Receptor-mediated endocytosis plays an important role in allowing mammalian cells to sense and respond to their environment. During the last ten years, significant progress has been made in understanding this fundamental process, but a number of questions remain unanswered. The primary goal of the research described in this proposal is to make accurate, temporally-resolved measurements of the related processes of ligand/receptor acidification, exposure to hydrolytic (lysosomal) enzymes, and sorting of ligands and receptors, and to relate these measurements to the biological role of the ligand being studied. The kinetics of acidification and the temperature dependence of both internalization and acidification will be determined for transferrin, epidermal growth factor (EGF), histocompatibility antigen H-2K, and dextran; the kinetics and temperature dependence of entry of endocytosed fluid into compartments containing proteolytic enzymes will be measured using fluorogenic enzyme substrates. Differences in these properties between cell types, or the same cell type under different conditions (such as iron starvation), will also be determined. Flow cytometric analysis and density gradient centrifugation will be used to determine the kinetics with which the pathways of endocytosis of transferrin, EGF, H2K and dextran diverge, and to determine some of the biochemical characteristics of the compartments involved.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032508-06
Application #
3281420
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1983-08-01
Project End
1990-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Carnegie-Mellon University
Department
Type
Schools of Arts and Sciences
DUNS #
052184116
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Brockman, S A; Murphy, R F (1994) Isolation and analysis of somatic cell mutants with defects in endocytic traffic. Methods Cell Biol 42 Pt B:131-48
Murphy, R F; Schmid, J; Fuchs, R (1993) Endosome maturation: insights from somatic cell genetics and cell-free analysis. Biochem Soc Trans 21 ( Pt 3):716-20
Wilson, R B; Mastick, C C; Murphy, R F (1993) A Chinese hamster ovary cell line with a temperature-conditional defect in receptor recycling is pleiotropically defective in lysosome biogenesis. J Biol Chem 268:25357-63
Cain, C C; Wilson, R B; Murphy, R F (1991) Isolation by fluorescence-activated cell sorting of Chinese hamster ovary cell lines with pleiotropic, temperature-conditional defects in receptor recycling. J Biol Chem 266:11746-52
Sipe, D M; Jesurum, A; Murphy, R F (1991) Absence of Na+,K(+)-ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures. J Biol Chem 266:3469-74
Sipe, D M; Murphy, R F (1991) Binding to cellular receptors results in increased iron release from transferrin at mildly acidic pH. J Biol Chem 266:8002-7
Roederer, M; Barry, J R; Wilson, R B et al. (1990) Endosomes can undergo an ATP-dependent density increase in the absence of dense lysosomes. Eur J Cell Biol 51:229-34
Bowser, R; Murphy, R F (1990) Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: early introduction of lysosomal enzymes into the endocytic pathway. J Cell Physiol 143:110-7
Yamashiro, C T; Kane, P M; Wolczyk, D F et al. (1990) Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton-translocating ATPase. Mol Cell Biol 10:3737-49

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