The extracellular portion of the insulin receptor β-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

Luca Benzi, P. Cecchetti, A. M. Ciccarone, S. Novelli, A. Paoli, A. Bertacca, M. Maffei, D. Maggi, G. Andraghetti, S. Del Prato, R. Cordera

Research output: Contribution to journalArticlepeer-review

Abstract

Objective: Chinese hamster ovary (CHO) cells transfected with human engineered insulin receptor (IR) cDNA to mutate Cys 860 to Ser (CHO-IRC860S) 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 β-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 (t1/2): 21 min vs 40 min, P <0.001) and more extensive (P <0.01) for IRC860S than for IRWT. 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 β-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.

Original languageEnglish
Pages (from-to)365-371
Number of pages7
JournalEuropean Journal of Endocrinology
Volume148
Issue number3
DOIs
Publication statusPublished - Mar 1 2003

ASJC Scopus subject areas

  • Endocrinology

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