title: Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts.
abstract: Epidermal growth factor (EGF) receptors extracted with Triton X-100 from human skin fibroblasts and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin fibroblasts and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = 16 h) than that (t1/2 = 9 h) of fibroblasts. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from 16 to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*
chemical_list: D011956:Receptors, Cell Surface; D003208:Concanavalin A; D004815:Epidermal Growth Factor; D066246:ErbB Receptors