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Opioid receptor random mutagenesis reveals a mechanism for G protein–coupled receptor activation

Abstract

The high resolution structure of rhodopsin has greatly enhanced current understanding of G protein–coupled receptor (GPCR) structure in the off-state, but the activation process remains to be clarified. We investigated molecular mechanisms of δ-opioid receptor activation without a preconceived structural hypothesis. Using random mutagenesis of the entire receptor, we identified 30 activating point mutations. Three-dimensional modeling revealed an activation path originating from the third extracellular loop and propagating through tightly packed helices III, VI and VII down to a VI-VII cytoplasmic switch. N- and C-terminal determinants also influence receptor activity. Findings for this therapeutically important receptor may apply to other GPCRs that respond to diffusible ligands.

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Figure 1: Identification of constitutively active mutant (CAM) δ-opioid receptors by random mutagenesis.
Figure 2: Functional activity of mutant receptors in the [35S]GTPγS binding assay.
Figure 3: Locations of activating mutations.
Figure 4: Schematic representation of activating determinants in the δ-opioid receptor.
Figure 5: Mechanism for δ-opioid receptor activation by an agonist.

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Acknowledgements

We wish to thank M. Valiquette and T. Groblewski for helpful discussions, M. Hibert for critical review of the manuscript and C. Iderne, M. Ballié and G. Recht for their help. We acknowledge support of the Human Frontier Science Program, the French Centre National de la Recherche Scientifique, the French Institut National de la Santé et de la Recherche Médicale, the Université Louis Pasteur, the Association de la Recherche pour le Cancer, the Institut UPSA de la Douleur, the Mission Interministérielle de Lutte contre la Drogue et la Toxicomanie and the US National Institutes of Health, National Institute on Drug Abuse.

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Correspondence to Brigitte L Kieffer.

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Décaillot, F., Befort, K., Filliol, D. et al. Opioid receptor random mutagenesis reveals a mechanism for G protein–coupled receptor activation. Nat Struct Mol Biol 10, 629–636 (2003). https://doi.org/10.1038/nsb950

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