Covalent binding of growth hormone to surface receptors on rat adipocytes.

GH specifically binds to receptors on the surface of adipocytes and produces a variety of biological effects in these cells. To gain insight into the nature of the GH receptors, [125I] human GH ([125I]hGH) was cross-linked to surface binding sites on intact rat adipocytes using the bifunctional reagent disuccinimidyl suberate. Plasma membranes were isolated, and after solubilization with sodium dodecyl sulfate (SDS), the proteins were subjected to electrophoresis on 5% or 7.5% polyacrylamide gel. Autoradiography of the 7.5% gels revealed three iodinated bands corresponding to apparent molecular weights of 56, 130, and more than 240 kilodaltons. The more than 240-kilodalton band contained approximately as much 125I as the 130-kilodalton species and about twice as much as the 56-kilodalton species. When run on the more porous 5% gel, the more than 240-kilodalton band resolved into two bands, corresponding to apparent molecular weights of 240 and 310 kilodaltons. Excess unlabeled human or bovine GH, but not ovine PRL, competed with [125I]hGH for binding and prevented the formation of all of the labeled bands. Treatment of the membranes and extracted proteins with dithiothreitol resulted in the generation of additional 130-kilodalton material at the expense of both the 310- and 240-kilodalton species, but failed to alter the amount of 125I that migrated with the 56-kilodalton species. The same pattern of labeling was seen regardless of whether protease inhibitors were present during isolation of membrane proteins or when membrane proteins were isolated under conditions that favored proteolysis, suggesting that the 56-kilodalton species is not a degradative product of the higher molecular weight species. When [125I]hGH was cross-linked to adipocytes in which total binding was decreased by hypophysectomy or starvation of the donor rats or by treatment of the cells with cycloheximide, there was a proportionate diminution in labeling of all species. It thus appears that the GH receptor contains a 130-kilodalton subunit, a portion of which is in disulfide linkage with higher molecular weight complexes and, in addition, contains a 56-kilodalton species. It cannot be determined from these studies if the various labeled protein complexes are components of a single or multiple classes of GH receptors in the adipocyte membrane.