Polymeric IgA and IgM (polymeric immunoglobulins, pIg) bind to a receptor on the basolateral surface of many types of epithelial cells and are endocytosed, transported across the cell in vesicles, and exocytosed into external secretions. In a related process, neonatal rats acquire immunity from their mothers by transporting milk IgG from the lumen of the small intestine to the basolateral surface of the intestinal cell and releasing it into the circulation. Transport of pIg and IgG are in opposite directions across the cell. The goal of the research is to determine the molecular mechanisms of transcellular transport of pIg and IgG. This is important for four reasons. First, transport of pIg into secretions is a major defense against infection. Second, failure to transport IgA may lead to IgA immune complex deposition, which occurs in several diseases. Third, intestinal transport of IgG in rats is a model for transplacental transport of IgG in humans, which is difficult to study. Fourth, transport of pIg and IgG is an excellent model for studying general mechanisms of protein sorting, and can thus provide insights relevant to a broad range of biomedical problems. Several approaches will be used. 1) The exons in the pIg receptor (pIg-R) gene will be located to understand the functional units of the protein. 2) The pIg-R has been expressed in polarized Madin Darby Canine Kidney (MDCK) cells, where it functions as in vivo. Its pathway will be studied by immunocytochemistry. 3) The cytoplasmic tail of pIg-R contains sorting signals that send pIg-R to the basolateral surface and causes endocytosis. This tail has been produced by expression in E. coli and its structure will be studied by X-ray diffraction. 4) The sorting signals in the tail will be studied by making various deletions as well as fusing it to other proteins and expressing these in MDCK cells. 5) Proteins that bind to this cytoplasmic tail will be isolated. 6) Cell-free assays for pIg-R transport will be developed. 7) The intestinal IgG receptor will be cloned and sequenced, and the cDNA will be expressed in MDCK cells to study the receptor's function.
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