Academic Journal
Functional characteristics of insulin receptors with a Thr-->Ser1200 mutation overexpressed in Chinese hamster ovary cells
| العنوان: | Functional characteristics of insulin receptors with a Thr-->Ser1200 mutation overexpressed in Chinese hamster ovary cells |
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| المؤلفون: | Schutt, M, Benecke, H, Drenckhan, M, Klein, HH |
| المصدر: | European Journal of Endocrinology ; page 125-131 ; ISSN 0804-4643 1479-683X |
| بيانات النشر: | Oxford University Press (OUP) |
| سنة النشر: | 2000 |
| مصطلحات موضوعية: | Endocrinology, General Medicine, Endocrinology, Diabetes and Metabolism |
| الوصف: | OBJECTIVE: To investigate the functional properties of insulin receptors with a Thr-->Ser(1200)-mutation that is associated with severe insulin resistance in humans. DESIGN AND 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. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | unknown |
| DOI: | 10.1530/eje.0.1430125 |
| الاتاحة: | http://dx.doi.org/10.1530/eje.0.1430125 https://eje.bioscientifica.com/view/journals/eje/143/1/125.xml |
| رقم الانضمام: | edsbas.61059547 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1530/eje.0.1430125 |
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