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  • Title: Identification of a high affinity growth hormone receptor in rat adipocyte membranes.
    Author: Carter-Su C, Schwartz J, Kikuchi G.
    Journal: J Biol Chem; 1984 Jan 25; 259(2):1099-104. PubMed ID: 6319377.
    Abstract:
    Affinity cross-linking techniques have been used to identify a growth hormone (GH) receptor in rat adipocyte membranes. Adipocytes were incubated with 125I-human GH (125I-hGH) for 2 h at 37 degrees C and washed once to remove unbound hGH. The bivalent cross-linking reagent disuccinimidyl suberate (0.4 mM) was added to the cells for 15 min at 15 degrees C. A plasma membrane-enriched fraction was prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The autoradiographs of gels containing samples treated with reductant revealed an intense band of apparent Mr = 134,000 which was not present when cells were incubated with an excess of hGH or bovine GH. In contrast, the intensity of the Mr = 134,000 band was not altered by the presence of a similar excess of insulin or rat prolactin during the binding step. Multiple lower molecular weight species displaying the same hormone sensitivity as the Mr = 134,000 species were also present but at much lower levels. In the absence of reductant, the affinity-labeled GH receptor migrated as a broad band of Mr = 116,000-125,000 and as a less intense band of Mr = 230,000. At low reductant concentrations, both of the hGH-labeled complexes exhibited larger apparent molecular weights (Mr (X 10(3) ) = 135 and 270), indicating the presence of intrachain disulfide bonds. At higher reductant concentrations, the Mr = 270,000 species disappeared as the Mr = 134,000 band increased in intensity. Use of a cleavable cross-linking reagent, ethylene glycol bis(succinimidyl succinate), in conjunction with two-dimensional gel electrophoresis, revealed that the Mr = 134,000 complex is composed of iodinated monomeric hGH (Mr = 22,000) bound to a membrane protein. The molecular weight of the reduced hGH receptor protein itself was calculated to be 112,000, assuming a 1:1 stoichiometry of hormone to receptor.
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