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HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants.

ABSTRACT: G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal ³⁸⁷LENLEAETAPLP³⁹⁸ motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the ³⁸⁷LENLEAETAPLP³⁹⁸ motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

SUBMITTER: Fritzwanker S

PROVIDER: S-EPMC8440528 | biostudies-literature |

REPOSITORIES: biostudies-literature

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HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants

Project description:G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

2022-12-07 | PXD027825 | Pride

Quantitative Detection of µ Opioid Receptor: Western Blot Analyses Using µ Opioid Receptor Knockout Mice.

Project description:Increasing evidence suggests that µ opioid receptor (MOP) expression is altered during the development of and withdrawal from substance dependence. Although anti-MOP antibodies have been hypothesized to be useful for estimating MOP expression levels, inconsistent MOP molecular weights (MWs) have been reported in studies using anti-MOP antibodies. In the present study, we generated a new anti-MOP antibody (N38) against the 1-38 amino acid sequence of the mouse MOP N-terminus and conducted Western blot analysis with wildtype and MOP knockout brain lysates to determine the MWs of intrinsic MOP. The N38 antibody detected migrating bands with relative MWs of 60-67 kDa in the plasma membrane fraction isolated from wildtype brain, but not from the MOP knockout brain. These migrating bands exhibited semi-linear density in the range of 3-30 µg membrane proteins/lane. The N38 antibody may be useful for quantitatively detecting MOP.

| S-EPMC3137187 | biostudies-literature

Structural insights into µ-opioid receptor activation.

Project description:Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for μOR activation, here we report a 2.1 Å X-ray crystal structure of the murine μOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2-adrenergic receptor (β2AR) and the M2 muscarinic receptor. Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

| S-EPMC4639397 | biostudies-literature

N-Phenethyl Substitution in 14-Methoxy-N-methylmorphinan-6-ones Turns Selective µ Opioid Receptor Ligands into Dual µ/δ Opioid Receptor Agonists.

Project description:Morphine and structurally-derived compounds are µ opioid receptor (µOR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective µOR ligands into dual µ/δOR agonists (3a and 4a). Contrary to N-methylmorphinans 1-4, the N-phenethyl substituted morphinans 1a-4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the µOR, we establish that N-methylmorphinans 1-4 and their N-phenethyl counterparts 1a-4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics.

| S-EPMC7101422 | biostudies-literature

Structure of the µ-opioid receptor-Gi protein complex.

Project description:The ?-opioid receptor (?OR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by ?OR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5?Å resolution cryo-electron microscopy structure of the ?OR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the ?OR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein ?-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.

| S-EPMC6317904 | biostudies-literature

Agonists at the ?-opioid receptor modify the binding of µ-receptor agonists to the µ-? receptor hetero-oligomer.

Project description:Background and purposeµ- and ?-opioid receptors form heteromeric complexes with unique ligand binding and G protein-coupling profiles linked to G protein ? z-subunit (G?(z) ) activation. However, the mechanism of action of agonists and their regulation of the µ-? receptor heteromer are not well understood.Experimental approachCompetition radioligand binding, cell surface receptor internalization in intact cells, confocal microscopy and receptor immunofluorescence techniques were employed to study the regulation of the µ-? receptor heteromer in heterologous cells with and without agonist exposure.Key resultsG?(z) enhanced affinity of some agonists at µ-? receptor heteromers, independent of agonist chemical structure. ?-Opioid agonists displaced µ-agonist binding with high affinity from µ-? heteromers, but not µ receptor homomers, suggestive of ?-agonists occupying a novel µ-receptor ligand binding pocket within the heteromers. Also, ?-agonists induced internalization of µ-opioid receptors in cells co-expressing µ- and ?-receptors, but not those expressing µ-receptors alone, indicative of µ-? heteromer internalization. This dose-dependent, Pertussis toxin-resistant and clathrin- and dynamin-dependent effect required agonist occupancy of both µ- and ?-opioid receptors. In contrast to µ-receptor homomers, agonist-induced internalization of µ-? heteromers persisted following chronic morphine exposure.Conclusions and implicationsThe µ-? receptor heteromer may contain a novel ?-agonist-detected, high-affinity, µ-receptor ligand binding pocket and is regulated differently from the µ-receptor homomer following chronic morphine exposure. Occupancy of both µ- and ?-receptor binding pockets is required for ?-agonist-induced endocytosis of µ-? receptor heteromers. ?-Opioid agonists target µ-? receptor heteromers, and thus have a broader pharmacological specificity than previously identified.

| S-EPMC2998692 | biostudies-literature

Cellular tolerance at the µ-opioid receptor is phosphorylation dependent.

