The extracellular portion of the insulin receptor b-subunit regulates the cellular trafficking of the insulin -insulin receptor complex. Studies on Chinese hamster ovary cells carrying the Cys 860 ! Ser insulin receptor mutation AbstractObjective: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IR C860S ) 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 b-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 IR C860S 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-IR WT ) 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 ðP , 0:01Þ for IR C860S than for IR WT . 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 ðP , 0:01Þ in CHO-IR WT as compared with CHO-IR C860S cells. Conclusions: Transmembrane and intracellular domain of IR seem to be determinants for IR internalization. Now we report that Cys 860 in the IR b-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin -IR complex.

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