Recent reports suggest that, after initial cell surface binding, insulin receptor complexes are internalized by absorptive endocytosis (1, 2). Following this, insulin is degraded at one or more intracellular sites, possibly involving lysosomes (3-7). The fate of the internalized receptor is unknown at present, although it is most likely degraded or recycled back to the plasma membrane or both. Gavin et al. (8) were the first to report that insulin could mediate the regulation of its own receptors; they found that, when IM-9 cultured lymphocytes were incubated with insulin, a marked reduction in insulin binding occurred. Since this initial report, it has been confirmed that insulin can lead to the loss of cell surface receptors in other cell types such as hepatocytes, adipocytes, and fibroblasts (9-11). Several workers have speculated that the internalization pathway for the insulin receptor may mediate the process of insulin-induced receptor loss. In order to examine the relationship between insulin internalization and receptor regulation, we have studied these processes in two cultured cell lines: normal human fibroblasts and the transformed cell line IM-9 lymphocytes.MATERIALS AND METHODS Materials. Minimal essential medium and trypsin were purchased from GIBCO. Fetal calf serum and bovine serum albumin were purchased from Reheis (Kankakee, IL). Hepes, N-tris[(hydroxymethyl)methylglycine] (Tricine), and chloroquine were purchased from Sigma. Na125I (carrier free) was Experimental Procedure. Binding experiments were performed with cultured human fibroblast monolayers in 60 X 15 mm plastic dishes as follows. After aspiration of growth media, monolayers were washed twice with 3 ml of cool (22°C) binding buffer (minimal essential medium, pH 7.4/25 mM Hepes/25 mM Tricine/1% bovine serum albumin). Then, 1251-labeled insulin (1251-insulin) at 0.3-0.4 mg/ml, insulin standards, other reagents, and buffer were added to a total volume of 2 ml and the dishes were incubated in a shaking water bath (50 oscillations per minute). After incubation, an aliquot of the buffer was withdrawn for estimation of total radioactivity, and the remaining buffer was aspirated and discarded. The monolayers were washed four times with 3 ml of ice-cold Hanks' buffer; then, 1.5 ml of 1 M NaOH was added to each dish. The dishes were rotated (100 oscillations per minute) for 15 min and the NaOH was replaced by another 1.5 ml for another 15 min. These two 1.5-ml portions of NaOH, containing all cell-associated radioactivity, were pooled and assayed in an automatic gamma counter with a 7.62-cm (3-inch) crystal. Thus, the amount bound contained all of the '25I-insulin bound from the original entire 2 ml incubation volume. All data are normalized to a cell concentration of 106 cells per dish.'25I-Insulin was prepared by a modification (12) of the method of Freychet et al. (13) as described (6).Cultured Lymphocytes. These cells were grown in suspension culture in modified minimal essential media as described (8). Cells were subcultured ever...

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