We have previously reported that prolonged incubations of Fao cells, a cell line derived from the well-differentiated Reuber H35 rat hepatoma, with 10-6 M insulin, induced a decrease in receptor number (down-regulation), an increase in receptor affinity for insulin, and a loss of insulin's biological effect (desensitization). In the present study, we have investigated the relationship between these changes in insulin binding and action and changes in the structure of the insulin receptor. Intact cells were surface labeled with Na125I and lactoperoxidase, and the 125I-labeled insulin receptor was immunoprecipitated using specific antibodies and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of gels done under reducing conditions demonstrated the α (M(r) = 135,000) and the β (M(r) = 95,000) subunits of the receptor. In nonreduced gels, free insulin receptor subunits were observed as well as four higher molecular weight bands with M(r) = 210,000, 270,000, 350,000 and 520,000. Two-dimensional gel electrophoresis revealed that these bands correspond to α-β heterodimer, α2 homodimer, and two α-β oligomers of high molecular weights, respectively. Cross-linking of 125I-insulin to intact cells with disuccinimidyl suberate revealed bands of M(r) = 125,000, 210,000, 250,000 and 320,000, indicating that most of the forms of the receptor could bind insulin. After incubation with 10-6 M insulin for 24 h, Fao cells revealed a marked decrease of the four oligomeric forms of the receptor, with little change in the level of the free α and β subunits. A similar decrease of the oligomeric forms of the insulin receptor and an increase in the free subunits was observed when normal Fao cells are treated with 7 mM dithiothreitol. In dithiothreitol-treated cells, 125I-insulin binding was increased and this increase was accounted for by a change in affinity. In contrast to Fao cells, down-regulation of the insulin receptor in IM-9 lymphocytes occurs without a change in receptor affinity. In these cells, surface labeling revealed a decrease in total receptors after down-regulation, but not change in the proportion of the oligomeric forms to the free subunits of the receptor. These data suggest the following in Fao hepatoma cells. 1) In the native state, the insulin receptor consists of free α and β subunits and several kinds of disulfide-linked oligomers of these subunits. 2) Insulin-induced down-regulation of the receptor produces a preferential loss of the oligomeric forms of the receptor. 3) The affinity of the insulin receptor correlates with the proportion of the oligomeric forms present; the greater the proportion of the oligomeric forms, the lower the receptor affinity.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
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