Ligand binding and processing. The perfused liver as a model system.
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Biomedical subjects
C A Renfrew
Publications and source records attributed to C A Renfrew.
Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors.
We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway.
Animals↗
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.
Amino Acid Sequence↗
Sequential processing of epidermal growth factor in early and late endosomes of rat liver.
We have used isolated perfused rat livers to examine the intracellular processing of 125I-epidermal growth factor (EGF) and to determine where in the endocytic pathway the hydrolases which degrade EGF are acting. Following uptake of 125I-EGF at 37 or 16 degrees C, subcellular fractions enriched in endosomes and lysosomes were isolated, and their 125I-EGF content was examined by reverse-phase high performance liquid chromatography. Three forms of EGF processed at their carboxyl termini are generated in endosomes. At 37 degrees C, EGF is first processed in early endosomes by a carboxypeptidase B-like protease and is further processed in late endosomes by a trypsin-like protease and then a carboxypeptidase B-like protease. At 16 degrees C, entry of EGF into late endosomes is slowed, and only the first processed form is generated over 60 min. Longer perfusions (180 min) at 16 degrees C result in some processing (7%) by proteases found in late endosomes. EGF-horseradish peroxidase cytochemistry confirmed that the additional processing detected at 180 min correlated with movement of EGF from tubulovesicular to multivesicular endosomes. These results, combined with in vitro incubations of EGF in isolated endosomal and lysosomal fractions, suggest that different proteases are active at selective points in the endocytic pathway and that the full complement of proteases needed for complete degradation of EGF is active only in lysosomes.
Amino Acid Sequence↗