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 language | English |
|---|---|
| Pages (from-to) | 365-371 |
| Number of pages | 7 |
| Journal | European Journal of Endocrinology |
| Volume | 148 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Mar 1 2003 |
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ASJC Scopus subject areas
- Endocrinology
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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. / Benzi, Luca; Cecchetti, P.; Ciccarone, A. M.; Novelli, S.; Paoli, A.; Bertacca, A.; Maffei, M.; Maggi, D.; Andraghetti, G.; Del Prato, S.; Cordera, R.
In: European Journal of Endocrinology, Vol. 148, No. 3, 01.03.2003, p. 365-371.Research output: Contribution to journal › Article
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TY - JOUR
T1 - 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
AU - Benzi, Luca
AU - Cecchetti, P.
AU - Ciccarone, A. M.
AU - Novelli, S.
AU - Paoli, A.
AU - Bertacca, A.
AU - Maffei, M.
AU - Maggi, D.
AU - Andraghetti, G.
AU - Del Prato, S.
AU - Cordera, R.
PY - 2003/3/1
Y1 - 2003/3/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037356116&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037356116&partnerID=8YFLogxK
U2 - 10.1530/eje.0.1480365
DO - 10.1530/eje.0.1480365
M3 - Article
C2 - 12611619
AN - SCOPUS:0037356116
VL - 148
SP - 365
EP - 371
JO - European Journal of Endocrinology
JF - European Journal of Endocrinology
SN - 0804-4643
IS - 3
ER -