Degradation of epidermal growth factor receptor in rat liver. Membrane topology through the lysosomal pathway.
We have generated and characterized three rabbit polyclonal antibodies that recognize different regions of the epidermal growth factor receptor (EGF-R) and used them to study the degradation of the receptor in the isolated perfused rat liver. Quantitative immunoblot analyses of rat liver homogenates prepared from tissue biopsies collected at various times after epidermal growth factor (EGF) addition showed that both the ectoplasmic and cytoplasmic domains of rat liver EGF-Rs were degraded with similar kinetics (t1/2 = 3.5-3.8 h at 25 degrees C with cycloheximide). No immunoreactive intermediate breakdown products were detected. EGF-stimulated degradation of both receptor domains was inhibited by the thiol protease inhibitor leupeptin, suggesting lysosome involvement in the hydrolysis of the whole molecule. To study this further, protease protection experiments were performed on endosome- and lysosome-enriched fractions isolated from leupeptin-treated livers. We found that the cytoplasmic domains of greater than 90% of the EGF-Rs in endosomal fractions were accessible to digestion when proteinase K was added to the intact vesicle populations, while the ectoplasmic domain was unaltered. In contrast, both the ectoplasmic and cytoplasmic domains of approximately 55% of the EGF-Rs present in lysosome-enriched fractions were inaccessible to proteinase K digestion in the absence of detergent. These findings suggest that movement of EGF-Rs from the limiting membrane of endosomes to the lumen of lysosomes permits the degradation of the entire EGF-R molecule within lysosomes.
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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Renfrew
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Hubbard
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D010450:Endopeptidases / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism* / Q000648:ultrastructure; D005110:Extracellular Space / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D051381:Rats; D013997:Time Factors
D008562:Membrane Glycoproteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010450:Endopeptidases