Abstract
We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.
Highlights
Insulin Down-regulates Insulin Receptor Number and Up-regulates Insulin Receptor Affinity in Cells Expressing a Tyrosine Kinasedefective Insulin Receptor*
Insulinstimulated receptor kinase activity toward poly(Glu-Tyr) was enhanced in HTC-IR cells but reduced by more than 50% in HTC-IR/M-1030 cells when compared to HTC-WT cells
Insulin Receptor Degradation Studies-In order to determine whether insulin accelerated receptor degradation, HTCIR and HTC-IR/M-1030 cells were placed into methioninefree medium, pulsed with [35S]methionine for 4 h, and chased with media containing unlabeled methionine for 24 h in the absence or presence of 1 ELMinsulin (Fig. 6)
Summary
HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 MM) at 37 ‘C! resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells These studies indicate, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor. We demonstrate that insulin can increase insulin receptor degradation and increase receptor affmity in HTC cells transfect,ed with both normal and mutant insulin receptors
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