Expression of δ, κ and μ human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins

被引:30
作者
Stanasila, L [1 ]
Massotte, D [1 ]
Kieffer, BL [1 ]
Pattus, F [1 ]
机构
[1] ESBS, UPR 9050, CNRS, Dept Recepteurs & Prot Membranaires, Illkirch Graffenstaden, France
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 260卷 / 02期
关键词
opioid receptor; GTP-binding protein; expression in Escherichia coli; reconstitution; pharmacology;
D O I
10.1046/j.1432-1327.1999.00187.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell(-1) or 0.5 pmol.mg(-1) membrane protein. Binding of [H-3]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, K-D, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of-endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.
引用
收藏
页码:430 / 438
页数:9
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