Affinity labelling of endothelin receptor and characterization of solubilized endothelin–endothelin‐receptor complex

Chick cardiac membranes were affinity labelled by cross‐linking to membrane‐bound ¹²⁵I‐endothelin‐1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the ¹²⁵I‐endothelin‐1‐labelled material in the presence or absence of 2‐mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose‐dependently inhibited by the addition of unlabelled endothelin‐1 (1–100 nM). Subtracting the molecular mass of ¹²⁵I‐endothelin‐1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa. To investigate further the molecular properties of endothelin receptor, the ¹²⁵I‐endothelin‐1–endothelin‐receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)‐[³H]PN200–110, a dihydropyridine derivative, and dihydropyridine‐sensitive Ca²⁺ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine‐sensitive Ca²⁺ channels from the chick brain did not immunoprecipitate the ¹²⁵I‐endothelin‐1–endothelin‐receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine‐sensitive Ca²⁺ channels and endothelin has its own specific 50‐kDa receptor.