Objective: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IRC60S) showed a defective insulin internalization without affecting insulin binding and IR autophosphorylation. Moreover, this mutation reduces insulin receptor substrate (IRS)-1 tyrosine phosphorylation and insulin-induced metabolic and mitogenic effects. Altogether, these observations support a role of the extracellular domain of IR P-subunit in insulin and receptor intracellular targeting as well as in insulin signaling. Design and methods: This study assesses in more details the effect of IRC860S mutation on the trafficking of the insulin-IR complex. In particular, IR internalization, phosphorylation, dissociation and recycling, as well as insulin degradation and retroendocytosis have been investigated in CHO cells overexpressing either wild type (CHO-IRWT) or mutated IRs. Results: the C860S mutation significantly decreases IR internalization both insulin stimulated and constitutive. In spite of a similar dissociation of internalized insulin-IR complex, recycling of internalized IR was significantly faster (half life (t(1/2)): 21 min vs 40 min, P < 0.001) and more extensive (P < 0.01) for IRC860S than for. On the other hand, insulin degradation and retroendocytosis were superimposable in both cell lines. As expected, insulin-induced phosphorylation was similar in both IRs, however dephosphorylation was much more rapid and was greater (P < 0.01) in CHO-IRWT as compared with CHO-IRC860S cells. Conclusions: Transmembrane and intracellular domain of IR seem to be determinants for IR internalization. Now we report that Cys 860 in the IR P-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.
|Autori:||BENZI L; CECCHETTI P.; CICCARONE AM.; NOVELLI S.; PAOLI A.; BERTACCA A.; MAGGI D.; ANDRAGHETTI G.; DEL PRATO S.; CORDERA R.|
|Titolo:||The extracellular portion of the insulin receptor bete-subunit regulates the cellular trafficking of the insulin-insulin receptor complex. Studies on Chinese hamster ovary cell carrying the Cys 860 —Ser insulin receptor mutation|
|Anno del prodotto:||2003|
|Digital Object Identifier (DOI):||10.1530/eje.0.1480365|
|Appare nelle tipologie:||1.1 Articolo in rivista|