Identification of a high affinity growth hormone receptor in rat adipocyte membranes.
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.
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011986:Receptors, Somatotropin; D013388:Succinimides / Q000494:pharmacology
D007328:Insulin; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011986:Receptors, Somatotropin; D013388:Succinimides; D013006:Growth Hormone; C019358:disuccinimidyl suberate