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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029133-10
Application #
3276635
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-04-01
Project End
1992-03-31
Budget Start
1990-04-01
Budget End
1992-03-31
Support Year
10
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Casciola-Rosen, L A; Hubbard, A L (1992) Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase. J Biol Chem 267:8213-21
Casciola-Rosen, L A; Renfrew, C A; Hubbard, A L (1992) Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors. J Biol Chem 267:11856-64
Berryman, M A; Porter, W R; Rodewald, R D et al. (1992) Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry. J Histochem Cytochem 40:845-57
Casciola-Rosen, L A; Hubbard, A L (1991) Hydrolases in intracellular compartments of rat liver cells. Evidence for selective activation and/or delivery. J Biol Chem 266:4341-7
Renfrew, C A; Hubbard, A L (1991) Degradation of epidermal growth factor receptor in rat liver. Membrane topology through the lysosomal pathway. J Biol Chem 266:21265-73
Renfrew, C A; Hubbard, A L (1991) Sequential processing of epidermal growth factor in early and late endosomes of rat liver. J Biol Chem 266:4348-56
Braiterman, L T; Chance, S C; Porter, W R et al. (1989) The major subunit of the rat asialoglycoprotein receptor can function alone as a receptor. J Biol Chem 264:1682-8
Mueller, S C; Hubbard, A L (1986) Receptor-mediated endocytosis of asialoglycoproteins by rat hepatocytes: receptor-positive and receptor-negative endosomes. J Cell Biol 102:932-42
Dunn, W A; Connolly, T P; Hubbard, A L (1986) Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. J Cell Biol 102:24-36
Hoppe, C A; Connolly, T P; Hubbard, A L (1985) Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway. J Cell Biol 101:2113-23

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