Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors.
The G-protein coupled receptor (GPCR) human mu opioid receptor (hMOR) fused to the carboxy-terminus of the enhanced green fluorescent protein (EGFP) has been successfully and stably expressed in Drosophila Schneider 2 cells under the control of an inducible metallothionein promoter. Polyclonal cells expressing EGFPhMOR display high-affinity, saturable, and specific binding sites for the opioid antagonist diprenorphine. Competition studies with opioid agonists and antagonists defined the pharmacological profile of a mu opioid receptor similar to that observed in mammalian cells, suggesting proper folding of EGFPhMOR in a high-affinity state in Drosophila cells. The functionality of the fusion protein was demonstrated by the ability of agonist to reduce forskolin-stimulated cyclic AMP production and to induce [35S]GTPgammaS incorporation. The EGFPhMOR protein had the expected molecular weight (70kDa), as demonstrated by protein immunoblotting with anti-EGFP and anti-C-terminus hMOR antibodies. However, quantitative EGFP fluorescence intensity analysis revealed that the total level of expressed EGFPhMOR is 8-fold higher than the level of diprenorphine binding sites, indicating that part of the receptor is not in a high-affinity state. This may in part be due to a population of receptors localized in intracellular compartments, as shown by the distribution of fluorescence between the plasma membrane and the cell interior. This study shows that EGFP is a valuable and versatile tool for monitoring and quantifying expression levels as well as for optimizing and characterizing an expression system. Optimization of the Drosophila Schneider 2 cell expression system will allow large-scale purification of GPCRs, thus enabling structural studies to be undertaken.
Perret
Bénédicte G
BG
Récepteurs et Protéines Membranaires, UPR CNRS 9050, Ecole Supérieure de Biotechnologie de Strasbourg, Bld Sébastien Brant, B.P. 10413, F-67400, Illkirch, France.
Wagner
Renaud
R
Lecat
Sandra
S
Brillet
Karl
K
Rabut
Gwénaël
G
Bucher
Bernard
B
Pattus
Franc
F
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D019327:Copper Sulfate / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004174:Diprenorphine / Q000378:metabolism / Q000494:pharmacology; D004330:Drosophila / Q000166:cytology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008668:Metallothionein / Q000235:genetics; D018613:Microscopy, Confocal; D009020:Morphine / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009271:Naltrexone / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D018847:Opioid Peptides; D037342:Pertussis Toxin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis / Q000235:genetics; D017450:Receptors, Opioid, mu / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D018076:DNA, Complementary; D008164:Luminescent Proteins; D009842:Oligopeptides; D018847:Opioid Peptides; D043562:Receptors, G-Protein-Coupled; D017450:Receptors, Opioid, mu; D011993:Recombinant Fusion Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D049452:Green Fluorescent Proteins; D004174:Diprenorphine; D005576:Colforsin; C030058:dermorphin; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; D009020:Morphine; D008668:Metallothionein; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D019327:Copper Sulfate