Functional characteristics of insulin receptors with a Thr-->Ser1200 mutation overexpressed in Chinese hamster ovary cells.
OBJECTIVE
To investigate the functional properties of insulin receptors with a Thr-->Ser(1200)-mutation that is associated with severe insulin resistance in humans.


METHODS
The effect of in situ insulin-stimulation on insulin receptor kinase activity was studied in Chinese hamster ovary cells with overexpressed human Ser(1200)-mutated, non-mutated, and ATP-binding site-mutated (Lys-->Arg(1030)) receptors using a microwell-based assay that only detects human (and not hamster) insulin receptors. Moreover, the fraction of anti-phosphotyrosine antibody-binding receptors following in situ stimulation was separated, and autophosphorylation and kinase activity resulting from in situ and/or in vitro activation evaluated in this fraction.


RESULTS
Although insulin-stimulated kinase activity of human-specific anti-insulin receptor antibody-binding receptors in cells with Ser(1200)-mutated insulin receptors represented only 3.3% of that reached in cells with non-mutated receptors, a clear insulin-induced increase in kinase activity was observed (3.4-fold; P<0.05). This increase was associated with a 2.3+/-0.6% (P<0.05) increase in anti-phosphotyrosine-binding receptors with a kinase activity representing 43+/-8% of that found in activated non-mutated receptors. In vitro autophosphorylation and kinase activation proceeded much more slowly in Ser(1200)-mutated receptors (t(1/2)): 100 min) compared with non-mutated receptors (t(1/2)): 1 min) and were inhibitable by lower alkaline phosphatase concentrations (EC(50): 3 U/ml and 70 U/ml respectively). No activation of insulin receptor kinase was observed with Arg(1030)-mutated receptors.


CONCLUSIONS
Overexpressed Ser(1200)-mutated human insulin receptors possess insulin-stimulated kinase activity and can be activated in situ and in vitro. They are characterized by a markedly slower autophosphorylation reaction, which, in a phosphatase-containing environment, results in a small fraction of phosphorylated and activated receptors.
Schütt
M
M
Department of Internal Medicine I, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
Benecke
H
H
Drenckhan
M
M
Klein
H H
HH
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics* / Q000502:physiology*; D012694:Serine; D013552:Swine; D013912:Threonine; D014162:Transfection
D007328:Insulin; D013912:Threonine; D012694:Serine; D007334:Insulin-Like Growth Factor I; D011494:Protein Kinases; D011972:Receptor, Insulin; D000469:Alkaline Phosphatase