The polymeric immunoglobulins, IgA and IgM (pIg) bind to a pIg receptor (pIgR) on the basolateral surface of many types of epithelial cells. The receptor and ligand are endocytosed and delivered to early endosomes. Here they are sorted into transcytotic vesicles, which can fuse with the apical surface and deliver the pIg into external secretions. The goal of this research is to determine the molecular mechanism of transcytosis of pIg. This is important for three 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, transport of Pig is an excellent model for studying general mechanisms of protein sorting in polarized epithelial cells, and can thus provide insights relevant to a broad range of biomedical problems. Several approaches will be used. 1) The transcytotic pathway of the Pigr will be studied by laser confocal fluoresence microscopy and EM immunocytochemistry. 2) The Pigr contains a C-terminal, 103 amino acid cytoplasmic domain. A 17 residue segment of this domain contains the sorting signals for delivery of the Pigr from the trans-Golgi network (TGN) to the basolateral surface, and for transcytosis of the pIgR after endocytosis. This segment will be systematically mutated to precisely define the requirements for these signals. 3) The three-dimensional structure of this segment will be studied by circular dichroism and NMR spectroscopy. 4) Proteins that interact with this 17 residue segment will be identified by affinity chromatography using a synthetic peptide corresponding to this segment as a ligand. Antibodies to these proteins will be raised for use in immunolocalization and functional studies. These proteins will also be cloned. 5) Other proteins that interact with the pIgR will be isolated by reversible cross-linking and co-immunoprecipitation. 6) The role of cytosolic proteins, especially GTPase, in budding of transcytotic vesicles will be studied in a reconstituted system. 7) Transport from the TGN to the basolateral surface will be reconstituted in a perforated cell system. 8) The composition of transcytotic and TGN derived vesicles will be compared.
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