Construction and characterization of a kappa opioid receptor devoid of all free cysteines.
We have constructed an optimized mutant of the kappa opioid receptor (KOR), which is devoid of its 10 free cysteines. It was necessary to test different amino acid replacements at various positions and we used a structural model and homology with other receptor family members as a guide. This mutant binds ligands and couples to the cognate G-proteins in a very similar fashion to wild-type KOR. The addition of the antagonist naloxone during cell growth greatly enhances heterogeneous expression of the mutant in mammalian cells, such that amounts similar to wild-type could be produced. We showed by fluorescence microscopy that naloxone stabilizes the mutant in the plasma membrane. This mutant, which now permits the insertion of single cysteines, was designed for use in spectroscopic studies of ligand-induced receptor conformational changes as well as to simplify folding studies.
Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
David
D
Ott
Renato
R
Frischknecht
Andreas
A
Plückthun
D000262:Adenylyl Cyclases / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D009270:Naloxone / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D051381:Rats; D017473:Receptors, Opioid, kappa / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection
D000596:Amino Acids; D008024:Ligands; D017473:Receptors, Opioid, kappa; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D000262:Adenylyl Cyclases; D003545:Cysteine