CELLULAR MECHANISMS OF MACROMOLECULAR TRANSPORT ACROSS EPITHELIA

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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.