title: C-terminus or juxtamembrane deletions in the insulin receptor do not affect the glucose-dependent inhibition of the tyrosine kinase activity.
abstract: We have previously shown, in rat-1 fibroblasts which stably overexpress high levels of human insulin receptor (HIR), that high glucose levels induce an inhibition of insulin receptor tyrosine kinase (IRK) activity [Berti, L., Mosthaf, L., Kellerer, M., Tippmer, S., Mushack, J., Seffer, E., Seedorf, K., Häring, H. (1994) J. Biol. Chem. 269, 3381-3386]. This effect appears to be mediated through activation of protein kinase C and phosphorylation of the receptor beta-subunit on threonine or serine residues. The aim of the present study was to determine whether the juxtamembrane region or the C-terminus tail of the receptor are involved in the IRK modulation by glucose. In these domains increased serine and threonine phosphorylation was observed after phorbol ester or insulin stimulation of cells, and a regulatory function for IRK activity seems conceivable. We used an antibody directed against one potential regulatory site in the C-terminus tail, i.e. PSer1315, to study the effect of glucose. An increased signal was detected in HIR from rat-1 fibroblasts treated with phorbol 12-myristate 13-acetate or glucose (25 mM). To investigate whether this site in the C-terminus is essential for glucose-dependent IRK inhibition, rat-1 fibroblasts stably overexpressing a C-terminus-truncated human insulin receptor lacking 43 amino acids (HIR delta CT) were studied in parallel with cells expressing the wild-type receptor. As described earlier, HIR delta CT has lost the ability to stimulate glucose uptake. Glucose (25 mM) inhibited the insulin effect on the autophosphorylation of both receptors to a similar extent. Thus, glucose (25 mM) stimulates phosphorylation of Ser1315, however, this appears not to mediate the inhibitory effect on IRK. To test whether serine residues 955/956 and 962/964 in the juxtamembrane region of the insulin receptor are involved in the inhibitory effect of glucose, 293 cells transiently transfected either with wild-type HIR or HIR with a juxtamembrane deletion spanning amino acids 954-965 [des-(954-965)-HIR] were studied in parallel. As described earlier, the des-(954-965)-HIR has lost the ability to stimulate PI-3 kinase. However, 25 mM glucose equally inhibited the insulin effect on tyrosine phosphorylation of the receptor. Together, the data suggest that the regulatory serine or threonine phosphorylation site(s) involved in the inhibitory effect of hyperglycemia are neither located in the C-terminus nor in the juxtamembrane region of the insulin receptor beta subunit.
authors.affiliation: Institut für Diabetesforschung, München, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Mosthaf
authors.forename: L
authors.initials: L
authors.lastname: Berti
authors.forename: M
authors.initials: M
authors.lastname: Kellerer
authors.forename: J
authors.initials: J
authors.lastname: Mushack
authors.forename: E
authors.initials: E
authors.lastname: Seffer
authors.forename: B
authors.initials: B
authors.lastname: Bossenmaier
authors.forename: M
authors.initials: M
authors.lastname: Coghlan
authors.forename: K
authors.initials: K
authors.lastname: Siddle
authors.forename: A
authors.initials: A
authors.lastname: Ullrich
authors.forename: H U
authors.initials: HU
authors.lastname: Häring
mesh_terms: D019325:3-O-Methylglucose; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D007333:Insulin Resistance; D008757:Methylglucosides / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D007328:Insulin; D008757:Methylglucosides; D011994:Recombinant Proteins; D019325:3-O-Methylglucose; D020794:Receptor Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D005947:Glucose