TY - JOUR
T1 - Insulin-induced internalization of the insulin receptor in the isolated rat adipose cell
T2 - Detection of the internalized 138-kilodalton receptor subunit using a photoaffinity 125I-insulin
AU - Wang, C. C.
AU - Sonne, O.
AU - Hedo, J. A.
AU - Cushman, S. W.
AU - Simpson, I. A.
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 1983
Y1 - 1983
N2 - A photoactive insulin analogue (N(ε)-B29-(2-nitro-4-azidophenylacetyl)insulin) which specifically and covalently labels the 138-kDa insulin receptor subunit, is used here to examine the effect of insulin on the subcellular distribution of insulin receptors in the isolated rat adipose cell. The photolabeled 138-kDa receptor subunit in the plasma and Golgi-enriched membrane fractions was quantitated by Na dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When intact cells are photolabeled, subsequent incubation for 30 min at 37°C with saturating native insulin induces a 30% loss of the labeled receptor subunit from the plasma membrane fraction. Greater than 50% of the lost receptor subunits can be specifically recovered in the Golgi-enriched membrane fraction. Qualitatively and quantitatively similar results are obtained when the 138-kDA receptor subunit is labeled in the membrane fractions following their preparation. However, the 138-kDa receptor subunit in the Golgi-enriched membrane fraction can only be labeled when the vesicles in this fraction are made permeable to the insulin analogue by the presence of 0.01% digitonin. The appearance of the 138-kDa receptor subunit in the Golgi-enriched membrane fraction is rapid, with a half-time of 2 min, and achieves a steady state within 10 min. This effect is also insulin concentration-dependent, with half-maximal and maximal effects at 6 and 30 nM, respectively, and is markedly, but not completely, inhibited at 16°C. These results suggest that insulin induces a rapid and insulin concentration- and temperature-dependent translocation of its own receptor from the plasma membrane to an intracellular membrane fraction in the isolated rat adipose cell, and that this translocation represents internalization of the insulin receptor through an endocytic like process.
AB - A photoactive insulin analogue (N(ε)-B29-(2-nitro-4-azidophenylacetyl)insulin) which specifically and covalently labels the 138-kDa insulin receptor subunit, is used here to examine the effect of insulin on the subcellular distribution of insulin receptors in the isolated rat adipose cell. The photolabeled 138-kDa receptor subunit in the plasma and Golgi-enriched membrane fractions was quantitated by Na dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When intact cells are photolabeled, subsequent incubation for 30 min at 37°C with saturating native insulin induces a 30% loss of the labeled receptor subunit from the plasma membrane fraction. Greater than 50% of the lost receptor subunits can be specifically recovered in the Golgi-enriched membrane fraction. Qualitatively and quantitatively similar results are obtained when the 138-kDA receptor subunit is labeled in the membrane fractions following their preparation. However, the 138-kDa receptor subunit in the Golgi-enriched membrane fraction can only be labeled when the vesicles in this fraction are made permeable to the insulin analogue by the presence of 0.01% digitonin. The appearance of the 138-kDa receptor subunit in the Golgi-enriched membrane fraction is rapid, with a half-time of 2 min, and achieves a steady state within 10 min. This effect is also insulin concentration-dependent, with half-maximal and maximal effects at 6 and 30 nM, respectively, and is markedly, but not completely, inhibited at 16°C. These results suggest that insulin induces a rapid and insulin concentration- and temperature-dependent translocation of its own receptor from the plasma membrane to an intracellular membrane fraction in the isolated rat adipose cell, and that this translocation represents internalization of the insulin receptor through an endocytic like process.
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M3 - Article
C2 - 6339507
AN - SCOPUS:0020536405
SN - 0021-9258
VL - 258
SP - 5129
EP - 5134
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 8
ER -