C-TERMINUS OR JUXTAMEMBRANE DELETIONS IN THE INSULIN-RECEPTOR DO NOT AFFECT THE GLUCOSE-DEPENDENT INHIBITION OF THE TYROSINE KINASE-ACTIVITY

We have previously shown, in rat-1 fibroblasts which stably overexpres s high levels of human insulin receptor (HIR), that high glucose level s induce an inhibition of insulin receptor tyrosine kinase (IRK) activ ity [Berti, L., Mosthaf, L., Kellerer, M., Tippmer, S., Mushack, J., S effer, E., Seedorf, K., Haring, H. (1994) J. Biol. Chem. 269, 3381-338 6]. This effect appears to be mediated through activation of protein k inase C and phosphorylation of the receptor beta-subunit on threonine or serine residues. The aim of the present study was to determine whet her the juxtamembrane region or the C-terminus tail of the receptor ar e involved in the IRK modulation by glucose. In these domains increase d serine and threonine phosphorylation was observed after phorbol este r or insulin stimulation of cells, and a regulatory function for IRK a ctivity seems conceivable. We used an antibody directed against one po tential regulatory site in the C-terminus tail, i.e. PSer1315, to stud y the effect of glucose. An increased signal was detected in HIR from rat-1 fibroblasts treated with phorbol 12-myristate 13-acetate or gluc ose (25 mM). To investigate whether this site in the C-terminus is ess ential for glucose-dependent IRK inhibition, rat-1 fibroblasts stably overexpressing a C-terrninus-truncated human insulin receptor lacking 43 amino acids (HIR Delta CT) were studied in parallel with cells expr essing the wild-type receptor. As described earlier, HIR Delta CT has lost the ability to stimulate glucose uptake. Glucose (25 mM) inhibite d the insulin effect on the autophosphorylation of both receptors to a similar extent. Thus, glucose (25 mM) stimulates phosphorylation of S er1315, however, this appears not to mediate the inhibitory effect on IRK. To test whether serine residues 955/956 and 962/964 in the juxtam embrane 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 earlie r, the des-(954-965)-HIR has lost the ability to stimulate PI-3 kinase . However, 25 mM glucose equally inhibited the insulin effect on tyros ine phosphorylation of the receptor. Together, the data suggest that t he regulatory serine or threonine phosphorylation site(s) involved in the inhibitory effect of hyperglycemia are neither located in the C-te rminus nor in the juxtamembrane region of the insulin receptor beta su bunit.