Structural basis for μ-opioid receptor binding and activation

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ～1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics.     

Stabilization of μ-opioid receptor facilitates its cellular translocation and signaling

The G protein-coupled μ-opioid receptor (μ-OR) mediates the majority of analgesia effects for morphine and other pain relievers. Despite extensive studies of its structure and activation mechanisms, the inherently low maturation efficiency of μ-OR represents a major hurdle to understanding its function. Here we computationally designed μ-OR mutants with altered stability to probe the relationship between cell-surface targeting, signal transduction, and agonist efficacy. The stabilizing mutation T315Y enhanced μ-OR trafficking to the plasma membrane and significantly promoted the morphine-mediated inhibition of downstream signaling. In contrast, the destabilizing mutation R165Y led to intracellular retention of μ-OR and reduced the response to morphine stimulation. These findings suggest that μ-OR stability is an important factor in regulating receptor signaling and provide a viable avenue to improve the efficacy of analgesics.     

Facilitation of μ-opioid receptor activity by preventing δ-opioid receptor-mediated codegradation

δ-opioid receptors (DORs) form heteromers with μ-opioid receptors (MORs) and negatively regulate MOR-mediated spinal analgesia. However, the underlying mechanism remains largely unclear. The present study shows that the activity of MORs can be enhanced by preventing MORs from DOR-mediated codegradation. Treatment with DOR-specific agonists led to endocytosis of both DORs and MORs. These receptors were further processed for ubiquitination and lysosomal degradation, resulting in a reduction of surface MORs. Such effects were attenuated by treatment with an interfering peptide containing the first transmembrane domain of MOR?(MOR(TM1)), which interacted with DORs and disrupted the MOR/DOR interaction. Furthermore, the systemically applied fusion protein consisting of MOR(TM1) and TAT at the C terminus could disrupt the MOR/DOR interaction in the mouse spinal cord, enhance the morphine analgesia, and reduce the antinociceptive tolerance to morphine. Thus, dissociation of MORs from DORs in the cell membrane is?a potential strategy to improve opioid analgesic therapies.     

Agonist-dependent μ-opioid receptor signaling can lead to heterologous desensitization

Desensitization of the µ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca²⁺ ([Ca²⁺]_i), we reported that [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of β-arrestins (βArrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by modulating PKCε activity. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating three putative PKC phosphorylation sites, Ser⁴⁴, Ser¹⁴⁴ and Ser³⁰² on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca²⁺]_i release, and this desensitization could be reversed by pretreating the cells with PKCε inhibitor or overexpressing Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors, such as the CB1.     

Ligand based conformational space studies of the μ-opioid receptor

BackgroundG protein-coupled receptors (GPCRs) comprise a family of membrane proteins that can be activated by a variety of external factors. The μ-opioid receptor (MOR), a class A GPCR, is the main target of morphine. Recently, enhanced sampling molecular dynamics simulations of a constitutively active mutant of MOR in its apo form allowed us to capture the novel intermediate states of activation, as well as the active state. This prompted us to apply the same techniques to wild type MOR in complex with ligands, in order to explore their contributions to the receptor conformational changes in the activation process.MethodsMOR was modeled in complex with agonists (morphine, BU72), a partial agonist (naloxone benzoylhydrazone) and an antagonist (naloxone). Replica exchange with solute tempering (REST2) molecular dynamics simulations were carried out for all systems. Trajectory frames were clustered, and the activation state of each cluster was assessed by two different methods.ResultsCluster sizes and activation indices show that while agonists stabilized structures in a higher activation state, the antagonist behaved oppositely. Morphine tends to drive the receptor towards increasing R165-T279 distances, while naloxone tends to increase the NPxxYA motif conformational change.ConclusionsDespite not observing a full transition between inactive and active states, an important conformational change of transmembrane helix 5 was observed and associated with a ligand-driven step of the process.General significanceThe activation process of GPCRs is widely studied but still not fully understood. Here we carried out a step forward in the direction of gaining more details of this process.     

