EXTRACELLULAR CYSTEINE RESIDUE-174 AND RESIDUE-255 ARE ESSENTIAL FOR ACTIVE EXPRESSION OF HUMAN ENDOTHELIN RECEPTOR-ET(B) IN ESCHERICHIA-COLI

The coding sequences for the non-isopeptide-selective human endothelin receptor ET(B) were introduced into the prokaryotic expression vector pKK233-2, and the resulting construct was used for transformation of competent E. coli JM105 cells. Specific binding was observed for bacte rial membrane fractions, using labeled ET-1 as a ligand. Site-directed mutagenesis was employed to individually modify the triplets for the cysteines of the first and second extracellular loops of ET(B) to alan ine codons. E. coli JM105 transformed with the mutated plasmids no lon ger displayed specific ET-1 binding to membranes, which suggests a cru cial role for these extracellular cysteines in agonist binding or rece ptor stability.