Demonstration of a 60K Molecular Weight Luteinizing Hormone-Releasing Hormone Receptor in Solubilized Adrenal Membranes by a Ligand-Immunoblotting Technique*

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Abstract
A new technique for the identification of LHRH receptors was developed and applied to demonstrate an adrenal LH binding protein. Solubilized membrane proteins were separated electrophoretically and transferred to nitrocellulose paper. This was followed by sequential incubations with LHRH, anti-LHRH antiserum, peroxidase-conjugated 2nd antibody and 4-chloro-1-naphthol. Using an antiserum directed towards the middle region of LHRH, a 60K MW band was visualized in rat adrenal and pituitary membranes. A band of slightly higher MW was present in membranes of bovine adrenal cortex but was absent in the medulla. The 60K band was not visualized when nonimmune rabbit serum was used. The 60K band was also not visualized when an antiserum requiring the NH2 and COOH termini of LHRH was used, suggesting that these regions of LHRH are not accessible to the antiserum after binding to the receptor. The existence of LHRH binding protein in adrenal cortical tissue with a molecular size similar to that of the pituitary receptor was demonstrated. Adrenal membrane binding sites were less clearly demonstrated by conventional 125I-ligand binding techniques as nonspecific binding was high. The ligand-immunoblotting technique is a sensitive, specific and rapid procedure with potential application in screening normal and tumor tissues for LHRH receptors and studying LHRH interactions with its receptor.