THE BOVINE ENDOTHELIN RECEPTOR HAS AN APPARENT MOLECULAR-WEIGHT OF 43000

In crosslinking experiments, [I-125]endothelin-1 was treated with N-hydroxysuccinimidyl-4-azidobenzoate, purified by HPLC, allowed to bind to bovine aortic membranes and then photoactivated. Autoradiography of sodium dodecyl sulfate polyacrylamide gel electrophoretograms of the products of this reaction showed that a component of apparent M(r) = 42 000 was specifically labelled by endothelin-1 under reducing conditions. Under nonreducing conditions, a small amount of I-125-labelled endothelin-1 specifically labelled a component of apparent M(r) = 45 900 in the absence of crosslinking agent. Non-radiolabelled endothelin analogues with a wide range of binding affinities inhibited specific labelling of the M(r) = 42 000 and 45 900 components in parallel over the concentration ranges which inhibited binding of radiolabelled endothelin. Specific labelling of these components was also observed in parallel in membranes from bovine heart and kidney. The components labelled in the presence and absence of crosslinker appear to be the same, and the small difference in apparent M(r) in the labelled components is likely due to a difference in conformational constraints arising from the two labelling processes, with a true, corrected M(r) of 43 400. Since the specific labelling of this component is related to physiologically relevant binding in several bovine tissues, we conclude that it is a component of the bovine endothelin receptor.