Inhibition of forebrain μ-opioid receptor signaling by low concentrations of rimonabant does not require cannabinoid receptors and directly involves μ-opioid receptors

Increasing number of publications shows that cannabinoid receptor 1 (CB(1)) specific compounds might act in a CB(1) independent manner, including rimonabant, a potent CB(1) receptor antagonist. Opioids, cannabinoids and their receptors are well known for their overlapping pharmacological properties. We have previously reported a prominent decrease in μ-opioid receptor (MOR) activity when animals were acutely treated with the putative endocannabinoid noladin ether (NE). In this study, we clarified whether the decreased MOR activation caused by NE could be reversed by rimonabant in CB(1) receptor deficient mice. In functional [(35)S]GTPγS binding assays, we have elucidated that 0.1mg/kg of intraperitoneal (i.p.) rimonabant treatment prior to that of NE treatment caused further attenuation on the maximal stimulation of Tyr-d-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO), which is a highly specific MOR agonist. Similar inhibitory effects were observed when rimonabant was injected i.p. alone and when it was directly applied to forebrain membranes. These findings are cannabinoid receptor independent as rimonabant caused inhibition in both CB(1) single knockout and CB(1)/CB(2) double knockout mice. In radioligand competition binding assays we highlighted that rimonabant fails to displace effectively [(3)H]DAMGO from MOR in low concentrations and is highly unspecific on the receptor at high concentrations in CB(1) knockout forebrain and in their wild-type controls. Surprisingly, docking computational studies showed a favorable binding position of rimonabant to the inactive conformational state of MOR, indicating that rimonabant might behave as an antagonist at MOR. These findings were confirmed by radioligand competition binding assays in Chinese hamster ovary cells stably transfected with MOR, where a higher affinity binding site was measured in the displacement of the tritiated opioid receptor antagonist naloxone. However, based on our in vivo data we suggest that other, yet unidentified mechanisms are additionally involved in the observed effects.     

An examination of the relationship between μ-opioid antinociceptive efficacy and G-protein coupling using pertussis and cholera toxins

The hypothesis that μ-opioid agonists having low antinociceptive efficacy might be more susceptible to interference with G-protein coupling than μ-opioid agonists having higher antinocicep-tive efficacy was tested. Supraspinal antinociceptive efficacy for the three μ-opioid agonists morphine, [D-Ala², NMePhe⁴, Gly⁵-ol]-enkephalin (DAMGO) and sufentanil in the mouse 55 °C warm-water tail-flick test was evaluated 18–24 h after intracerebroventricular (i.c.v.) administration of β-funaltrexamine (β-FNA). The β-FNA pretreatment (0.2–2.0 nmol) attenuated antinociception in the order morphine > DAMGO > sufentanil, consistent with previous reports of their relative antinociceptive efficacy. The association of efficacy with G-protein coupling was then assessed by determining sensitivity to i.c.v. (0.1–3.0 μg) pertussis toxin (PTX) or cholera toxin (CTX). The effect of PTX on equiantinociceptive doses was in the inverse order of agonist efficacy. CTX augmented sufentanil-induced antinociception. Morphine- and DAMGO-induced antinociception were unaffected by CTX. These data suggest that: (i) highly efficacious μ agonists (viz., sufentanil) couple more efficiently to PTX-sensitive inhibitory G_i-proteins than do agonists of lower efficacy (viz., morphine, DAMGO) and (ii) highly efficacious μ agonists have greater capacity to utilize CTX-sensitive stimulatory G_s-proteins than do μ-agonists with lower efficacy.     

β-Arrestin 1 and 2 similarly influence μ-opioid receptor mobility and distinctly modulate adenylyl cyclase activity

β-Arrestins are known to play a crucial role in GPCR-mediated transmembrane signaling processes. However, there are still many unanswered questions, especially those concerning the presumed similarities and differences of β-arrestin isoforms. Here, we examined the roles of β-arrestin 1 and β-arrestin 2 at different levels of μ-opioid receptor (MOR)-regulated signaling, including MOR mobility, internalization of MORs, and adenylyl cyclase (AC) activity. For this purpose, naïve HEK293 cells or HEK293 cells stably expressing YFP-tagged MOR were transfected with appropriate siRNAs to block in a specific way the expression of β-arrestin 1 or β-arrestin 2. We did not find any significant differences in the ability of β-arrestin isoforms to influence the lateral mobility of MORs in the plasma membrane. Using FRAP and line-scan FCS, we observed that knockdown of both β-arrestins similarly increased MOR lateral mobility and diminished the ability of DAMGO and endomorphin-2, respectively, to enhance and slow down receptor diffusion kinetics. However, β-arrestin 1 and β-arrestin 2 diversely affected the process of agonist-induced MOR endocytosis and exhibited distinct modulatory effects on AC function. Knockdown of β-arrestin 1, in contrast to β-arrestin 2, more effectively suppressed forskolin-stimulated AC activity and prevented the ability of activated-MORs to inhibit the enzyme activity. Moreover, we have demonstrated for the first time that β-arrestin 1, and partially β-arrestin 2, may somehow interact with AC and that this interaction is strongly supported by the enzyme activation. These data provide new insights into the functioning of β-arrestin isoforms and their distinct roles in GPCR-mediated signaling.     

