引入细胞中。 跨上皮转运 尽管大多数调查人员都把重点放在 ras家族的较大的异源三聚体G蛋白（称为rabs）， 越来越多的证据支持较大的异源三聚体G 蛋白质在一些囊泡运输过程中。 我们的初步证据 表明一类异源三聚体G蛋白，Gs，可能调节 二聚体伊加通过聚IG受体的跨上皮转运。 我们将首先确定这种参与是否是伊加特有的 转运，或者是胞吞转运的一般性质 通路 使用模型上皮细胞系（MDCK细胞），我们将 突变GS α亚基中的横切面， 积极的，或本质上不活跃，并作为显性阴性 内源性GalphaS的抑制剂。 然后我们将研究 这些突变体蛋白对三种跨上皮转运的影响 不同的标记物：1）辣根过氧化物酶（液相）2）植物 蓖麻毒素，它与许多膜上的末端半乳糖残基结合， 蛋白质和脂质，a（整体膜流动的标志物）; 3）二聚体伊加 （受体介导的转胞吞作用的标志物）。 最后一个目标是 鉴定和表征与多聚蛋白相互作用的细胞蛋白 IG受体及其转运功能。 这些实验将 提供了有用的洞察机制，大分子， 特别是免疫球蛋白，通过上皮细胞转运， 这是粘膜宿主防御中的关键成分。

Mucosal epithelia serve as an interface between the antigen-laden luminal compartment that is contiguous with the external environment and the antigen-sensitive cells of the mucosal immune system. in the serosal compartment. In mucosal tissues, antigens are continuously sampled by uptake across the epithelium. Similarly, antibodies that are produced in response to specific antigens are exported across the epithelium and into mucosal secretions where thy have their effect. This transepithelial transport of macromolecules is a highly specialized form of membrane traffic, and it is the primary goal of this proposal to examine the molecular mechanisms that underlay this process. Perhaps the best understood example of transepithelial transport is that of the polymeric immunoglobulins IgA and IgM, which is mediated by the poly Ig receptor (secretory component). Preliminary data suggest that transport of the receptor across epithelia is significantly enhanced upon binding of dimeric IgA, and is largely independent of receptor phosphorylation. The data further suggest that this difference is behavior between ligand- occupied and unoccupied receptors may be due to dimerization/oligomerization of the receptor in the presence of ligand. One aim of this proposal is to heterotrimeric GTP binding proteins in transepithelial transport. Although most investigators have focused on larger, heterotrimeric G proteins of the ras family (known as rabs), increasing evidence has supported a role for larger, heterotrimeric G proteins in some vesicular transport processes. Our preliminary evidence suggests that one class of heterotrimeric G proteins, Gs, may regulate the transepithelial transport of dimeric IgA via the poly Ig receptor. We will first determine whether this involvement is specific for IgA transport, or is a general property of the transcytotic transport pathway. Using a model epithelial cell line (MDCK cells), we will transect in mutant GS alpha subunits that are either constitutively active, or are constitutively inactive and serve as dominant negative inhibitors of endogenous GalphaS. We will then examine the effects of these mutants proteins on the transepithelial transport of three different markers: 1) Horseradish peroxidase (fluid phase) 2) The plant toxin ricin, which binds to terminal galactose residues on many membrane proteins and lipids, a (a marker of bulk membrane flow); 3) Dimeric IgA (a marker for receptor-mediated transcytosis). The last aim is to identify and characterize cellular proteins that interact with the poly Ig receptor and function in its transport. These experiments will provide useful insight into the mechanisms by which macromolecules, in particular immunoglobulins, are transported across epithelia, a process that is a key component in mucosal host defense.