Characterization of delta, kappa, and mu human opioid receptors overexpressed in baculovirus-infected insect cells.
The cDNAs encoding human delta (hDOR), kappa (hKOR) and micro (hMOR) opioid receptors were cloned in the baculovirus Autographa californica (AcMNPV) under the control of the polyhedrin promoter with or without an amino-terminal hexahistidine tag. Expression levels were optimized in Spodoptera frugiperda (Sf9) cells and were in the following order hMOR > hDOR > hKOR. The receptors bound antagonists with affinity values similar to those published previously for the receptors expressed in mammalian cells. They also retained selectivity toward specific antagonists. The three receptors bound peptidic agonists with low affinity, suggesting that they might not be functionally coupled to intracellular effectors. Introduction of an amino-terminal hexahistidine tag decreased the levels of expression markedly. Only hMOR-his was expressed at a level allowing binding study, but no difference could be detected in the affinities of both agonists and antagonists compared with the nontagged protein. hMOR expression was also optimized in High Five cells leading to a further increase in protein production. The pharmacological profile was similar to the one obtained when the receptor was expressed in Sf9 cells. Our results show that the baculovirus expression system is suitable for large scale production of human opioid receptors.
Massotte
D
D
Département des Récepteurs et Protéines membranaires, CNRS Unité Propre de Recherche 9050, F-64700 Illkirch-Graffenstaden, France. massotte@esbs.u-strasbg.fr
Baroche
L
L
Simonin
F
F
Yu
L
L
Kieffer
B
B
Pattus
F
F
D000701:Analgesics, Opioid / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004174:Diprenorphine / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009292:Narcotic Antagonists / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D018847:Opioid Peptides; D017465:Receptors, Opioid, delta / Q000235:genetics* / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000235:genetics* / Q000378:metabolism; D017450:Receptors, Opioid, mu / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D000701:Analgesics, Opioid; D004745:Enkephalins; D008024:Ligands; D009292:Narcotic Antagonists; D009842:Oligopeptides; D018847:Opioid Peptides; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004174:Diprenorphine; C030058:dermorphin