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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.

Abstract : 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.
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https://hal.archives-ouvertes.fr/hal-00174046
Contributor : Sandra Lecat Connect in order to contact the contributor
Submitted on : Friday, September 21, 2007 - 1:58:05 PM
Last modification on : Friday, October 29, 2021 - 1:38:02 PM

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  • HAL Id : hal-00174046, version 1
  • PUBMED : 12963349

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Bénédicte G Perret, Renaud Wagner, Sandra Lecat, Karl Brillet, Gwénaël Rabut, et al.. 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.. Protein Expression and Purification, Elsevier, 2003, 31 (1), pp.123-32. ⟨hal-00174046⟩

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