Identification of a high affinity growth hormone receptor in rat adipocyte membranes

• Published in: The Journal of biological chemistry Vol. 259; no. 2; pp. 1099 - 1104
• Main Authors: Carter-Su, C, Schwartz, J, Kikuchi, G
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.01.1984; American Society for Biochemistry and Molecular Biology
• Subjects: Adipose Tissue - metabolism; Animals; Biological and medical sciences; Cell Membrane - metabolism; Cell physiology; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Growth Hormone - metabolism; Hormonal regulation; Insulin - metabolism; Male; Membrane Proteins - metabolism; Molecular and cellular biology; Molecular Weight; Rats; Rats, Inbred Strains; Receptors, Cell Surface - metabolism; Receptors, Somatotropin; Succinimides - pharmacology; Adipocyte; Protein hormone; Vertebrata; Membrane receptor; Mammalia; Rat; Affinity labelling; STH; Rodentia; Membrane; Hormonal receptor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)43571-X

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.