Insulin inhibition of protein degradation in cells expressing wild-type and mutant insulin receptors.
The mechanism by which insulin decreases protein degradation is unknown. We examined insulin binding and degradation (125I[A14]insulin) and protein degradation (3H-leucine labeling) in Chinese hamster ovary (CHO) cells transfected with wild-type (WI) and mutant human insulin receptors. The deltaExon-16 mutant is missing the juxtamembrane domain that mediates endocytosis. The delta343 mutant receptor lacks the tyrosine kinase structural domain but retains the juxtamembrane internalization domain. The mutant deltaNPEY lacks the single NPEY sequence located 16 residues after the end of the transmembrane domain. Null transfected cells (NEO) not expressing human receptors were studied as controls. The WT and deltaNPEY cells equivalently internalized and degraded insulin; delta343 cells internalized and degraded insulin, but at a reduced rate; deltaExon-16 cells internalized and degraded significantly less insulin than the other mutants; NEO cells showed essentially no internalization and degradation. In contrast, all cell types showed the same efficacy at inhibition of protein degradation, albeit at different potencies. These results suggest insulin actions are mediated by multiple and redundant effector systems, but that receptor tyrosine kinase activity is not required for inhibition of protein degradation.
Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska 68105, USA. fghamel@unmc.edu
F G
FG
Hamel
J
J
Fawcett
C I
CI
Andersen
P
P
Berhanu
R G
RG
Bennett
W C
WC
Duckworth
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology*; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; D007328:Insulin; D011506:Proteins; C067713:lactacystin; D014443:Tyrosine; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D007930:Leucine; D000111:Acetylcysteine