Visualization of the rat ovarian lutropin receptor by ligand blotting

Abstract

A ligand blotting technique was developed to identify the lutropin receptor after size separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The separated proteins were transferred to a nitrocellulose sheet, which was subsequently incubated with 125I-labeled human choriogonadotropin (125I-hCG), and subjected to autoradiography. An Mr 90,000 band was specifically and intensely labeled with 125I-hCG. The band was not observed, when the hormone incubation was performed in the presence of an excess of unlabeled hCG or human lutropin. The presence of rat follitropin or rat prolactin did not, however, abolish the labeling. No specific labeling was found when down-regulated ovarian tissue or rat liver was used as starting material. The Mr 90,000 band disappeared when the protein samples were treated with reducing agent, showing that integrity of receptor disulfide bonds is essential for the hormone-receptor interaction. In addition, a receptor-positive murine Leydig tumor cell line produced an Mr 90,000-92,000 band in ligand blotting, thus demonstrating the similarity between rat and murine lutropin receptors. These results provide strong evidence that the lutropin receptor is an Mr 90,000 protein.