Altered degradation of epidermal growth factor in a diphtheria toxin-resistant clone of KB cells.
We have investigated the cellular fate of epidermal growth factor (EGF) in KB cells and a variant, KB-R2A, that was isolated based on its resistance to diphtheria toxin and subsequently was shown to be resistant to infection by RNA viruses (Moehring and Moehring, 1972, Infect. Immunity. 6:487-492). Both cell lines bind 125I-EGF and internalize the cell-bound hormone at the same rate. However, when the degradation of internalized 125I-EGF was measured by the release of low molecular weight (mw) hydrolysis products into the medium, the toxin-resistant KB-R2A cells degraded the hormone at a drastically reduced rate; 50% and 3% of the cell-bound 125I-EGF was degraded and released by 80 min in the KB and KB-R2A cells, respectively. To investigate the fate of cell-associated EGF prior to release into the medium, the radioactivity in extracts of cells labeled with 125I-EGF was fractionated by native gel electrophoresis. In KB cells three peaks of radioactivity other than native 125I-EGF were resolved. Time course and subcellular fractionation studies showed that the first processed product appeared while the hormone was located in the endocytic vesicles and the appearance of the other two peaks correlated with the arrival of the hormone in the lysosomal compartment. KB-R2A cells also produced the first intermediate but they produced only very low amounts of the other two peaks. These studies show that endocytic vesicles in both cell lines contain enzymes capable of processing EGF prior to the arrival of the hormone in the lysosomes and show that the KB-R2A cells have a lesion that prevents the complete degradation of the hormone. We propose that the KB-R2A cell line has a defective mechanism for the intracellular processing of a number of ligands that are internalized by the process of receptor-mediated endocytosis and that this defect is located beyond the initial endocytic step.
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002999:Clone Cells / Q000187:drug effects; D003080:Cold Temperature; D003470:Culture Media; D004167:Diphtheria Toxin / Q000494:pharmacology*; D004351:Drug Resistance; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D051379:Mice; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D003470:Culture Media; D004167:Diphtheria Toxin; D004815:Epidermal Growth Factor