Functional characteristics of insulin receptors with a Thr -> Ser(1200) mutation overexpressed in Chinese hamster ovary cells

Objective: To investigate the functional properties of insulin receptors wi th a Thr-->Ser(1200)-mutation that is associated with severe insulin resist ance in humans. Design and methods: The effect of in situ insulin-stimulation on insulin re ceptor kinase activity was studied in Chinese hamster ovary cells with over expressed human Ser(1200)-mutated, non-mutated, and ATP-binding site-mutate d (Lys-->Arg(1030)) receptors using a microwell-based assay that only detec ts human (and not hamster) insulin receptors. Moreover, the fraction of ant i-phosphotyrosine antibody-binding receptors following in situ stimulation was separated, and autophosphorylation and kinase activity resulting from i n 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)-mutate d 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-mutat ed 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 al kaline phosphatase concentrations (EC50: 3 U/ml and 70 U/ml respectively). No activation of insulin receptor kinase was observed with Arg(1030)-mutate d receptors. Conclusions: Overexpressed Ser(1200)-mutated human insulin receptors posses s insulin-stimulated kinase activity and can be activated in situ and in vi tro. They are characterized by a markedly slower autophosphorylation reacti on, which, in a phosphatase-containing environment, results in a small frac tion of phosphorylated and activated receptors.