The conformational transition during G protein–coupled receptor (GPCR) and G protein interaction

The precise structural mechanism of G protein–coupled receptor (GPCR)–G protein coupling has been of significant research interest because it provides fundamental knowledge on cellular signaling and valuable information for GPCR-targeted drug development. Over the last decade, several GPCR–G protein complex structures have been identified. However, these structures are mere snapshots of guanosine diphosphate (GDP)-released stable GPCR–G protein complexes, which have limited the understanding of the allosteric conformational transition during receptor binding to GDP release and the GPCR–G protein coupling selectivity. Recently, deeper insights into the mechanism underlying stepwise conformational changes during GPCR–G protein coupling were obtained using hydrogen/deuterium exchange mass spectrometry, hydroxyl radical footprinting-mass spectrometry, X-ray crystallography, cryoelectron microscopy, and molecular dynamics simulation techniques. This review summarizes these recent developments.     

Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists

The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.     

Endomorphin analogues with mixed μ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking β-arrestin2 recruitment activity

Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH₂) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH₂) modified at position 3, with various phenylalanine analogues (Xaa = Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high μ-opioid (MOP) receptor binding affinity (K_iMOP = 0.13–0.81 nM), modest MOP-selectivity (K_{iδ-opioid (DOP)}/K_iMOP = 3.5–316), and potent functional MOP agonism (GPI, IC₅₀ = 0.274–249 nM) without DOP and κ-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH₂) and 9 (Dmt-Pro-1-Nal-NH₂) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of β-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine.     

Selective interactions of spinophilin with the C-terminal domains of the δ- and μ-opioid receptors and G proteins differentially modulate opioid receptor signaling

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218-1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139-148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and μ-opioid receptors (δ-ΟR, μ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both μ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and μ-OR is narrowed to a region encompassing amino acids 151-444. Spinophilin, RGS4, Gα and Gβγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-μ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the μ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the μ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and μ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.     

Mechanisms of selective G protein–coupled receptor localization and trafficking

The trafficking of G protein–coupled receptors (GPCRs) to different membrane compartments has recently emerged as being a critical determinant of the signaling profiles of activation. GPCRs, which share many structural and functional similarities, also share many mechanisms that traffic them between compartments. This sharing raises the question of how the trafficking of individual GPCRs is selectively regulated. Here, we will discuss recent studies addressing the mechanisms that contribute to selectivity in endocytic and biosynthetic trafficking of GPCRs.     

Membrane partitioning of various δ-opioid receptor forms before and after agonist activations: The effect of cholesterol

Lipid rafts depicted as densely packed and thicker membrane microdomains, based on the dynamic clustering of cholesterol and sphingolipids, may help as platforms involved in a wide variety of cellular processes. The reasons why proteins segregate into rafts are yet to be clarified. The human delta opioid receptor (hDOR) reconstituted in a model system has been characterised after ligand binding by an elongation of its transmembrane part, inducing rearrangement of its lipid microenvironment [Alves, Salamon, Hruby, and Tollin (2005) Biochemistry 44, 9168-9178]. We used hDOR to understand better the correlation between its function and its membrane microdomain localisation. A fusion protein of hDOR with the Green Fluorescent Protein (DOR?) allows precise receptor membrane quantification. Here we report that (i) a fraction of the total receptor pool requires cholesterol for binding activity, (ii) G-proteins stabilize a high affinity state conformation which does not seem modulated by cholesterol. In relation to its distribution, and (iii) a fraction of DOR? is constitutively associated with detergent-resistant membranes (DRM) characterised by an enrichment in lipids and proteins raft markers. (iv) An increase in the quantity of DOR? was observed upon agonist addition. (v) This DRM relocation is prevented by uncoupling the receptor-G-protein interaction.     

