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Engineering and functional immobilization of opioid receptors


Ott, D; Neldner, Y; Cèbe, R; Dodevski, I; Plückthun, A (2005). Engineering and functional immobilization of opioid receptors. Protein Engineering Design and Selection : PEDS, 18(3):153-160.

Abstract

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs.

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the mu-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable kappa-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type mu-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs.

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7 citations in Web of Science®
6 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:March 2005
Deposited On:07 Jul 2008 09:40
Last Modified:05 Apr 2016 12:24
Publisher:Oxford University Press
ISSN:1741-0126
Publisher DOI:10.1093/protein/gzi012
PubMed ID:15790572
Permanent URL: http://doi.org/10.5167/uzh-2701

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