Insulin internalization in the absence of the insulin receptor tyrosine kinase domain is insufficient for mediating intracellular biological effects.
The intracellular portion of the human insulin receptor (hIR) beta-subunit contains distinct functional domains including the exon 16-encoded juxtamembrane (JM) domain that mediates endocytosis, and the tyrosine kinase (TK) domain that mediates insulin's metabolic and mitogenic actions. To explore the functional relationship between these domains and to determine the role of insulin internalization in insulin action, we constructed and studied the endocytic and signaling properties of an hIR mutant truncated at Glu-1012. This truncation removes the carboxyl-terminal 343 amino acids containing essentially all of the TK domain but leaves behind the exon 16-encoded JM domain that is necessary for endocytosis, plus an additional 35 amino acids downstream. The wild-type (hIR-WT) and mutant (hIR delta 343) receptors were stably expressed in CHO cells and their abilities to mediate various insulin-stimulated functions were comparatively analyzed. In cells expressing hIR-WT, insulin markedly enhanced tyrosine phosphorylation of the beta-subunit and of the endogenous 185 kDa substrate whereas these effects were completely absent in cells expressing hIR delta 343. The hIR delta 343 receptors retained the ability to internalize a significant amount of surface-bound insulin at 37 degrees C. However, they were unable to mediate either the short or long-term biological effects of insulin as determined by assaying insulin-stimulated glucose uptake (assessed by 2-deoxyglucose uptake), protooncogene expression (measured by Northern blot analysis of c-fos mRNA) and DNA synthesis (measured by 3H-thymidine incorporation). These results indicate that the hIR beta-subunit JM and TK domains can be functionally uncoupled, and that insulin internalization in the absence of hIR TK domain and kinase activity is insufficient for mediating intracellular insulin action.
Schranz
D B
DB
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
Rohilla
A M
AM
Anderson
C
C
Wood
W M
WM
Berhanu
P
P
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D004705:Endocytosis; D005091:Exons; D018698:Glutamic Acid; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D013936:Thymidine / Q000378:metabolism; D014162:Transfection
D016895:Culture Media, Serum-Free; D007328:Insulin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D018698:Glutamic Acid; D004247:DNA; D011972:Receptor, Insulin; D013936:Thymidine