CELLULAR MECHANISMS OF MACROMOLECULAR TRANSPORT ACROSS EPITHELIA

粘膜上皮起着连接带抗原的管腔的作用 与外部环境和 粘膜免疫系统中对抗原敏感的细胞。在浆膜中 车厢。在粘膜组织中，通过连续采样抗原 上皮细胞的摄取。类似地，产生的抗体 作为对特定抗原的反应，上皮细胞和 转化为粘膜分泌物，在那里起作用。这 大分子的跨皮运输是一种高度专门化的形式。 膜流量，这是这项提议的主要目标 研究这一过程背后的分子机制。也许是因为 最好理解的跨上皮运输的例子是 多聚免疫球蛋白介导的聚合免疫球蛋白IgA和IgM 受体(分泌成分)。初步数据显示，运输 跨上皮细胞的受体结合后显著增强 二聚体的免疫球蛋白A，并且在很大程度上不依赖于受体的磷酸化。 数据进一步表明，这种差异是配体之间的行为- 被占用和未被占用的受体可能是由于 在配体存在的情况下受体的二聚化/寡聚化。 这项建议的目的之一是将异源三聚体GTP结合蛋白 跨上皮运输。尽管大多数调查人员都专注于 Ras家族中较大的异三聚体G蛋白(称为RABS)， 越来越多的证据支持更大的、异三聚体G的作用 蛋白质在一些囊泡运输过程中。我们的初步证据 提示一类异源三聚体G蛋白Gs可能调节 二聚体免疫球蛋白A通过多聚Ig受体的跨皮转运。 我们将首先确定这种参与是否是针对IgA的 运输，或者说是跨细胞运输的一般性质 路径。使用模型上皮细胞系(MDCK细胞)，我们将 在突变的GSα亚基中的横断面 积极的，或在构成上不活跃并作为显性否定的 内源性GalphaS抑制剂。然后，我们将研究 这些突变蛋白对三者跨上皮转运的影响 不同的标记：1)辣根过氧化物酶(液体相)2)植物 毒素蓖麻毒素，与许多膜上的末端半乳糖残基结合 蛋白质和脂类，(大块膜流的标志)；3)二聚体IgA (受体介导的跨细胞作用的标志)。最后一个目标是 鉴定和鉴定与多聚物相互作用的细胞蛋白 免疫球蛋白受体及其转运功能。这些实验将 提供有用的洞察力，了解大分子在 特定的免疫球蛋白通过上皮细胞运输，这是一个过程 这是粘膜宿主防御的关键组成部分。

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.