title: Extracellular cysteine residues 174 and 255 are essential for active expression of human endothelin receptor ETB in Escherichia coli.
abstract: The coding sequences for the non-isopeptide-selective human endothelin receptor ETB 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 bacterial 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 ETB to alanine codons. E. coli JM105 transformed with the mutated plasmids no longer displayed specific ET-1 binding to membranes, which suggests a crucial role for these extracellular cysteines in agonist binding or receptor stability.
authors.affiliation: Research Laboratories of Schering AG, Berlin, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Haendler
authors.forename: U
authors.initials: U
authors.lastname: Hechler
authors.forename: A
authors.initials: A
authors.lastname: Becker
authors.forename: W D
authors.initials: WD
authors.lastname: Schleuning
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017466:Receptors, Endothelin / Q000096:biosynthesis* / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D007457:Iodine Radioisotopes; D017466:Receptors, Endothelin; D003545:Cysteine