Identification and characterization of corticotropin receptors in bovine and human adrenals

Abstract

Corticotropin (ACTH) receptors have been characterized by covalent cross-linking of radiolabeled ACTH ([ 125I]ACTH) with the bifunctional cross-linking reagent disuccinimidyl suberate to cultured bovine adrenal fasciculata reticularis cells (BAC), and to crude plasma membrane fractions prepared from both human and bovine adrenals. Incubation of BAC with [ 125I]ACTH at 20°C followed by cross-linking resulted in the specific labeling of two binding proteins with apparent M r of 154,000 and 43,000 as measured by SDS-PAGE under reducing and non-reducing conditions. In addition, in some experiments another band with an apparent M r of 124,000 was observed. All of these bands disappeared when the incubation was performed in the presence of an excess of unlabeled ACTH. When BAC were incubated with [ 125I]ACTH in the presence of 100 μM phenylarsine oxide at 20°C, a condition which prevents the internalization of the ACTH-receptor complex, the bulk of the radioactivity was present in the 43,000 band. After [ 125I]ACTH cross-linking to BAC, subcellular preparations followed by SDS-PAGE revealed that the 20,000 g fraction contained mainly the 43,000 M r form. Cross-linking of [ 125I]ACTH to plasma membrane-enriched fractions prepared from human and bovine adrenals resulted only in the labeling of the 43,000 protein. These results indicate that the ACTH receptor present at the cell surface is a macromolecule of 43,000, and suggest that the 154,000 form probably represents association of the ACTH-receptor complex to another macromolecule. The 154,000 protein would be formed during or after internalization of the ACTH-receptor complex.