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. 2014 Feb 13;506(7487):191-6.
doi: 10.1038/nature12944. Epub 2014 Jan 12.

Molecular control of δ-opioid receptor signalling

Gustavo Fenalti  1 , Patrick M Giguere  2 , Vsevolod Katritch  3 , Xi-Ping Huang  4 , Aaron A Thompson  3 , Vadim Cherezov  3 , Bryan L Roth  4 , Raymond C Stevens  3
Affiliations

Molecular control of δ-opioid receptor signalling

Gustavo Fenalti et al. Nature. .

Abstract

Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8 Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion in mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the seven-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn 131 to an alanine or a valine augments constitutive β-arrestin-mediated signalling. Asp95Ala, Asn310Ala and Asn314Ala mutations transform classical δ-opioid antagonists such as naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signalling, revealing that sodium-coordinating residues act as 'efficacy switches' at a prototypic G-protein-coupled receptor.

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Figures

Fig. 1
Fig. 1. Interactions in the 7TM core of BRIL-δOR(ΔN/ΔC)-naltrindole
(a) BRIL-δOR(ΔN/ΔC)-naltrindole structure (light green, BRIL fusion is omitted) and residues in the allosteric sodium site (green sticks). Sodium is shown as a blue sphere; red and pink spheres are waters in the first and second coordination shells, respectively. Naltrindole is shown as orange sticks. (b) Interactions in the sodium site. Orange transparent surface depicts hydrophobic residues below the allosteric sodium site. Hydrogen bonds are shown as grey dotted lines. (c) ‘Sliced’ surface representation of BRIL-δOR(ΔN/ΔC)-naltrindole showing the continuous pathway connectivity between the orthosteric and allosteric sodium site. Receptor surface and interior are colored green and black, respectively. (d) 2mFo- DFcelectron density map (grey mesh) contoured at 2 σ around residues, waters and sodium in the allosteric site. Hydrogen bonds in the first sodium ion coordination shell are shown as black dotted lines; all other hydrogen bonds are shown as yellow dotted lines. Yellow arrow indicates the increased β-arrestin constitutive activity of Asn1313.35Ala and Asn1313.35Val mutants. Red arrow indicates the potent β-arrestin biased activation and concomitant abrogation of Gi signaling in response to the ligand naltrindole in the Asp952.50Ala mutant. The “efficacy switch” residues that resulted in β-arrestin bias when mutated are highlighted in orange (Asn1313.35) and red (Asp952.50, Asn3107.45 and Asn3147.49) in panels a-d.
Fig. 2
Fig. 2. Structure of the human δ-OR ICL3
(a) 2mFo- DFcelectron density map (grey mesh) of BRIL-δOR(ΔN/ΔC)-naltrindole ICL3 contoured at 1σ. Polar and ionic interactions of Arg2576.31 shown by orange dashed lines (b) ICL3 loop comparison between BRIL-δOR(ΔN/ΔC)-naltrindole (green) and NOP (light grey) structures. Hydrophobic residues in the ICL3 hydrophobic cluster are shown as orange spheres and OLA molecules are represented by yellow sticks. (c) ICL3 comparison of BRIL-δOR(ΔN/ΔC)-naltrindole (green) with A2AAR (magenta; PDB ID 3PWH) and Rhodopsin (white; PDB ID 3CAP). (d) Details of the hydrogen bonds between Arg2395.66, Leu246ICL3, Val1503.54 and water molecule (red sphere) shown by grey dashed lines
Fig. 3
Fig. 3. Effect of sodium site mutations on allosteric sodium effect and β-arrestin constitutive activity
The effects of graded doses of sodium on DADLE affinity on (a) WT and (b) sodium site mutant D95A were determined, results normalized and then pooled for analysis in Prism using the allosteric model with fitted parameters summarized in Table 1. (c) The basal activity of N131A and N131V is compared to WT receptor over a range of DNA dosages (0.5 – 5.0 µg per 15-cm dish) and shows that receptors were expressed at comparable levels (WT 226 – 758 fmol/mg; N131A 77 – 553 fmol/mg; N131V 176 – 715 fmol/mg). Basal activity of N131A and N131V exceeded the activity achieved with a saturating concentration of DADLE (10 µM) at WT δ-OR (open square). Results represent average ± SEM from a minimum of 64 replicates from a representative assay; error bars are smaller than the corresponding symbols. (d) The sodium site mutations N131A and N131V responded to DADLE (10 µM) with a modest degree of stimulation (*p<0.01 (t-test) vs. no drug addition). Receptors were transfected at a low level in these experiments, allowing the detection of activation by a saturating concentration of DADLE.
Fig. 4
Fig. 4. Sodium-coordinating residues form an efficacy switch regulating biased signaling
Mutation of the sodium-anchoring residues Asp95, Asn310 and Asn314 promotes efficacy switching of the cyclopentene-containing antagonist naltrindole into a potent β-arrestin-biased agonist. Normalized concentration-responses of δ-OR-mediated Gαi signaling induced by (a) BW373U86 and (c) naltrindole, and δ-OR mediated β-arrestin recruitment with (b) BW373U86 and (d) naltrindole were quantified as in Methods. Results represent average ± SEM of four independent experiments each in quadruplicate and are presented as % of activation by BW373U86. Receptors were all transfected with 15 µg DNA revealing weak partial agonist activity of naltrindole at Gαi signaling as previously described,.
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