The processing pathway of endothelin-1 production.

Abstract

Production of endothelin (ET-1) is believed to be a three-step process, consisting of an initial proteolytic cleavage by signal peptidase of preproET-1, a second cleavage of proET-1 to big ET-1-Lys-Arg by dibasic amino acid-specific convertase and C-terminal trimming, and finally the processing of Big ET-1 to ET-1 by endothelin-converting enzyme (ECE). To clarify the relationships between the second processing step and the third, we introduced point mutation into ET-1 cDNA to replace the Arg in the -4 position of the recognition motifs of furin-like convertase in human preproET-1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in CHO-K1 cells, they failed to be processed at the mutated processing signal, suggesting the involvement of the enzyme with furin-like specificity in the processing at dibasic amino acid motifs. Co-transfection of mutant preproET-1 cDNA and ECE-1 cDNA revealed that cleavage at Arg92 is essential for cleavage by ECE-1 but that cleavage at Arg52 is not. Although without cleavage at Arg52 a high molecular weight form of ET-1, designated Large ET-1, is produced by processing by ECE-1, it did not evoke a Ca2+ transient in ETA receptor-expressing cells. In conclusion, the cleavage by furin-like convertase is essential for the production of active ET-1.