The long-term objective of this research is to understand the dynamics and mechanisms of receptor-mediated endocytosis. Three different ligand-receptor systems in one cell, the rat hepatocyte are being studied: the asialoglycoprotein, epidermal growth factor (EGF) and polymeric immunoglobulin A (pIgA) systems. The first two ligands were traced through the lysosomal pathway and three pre-lysosomal (endosomal) subcompartments identified biochemically (by subcellular fractionation) and morphologically (with cytochemical EM tracers). The transcellular route of pIgA (traced by morphology) overlaps part of the lysosomal pathway, but segregates ligand into unique transport vesicles prior to secretion into bile. The role of acidification in ligand-receptor and ligand-ligand sorting will be studied by selectively labeling the contents of each endosomal element and the transport vesicles with a pH-sensitive (fluorescein) conjugate of each ligand in isolated perfused livers. ATP-dependent pH changes of the fluorescein derivative in enriched subcellular fractions will be measured. Specific endosomal subcompartments and transport vesicles will be isolated by combinations of gradient fractionation, free-flow electrophoresis and immunoadsorption with antibodies to cytoplasmic domains of each receptor. Protein compositions will be analyzed using SDS-PAGE, 125I-lectins, antibodies to specific membrane proteins and enzyme activities, and compared to all relevant organelles in the pathways (eg lysosomes, sinusoidal or bile front membranes, other endosomes). Use of hybridoma technology will determine whether unique antigens are present in any or each endosomal subcompartment and/or the transport vesicles. Newly-found low-affinity EGF receptors, in a biochemically distinct location from high-affinity receptors, are activated by exposure of hepatocytes to EGF and then rapidly degraded in lysosomes. The two types of receptors will be further compared in both hepatocytes and NRK cells. Immuno-EM will be used to localize each. Differences in phosphorylation states (using 32Pi and phosphopeptide analysis), lipid and/or cytoskeletal interactions (using differential extractions) will be examined. Pulse-chase protocols with radiolabeled amino acids, followed by subcellular fractionation and immunochemical detection, will establish biosynthetic relationships. Finally, monoclonal and polyclonal antibodies to the cytoplasmic and ectoplasmic domains of the EGF receptor will be used in kinetic experiments to follow the receptor's fate in non-mitogenic hepatocytes and mitogenic NRK cells after short and long exposure to EGF.
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