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J Biol Chem. 1980 Feb 25;255(4):1722-31.

The subunit structure of the high affinity insulin receptor. Evidence for a disulfide-linked receptor complex in fat cell and liver plasma membranes.

Abstract

125I-Insulin equilibrated with high affinity fat cell and liver plasma membrane receptors was cross-linked to the membrane by addition of disuccinimidyl suberate. Autoradiographic analysis of the 125I-insulin-linked membranes subsequent to dodecyl sulfate electrophoresis in the absence of reductant revealed the presence of one labeled species which migrated with an apparent molecular weight of 300,000. Electrophoresis of these membranes in the presence of dithiothreitol resulted in the appearance of one major labeled band of about Mr = 125,000 concomitant with the loss of label in the Mr = 300,000 region. Another radioactive species which migrated in the Mr = 225,000 region on the reduced gels contained a much smaller amount of label. The degree to which the Mr = 125,000 membrane component was cross-linked to 125I-insulin in these experiments paralleled the extent of occupancy of high affinity membrane receptors by the 125I-insulin. 125I-Insulin-linked plasma membranes derived from adipocytes alkylated with N-ethylmaleimide prior to homogenization to prevent spontaneous sulfhydryl oxidation also exhibited the Mr = 300,000 and 125,000 labeled bands upon electrophoresis in the absence and presence of reductant, respectively. These same radioactive species were observed when 125I-insulin was covalently corss-linked to intact adipocytes. These data indicate that the labeled monomer with an apparent molecular weight of 125,00 represents a high affinity insulin receptor subunit which exists in the native adipocyte plasma membrane in a disulfide-linked complex. The amount of cross-linked label present in the reduced form of the receptor (Mr = 125,000) was only 20 to 30% of that present in the oxidized species (Mr = 300,000). Since the 125I-insulin used in these studies is labeled predominantly on the A chain, this large loss of label upon reduction indicates that it is predominantly the B chain of insulin that is covalently linked to the receptor protein by disuccinimidyl suberate. 125I-Proinsulin which lacks an A chain NH2 terminus could also be cross-linked to the receptor, indicating that the A1 glycine is not critical to the cross-linking reaction. 125I-Insulins modified either at lysine B29 with an acetyl group or at phenylalanine B1 with a phenylthiocarbamoyl group were both readily cross-linked to the receptor with roughly equal efficiency. It is concluded that the B1 terminal amino and B29 lysine amino groups are both accessible for cross-linking of insulin to the receptor by disuccinimidyl suberate.

PMID:
6986378
[PubMed - indexed for MEDLINE]
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