DESCRIPTION: The long term objective of this proposal is to elucidate the molecular regulation of vesicle-mediated transcytosis in hepatocytes and how regulation is altered by liver disease. The polymeric IgA receptor (pIgA-R), a component of the transcytotic carriers (TCs), is used as a marker protein because its maturation can be monitored precisely and the PI has already shown that transcytosis of the pIgA-R is disrupted during mechanical cholestasis, a clinically relevant condition. The general hypothesis is that rab proteins, a group of low molecular weight GTP-binding proteins, associate with TCs and participate in the formation of the "molecular sorting complex" which regulates the transcytosis of the pIgA-R. Specific aim 1 will investigate the distribution of the pIgA-R relative to distinct rab proteins associated with the transcytotic pathway in control versus cholestatic livers using immunofluorescence, immunoelectron microscopy, and cell fractionation. Specific aim 2 will identify protein components of TCs isolated from control and cholestatic livers using sucrose density gradient centrifugation and immunoisolation. Colocalization of rab proteins, pIgA-Rs, and other relevant proteins will be assessed by immunoblot analysis and immunocytochemistry. Specific aim 3 will investigate the functional role of pIgA-R and distinct rab proteins in regulating transcytosis. To do this, transcytosis in cultured hepatocytes will be perturbed with pharmacological agents and by microinjection of peptides and peptide-specific antibodies to the pIgA-R and relevant rab proteins. Changes in cellular distribution of the pIgA-R, its ligand, and rab proteins and the extent to which the manipulations alter transcytosis of ligand will be analyzed by confocal microscopy.