Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor.
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.
Sbraccia
P
P
Division of Diabetes and Endocrine Research, Mount Zion Hospital and Medical Center, California.
Wong
K Y
KY
Brunetti
A
A
Rafaeloff
R
R
Trischitta
V
V
Hawley
D M
DM
Goldfine
I D
ID
D000818:Animals; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D007328:Insulin; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin