The extracellular portion of the insulin receptor beta-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.
OBJECTIVE
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 beta-subunit in insulin and receptor intracellular targeting as well as in insulin signaling.


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 (P<0.01) 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 (P<0.01) 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 beta-subunit ectodomain may be of relevance in ensuring a proper internalization and intracellular trafficking of the insulin-IR complex.
Benzi
L
L
Institute of Clinical Physiology, National Research Council, Pisa, Italy. metabpi@immr.med.unipi.it
Cecchetti
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Ciccarone
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Novelli
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Paoli
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Bertacca
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Maffei
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Maggi
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Andraghetti
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Del Prato
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Cordera
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D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005110:Extracellular Space / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D007328:Insulin; D019000:Phosphotyrosine; D011972:Receptor, Insulin