Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the M(r)-190000 pro-receptor

J. A. Hedo, Ian Simpson

Research output: Contribution to journalArticle

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Abstract

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of M(r) 190000. More than 80% of the M(r)-190000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t( 1/2 ) approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of M(r) 135000 (α) and 95000 (β) rose steadily during the chase and reached a maximum at 6 h. The M(r)-190000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the M(r)-190000 precursor into the α and β subunits was completely inhibited. Our results suggest that the M(r)-190000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.

Original languageEnglish (US)
Pages (from-to)71-78
Number of pages8
JournalBiochemical Journal
Volume232
Issue number1
DOIs
StatePublished - Jan 1 1985

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Biosynthesis
Insulin Receptor
Golgi Apparatus
Cell membranes
Rats
Cell Membrane
Mannose
Endoplasmic Reticulum
Processing
Insulin
Monensin
Subcellular Fractions
Centrifugation
Ionophores
Radioactivity
Electrophoresis
Autoradiography
Sodium Dodecyl Sulfate
Detergents
Labels

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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abstract = "We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of M(r) 190000. More than 80{\%} of the M(r)-190000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t( 1/2 ) approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of M(r) 135000 (α) and 95000 (β) rose steadily during the chase and reached a maximum at 6 h. The M(r)-190000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the M(r)-190000 precursor into the α and β subunits was completely inhibited. Our results suggest that the M(r)-190000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.",
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Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the M(r)-190000 pro-receptor. / Hedo, J. A.; Simpson, Ian.

In: Biochemical Journal, Vol. 232, No. 1, 01.01.1985, p. 71-78.

Research output: Contribution to journalArticle

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