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

J. A. Hedo, I. A. Simpson

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

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 - 1985

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the M(r)-190000 pro-receptor'. Together they form a unique fingerprint.

Cite this