Differential inactivation of vasopressin receptor subtypes in isolated membranes and intact cells by N-ethylmaleimide

Vasopressin receptors in plasma membranes and on cell monolayers were treated with sulfhydryl reagents. Specific binding of [3H]AVP to renal V2 receptors in membranes from bovine and porcine kidney and on LLC-PK1 cells was markedly (80-90%) reduced after treatment with NEM but that to V1 receptors on rat liver membranes and A7r5 smooth muscle cells only slightly (10-30%). Inactivation of receptors by NEM reduced the number of binding sites without altering the affinity of unmodified receptor molecules. High affinity ligands (agonists and antagonists), in complex with the V2 receptor, protected against its inactivation. The results suggest that one or more cysteine residues are located in the ligand-binding site of the V2 receptor, and are essential for hormone binding. Furthermore, it is possible to use NEM to differentiate between vasopressin isoreceptors.