Abstract. The intracellular sorting of EGF-receptor complexes (EGFoRC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37"C. The initial rate of EGF recycling was reduced at 18°C. A significant pool of internalized EGF was incapable of recycling at 18°C but began to recycle when cells were warmed to 37°C. The relative rate of EGF outflow at 370C from ceils exposed to an 18°C temperature block was slower (tla = 20 min) than the rate from cells not exposed to a temperature block (tv2 = 5-7 min). These data suggest that there might be both short-and long-time cycles of EGF recycling in A431 cells. Examination of the intraceUular EGF-RC dissociation and dynamics of short-and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of t25I-EGF. Since EGF-RC degradation was also blocked at 18°C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.

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A 160,000 mol wt precursor of the epidermal growth factor (EGF) receptor has been identified in human A-431 carcinoma cells and skin fibroblasts. The presence of one discrete precursor band indicates the presence of a slow processing step. We have determined that this slow processing step involves the conversion of high mannose N-linked oligosaccharides on the receptor precursor to primarily complex oligosaccharides on the mature form of the receptor. This is shown by 1) the presence of fucose, a characteristic terminal sugar of complex oligosaccharides, in only the mature receptor and by 2) the susceptibility of the precursor to digestion with endoglycosidase H, which cleaves high mannose N-linked oligosaccharides, but not complex oligosaccharides from glycoproteins. The precursor to mature receptor transition half-time is 1.7 h in A-431 cells. This long transition half-time causes an accumulation of approximately 7.2 X 10(5) precursor molecules per cell (approximately 12% of the total population of EGF receptors). The net quantity of mature EGF receptors, but not of receptor precursors, is reduced when EGF is added to the culture medium of A-431 cells. The presence of EGF in the growth medium also decreases electrophoretic migration (as a result of increased phosphate incorporation) of the mature receptor, but not that of the precursor. The EGF-insensitive state of the precursor is most likely due to its intracellular location.

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125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.