Molecular control of δ-opioid receptor signalling

Nature (Impact Factor: 41.46). 01/2014; 506(7487). DOI: 10.1038/nature12944
Source: PubMed


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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    • "The insertion of b 562 RIL into ICL3 of the smoothened receptor has also been proposed as a reason for the lack of structural rearrangements at the cytoplasmic surface upon agonist binding (Wang et al., 2013b). Finally, comparison of the murine d-opioid receptor structure solved with an ICL3 T4L fusion (Granier et al., 2012) and the human d-opioid receptor with an N-terminal b 562 RIL fusion (Fenalti et al., 2014) shows a high degree of structural similarity, with the main deviations occurring proximal to the sites of fusion. "
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    ABSTRACT: G protein-coupled receptor (GPCR) structural biology has progressed dramatically in the last decade. There are now over 120 GPCR crystal structures deposited in the Protein Data Bank of 32 different receptors from families scattered across the phylogenetic tree, including Class B, C, and Frizzled GPCRs. These structures have been obtained in combination with a wide variety of ligands, and captured in a range of conformational states. This surge in structural knowledge has enlightened research into the molecular recognition of biologically active molecules, the mechanisms of receptor activation, the dynamics of functional selectivity, and fuelled structure- based drug design efforts for GPCRs. Here we summarize the innovations in both protein engineering/molecular biology and crystallography techniques that have led to these advances in GPCR structural biology, and discuss how they may influence the resulting structural models. We also provide a brief molecular pharmacologist's guide to GPCR X-ray crystallography, outlining some key aspects in the process of structure determination, with the goal to encourage non-crystallographers to interrogate structures at the molecular level. Finally we show how chemogenomics approaches can be used to marry the wealth of existing receptor pharmacology data with the expanding repertoire of structures, providing a deeper understanding of the mechanistic details of GPCR function. The American Society for Pharmacology and Experimental Therapeutics.
    Molecular pharmacology 07/2015; 88(3). DOI:10.1124/mol.115.099663 · 4.13 Impact Factor
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    • "The several recent crystal structures that show interacting parallel receptors in the crystal unit cell (Fig. 3) have been used as an argument in favor of GPCR dimerization, although the possibility exists that these are crystallographic artifacts and/or they do not necessarily represent physiologically relevant interfaces. Two of the five available opioid receptor crystal structures (Fenalti et al., 2014; Granier et al., 2012; Manglik et al., 2012; Thompson et al., 2012; Wu et al., 2012), specifically the structures of μ (Manglik et al., 2012) and κ (Wu et al., 2012) receptors, also reveal parallel arrangements of interacting receptors. As shown in Fig. 3, these correspond to two different interfaces in the case of μ receptor, one of which is also seen in the κ receptor crystal "
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    ABSTRACT: Opioid receptors are important drug targets for pain management, addiction, and mood disorders. Although substantial research on these important subtypes of G protein-coupled receptors has been conducted over the past two decades to discover ligands with higher specificity and diminished side effects, currently used opioid therapeutics remain suboptimal. Luckily, recent advances in structural biology of opioid receptors provide unprecedented insights into opioid receptor pharmacology and signaling. We review here a few recent studies that have used the crystal structures of opioid receptors as a basis for revealing mechanistic details of signal transduction mediated by these receptors, and for the purpose of drug discovery. Copyright © 2015. Published by Elsevier B.V.
    European journal of pharmacology 05/2015; 763. DOI:10.1016/j.ejphar.2015.05.012 · 2.53 Impact Factor
    • "The main technological advancements that enabled structural studies of GPCRs include (i) development of methods for heterologous expression in insect cells, (ii) discovery of a new method of membrane protein crystallization in lipidic cubic phase (Landau & Rosenbusch, 1996) and subsequent development of tools, instruments, and protocols for automation of crystallization and crystal detection, (iii) development of receptor stabilization technologies, including antibodies toward the intracellular side of GPCRs in Fab form (Rasmussen et al., 2007) and insertion of the T4 phage lysozyme domain (T4L) in the third intracellular (i3) loop (Cherezov et al., 2007; Rosenbaum et al., 2007), and (iv) development of microcrystallography. This toolbox for GPCR crystallization has rapidly expanded with (i) development of an alanine scanning mutagenesis approach enabling receptor stabilization in different functional states Serrano-Vega, Magnani, Shibata, & Tate, 2008; Warne et al., 2008), (ii) identification of other exogenous soluble fusion domains such as the thermostabilized apocytochrome b562RIL (BRIL) (Chun et al., 2012; Liu et al., 2012) and the rubredoxin (Tan et al., 427 The Ion Channel-Coupled Receptor Assay 2013), (iii) introduction of smaller camelid antibodies called nanobodies (Rasmussen, DeVree, et al., 2011a), and (iv) insertion of soluble domains in the N-terminus (Fenalti et al., 2014; Rasmussen, Choi, et al., 2011; Siu et al., 2013; Wang et al., 2013; Wu et al., 2014) and in the i2 loop (Hollenstein et al., 2013). Alone or in combination, these methods led to the structural determination of 25 other GPCRs from four different classes over the past 7 years. "
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    ABSTRACT: Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-"uncoupled" receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L). © 2015 Elsevier Inc. All rights reserved.
    Membrane Proteins—Production and Functional Characterization, 03/2015: chapter 20: pages 425-54;
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