INVIVO EXPRESSION OF MUTANT PREPROENDOTHELINS - HIERARCHY OF PROCESSING EVENTS BUT NO STRICT REQUIREMENT OF TRP-VAL AT THE PROCESSING SITE

Endothelin-1 (ET-1), a 21-residue vasoconstrictor peptide, originates in human cells from a 212-amino acid precursor (preproET-1). Big ET-1, an intermediate form of 38 amino acids, is generated by cleavage at b asic-pair residues of proET-1, while a specific ''ET-converting enzyme '' was proposed to process the unusual Trp-Val site at positions 21 an d 22 of big ET-1. We have previously shown that expression of syntheti c RNA encoding human preproET-1 in Xenopus oocytes results in secretio n of putative ET-1 and big ET-1. Here, to further dissect the processi ng pathway of preproET-1, we designed and expressed in oocytes a set o f preproET-1 mutants. Four mutants affecting the Trp-Val site always o riginated putative ET-1(s) at levels comparable to the wild type, sugg esting that there is only a conformational requirement for cleavage at this site. An Arg --> Ile mutation at the basic-pair site after the C terminus of big ET-1 fully inhibited the formation of both big ET-1 a nd ET-1, indicating that processing at this site is an early event and that big ET-1 is an obligate intermediate for the synthesis of ET-1 i n vivo. Also, a truncated mutant bearing a stop codon after the C term inus of the big ET-1 sequence was totally stable and further processed into mature big ET-1 and ET-1, indicating that the second part of the precursor is not necessary for maturation.