The neonatal Fc receptor (FcRn) transports IgG across epithelial cell barriers to provide maternal antibodies to offspring and recycles serum IgG to protect it from catabolism. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We solved crystal structures of rat FcRn alone and bound to Fc. The FcRn/Fc structure revealed an oligomeric ribbon of FcRn dimers bridged by Fcs, which we propose forms inside a transport vesicle. Formation of the ribbon by FcRn dimers on parallel adjacent membranes of the vesicle would impart morphological changes and result in clustering of FcRn cytoplasmic tails that could communicate to cytoplasmic motors that ligand is bound inside the vesicle. To test this hypothesis and to study the general properties of FcRn-mediated transport of IgG, we expressed rat FcRn in polarized MDCK cells. The transfected cells transcytose apically internalized IgG into the basolateral media. We will use this system to address the following specific aims: (1) Characterize intracellular trafficking pathways of wildtype proteins and oligomeric ribbon-disrupting mutants in FcRn-expressing MDCK cells. We will use confocal fluorescence imaging to study intracellular trafficking of FcRn and Fc and monitor FcRn dimerization during trafficking using a FRET assay involving cyan and yellow forms of GFP attached to FcRn. The trafficking of two types of mutants that disrupt the oligomeric ribbon but still form FcRn/Fc complexes will be investigated: (i) a heterodimeric Fc (hdFc) with only one functional FcRn binding site, and (ii) FcRn mutants that cannot dimerize. (2) Electron microscopic imaging of FcRn-expressing MDCK cells and FcRn-containing synthetic membranes. We will use electron tomography to derive 3D reconstructions of transport vesicles within the FcRn-expressing MDCK cells to a resolution of ~6 nm and directly visualize the oligomeric ribbon by reconstructing an ~2 nm resolution 3D image of the adjacent membranes of FcRn-containing synthetic vesicles that are bridged by wt Fc. (3) Test whether antigens bound to IgG can be transcytosed or recycled. We will determine the ability of antigens to undergo FcRn-mediated transport as a function of antigen size. We predict that large antigens such icosahedral viruses would be excluded from vesicles containing the oligomeric ribbon, perhaps representing a mechanism to ensure that pathogens are not transported to offspring along with maternal antibodies. (4) Identify signals instead of or in addition to the oligomeric ribbon that route FcRn- I IgG-containing vesicles to their correct destination. ? ? ? ? ? ?
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