Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the μ-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 κ-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 μ-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.
Bibliographical noteFunding Information:
The authors thank Drs Stephen F.Marino, Tomoaki Matsuura and Manca Kenig for valuable discussions. This work was supported by the National Center for Competence in Research in Structural Biology and by TopNano21 Project (KTI, Berne, Switzerland).
- Functional immobilization
- Opioid receptors
- μ-opioid receptor