Trp-21 is important in the processing and secretion of big endothelin-1.
To investigate the intracellular processing of endothelin-1 (ET-1), the synthesis and secretion of preproET-1 (PPET-1) was studied in transiently transfected COS-7 cells. Replacements at the highly conserved C-terminal region of ET-1 were made by site-directed mutagenesis, with codons for residues Ile-20 and Trp-21 being replaced by one for Ala in the PPET-1 cDNA. The mutant and wildtype PPET-1 cDNAs were expressed in COS-7 cells following transient transfection, and cell media and extracts, collected after 48 h, were assayed for ET-1 and big ET-1 by radioimmunoassay. The concentration of immunoreactive ET-1 in the medium obtained from the Ala-21-ET-1 mutant was 10-fold lower than that from PPET-1 wildtype and (Ala-20-ET-1) PPET-1 transfected cells. Moreover, Ala-21-big ET-1 accumulated in the cells transfected with the cDNA for (Ala-21-ET-1) PPET-1, whereas there was no significant accumulation of ET-1-like or big ET-1-like immunoreactivity in the cells transfected with cDNAs for PPET-1 wildtype and (Ala-20-ET-1) PPET-1. These results suggest that Trp-21 is involved in intracellular processing and secretion of big ET-1.
Ergul
A
A
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Puett
D
D
D000409:Alanine; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics; D019332:Endothelin-1; D016232:Endothelins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D014364:Tryptophan
D018076:DNA, Complementary; D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D014364:Tryptophan; D000409:Alanine