Methamphetamine-elicited alterations of dopamine- and serotonin-metabolite levels within μ-opioid receptor knockout mice: a microdialysis study

μ-Opioid receptors (μ-ORs) modulate methamphetamine (MA)-induced behavioral responses, increased locomotor activity and stereotyped behavior in the mouse model. We investigated the changes in dopamine (DA) and serotonin (5-HT) metabolism in the striatum following either acute or repeated MA treatment using in vivo microdialysis. We also studied the role of μ-ORs in the modulation of MA-induced DA and 5-HT metabolism within μ-OR knockout mice. Subsequent to either acute or repeated intraperitoneal administration of MA, wild-type mice revealed decreases in extracellular concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in a dose-dependent manner. Moreover, wild-type mice had reductions in basal concentrations of DOPAC and HVA following repeated MA treatment with a higher dose. The effects of acute, repeated or challenge MA administration upon extracellular levels of DOPAC and HVA within μ-OR knockout mice significantly differed from the wild-type controls. The duration of recovery to the basal levels of extracellular DA and 5-HT metabolites induced by MA were much longer in wild-type mice than for μ-OR knockout mice. These findings suggest that μ-ORs play a modulatory role in MA-induced DA and 5-HT metabolism in the mouse striatum. This possible mechanism of MA-induced behavioral change as modulated by μ-OR merits further study.     

The regulator of G protein signaling (RGS) domain of G protein–coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function

The G protein–coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function.     

Methamphetamine-induced changes in the striatal dopamine pathway in μ-opioid receptor knockout mice

Background  

Repeated exposure to methamphetamine (METH) can cause not only neurotoxicity but also addiction. Behavioral sensitization is widely used as an animal model for the study of drug addiction. We previously reported that the μ-opioid receptor knockout mice were resistant to METH-induced behavioral sensitization but the mechanism is unknown.     

Membrane phosphoinositides and protein–membrane interactions

Proteins with polybasic clusters bind to negatively charged phosphoinositides at the cell membrane. In this review, I have briefly discussed the types of phosphoinositides naturally found on membrane surfaces and how they recruit protein complexes for carrying out the process of signal transduction. A large number of researchers from around the world are now focusing their attention on protein–membrane binding, as these interactions have started to offer us a much better insight into the process of cell signaling. The main areas discussed in this brief review article include the phosphoinositide binding specificities of proteins and the role of their lipid binding in signaling processes downstream of membrane recruitment.     

The influence of μ-opioid receptor agonist and antagonist peptides on peripheral blood mononuclear cells (PBMCs)

Milk is one of the main source of biologically-active peptides that may function as regulatory substances called food hormones. After passing the gut-blood barrier, the μ-opioid receptor agonist and antagonist peptides may become the new factors influencing various functions of the human organism. The aim of the conducted research was to determine the influence of μ-opioid receptor agonist peptides: human and bovine β-casomorphin-7 (h/bBCM-7) and antagonistic peptides: casoxin-6 and- D (CXN-6/D) on proliferation and cytokine secretion of human peripheral blood mononuclear cells (PBMCs). The PBMCs proliferation was measured by the use of the BrdU test, which assesses the DNA synthesis activity and the WST-1 test which assesses the activity of mitochondrial dehydrogenase enzymes. The influence of all the investigated peptides on secretion of IL-4, IL-8, IL-13 and IFN-γ was determined by the use of the ELISA tests. Incubating the cells with the peptides has not caused any changes to their enzymatic activity, which has been proved by a WST-1 test. When using a BrdU test, however, it has been observed that there appear changes to proliferation of PBMCs correlated to amounts of bromodeoxyuridine incorporated into the cellular DNA. Moreover, changes to secretion of IL-4 and IL-13 by the cells under the influence of agonists were detected, as well as changes to secretion of IFN-gamma under the influence of all the examined substances. The obtained results provide information on immunomodulatory effects of food-derived opioid peptides, which may be of clinical significance especially in the case of allergic diseases in newborns.