Project description:Phosphorylation of the ?-opioid receptor (MOR) is known as a key step in desensitization and internalization but the role in the development of long-term tolerance at the cellular level is not known. Viral expression of wild type (exWT) and mutant MORs, where all phosphorylation sites on the C-terminus (Total Phosphorylation Deficient (TPD)) were mutated to alanine, were examined in locus coeruleus neurons in a MOR knockout rat. Both receptors activated potassium conductance similar to endogenous receptors in wild type animals. The exWT receptors, like endogenous receptors, acutely desensitized, internalized and, after chronic morphine treatment, displayed signs of tolerance. However, TPD receptors did not desensitize or internalize with agonist treatment. In addition the TPD receptors did not develop cellular tolerance following chronic morphine treatment. Thus C-terminal phosphorylation is necessary for the expression of acute desensitization, trafficking and one sign of long-term tolerance to morphine at the cellular level.

| S-EPMC5873894 | biostudies-literature

Dynamic and Kinetic Elements of µ-Opioid Receptor Functional Selectivity.

Project description:While the therapeutic effect of opioids analgesics is mainly attributed to µ-opioid receptor (MOR) activation leading to G protein signaling, their side effects have mostly been linked to ?-arrestin signaling. To shed light on the dynamic and kinetic elements underlying MOR functional selectivity, we carried out close to half millisecond high-throughput molecular dynamics simulations of MOR bound to a classical opioid drug (morphine) or a potent G protein-biased agonist (TRV-130). Statistical analyses of Markov state models built using this large simulation dataset combined with information theory enabled, for the first time: a) Identification of four distinct metastable regions along the activation pathway, b) Kinetic evidence of a different dynamic behavior of the receptor bound to a classical or G protein-biased opioid agonist, c) Identification of kinetically distinct conformational states to be used for the rational design of functionally selective ligands that may eventually be developed into improved drugs; d) Characterization of multiple activation/deactivation pathways of MOR, and e) Suggestion from calculated transition timescales that MOR conformational changes are not the rate-limiting step in receptor activation.

| S-EPMC5595830 | biostudies-literature

SpliceDB: database of canonical and non-canonical mammalian splice sites.

Project description:A database (SpliceDB) of known mammalian splice site sequences has been developed. We extracted 43 337 splice pairs from mammalian divisions of the gene-centered Infogene database, including sites from incomplete or alternatively spliced genes. Known EST sequences supported 22 815 of them. After discarding sequences with putative errors and ambiguous location of splice junctions the verified dataset includes 22 489 entries. Of these, 98.71% contain canonical GT-AG junctions (22 199 entries) and 0.56% have non-canonical GC-AG splice site pairs. The remainder (0.73%) occurs in a lot of small groups (with a maximum size of 0.05%). We especially studied non-canonical splice sites, which comprise 3.73% of GenBank annotated splice pairs. EST alignments allowed us to verify only the exonic part of splice sites. To check the conservative dinucleotides we compared sequences of human non-canonical splice sites with sequences from the high throughput genome sequencing project (HTG). Out of 171 human non-canonical and EST-supported splice pairs, 156 (91.23%) had a clear match in the human HTG. They can be classified after sequence analysis as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors corrected to AT-AC), one case was produced from a non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two other cases left of supported non-canonical splice pairs. The information about verified splice site sequences for canonical and non-canonical sites is presented in SpliceDB with the supporting evidence. We also built weight matrices for the major splice groups, which can be incorporated into gene prediction programs. SpliceDB is available at the computational genomic Web server of the Sanger Centre: http://genomic.sanger.ac. uk/spldb/SpliceDB.html and at http://www.softberry. com/spldb/SpliceDB.html.

| S-EPMC29840 | biostudies-literature

The µ-opioid system promotes visual attention to faces and eyes.

Project description:Paying attention to others' faces and eyes is a cornerstone of human social behavior. The µ-opioid receptor (MOR) system, central to social reward-processing in rodents and primates, has been proposed to mediate the capacity for affiliative reward in humans. We assessed the role of the human MOR system in visual exploration of faces and eyes of conspecifics. Thirty healthy males received a novel, bidirectional battery of psychopharmacological treatment (an MOR agonist, a non-selective opioid antagonist, or placebo, on three separate days). Eye-movements were recorded while participants viewed facial photographs. We predicted that the MOR system would promote visual exploration of faces, and hypothesized that MOR agonism would increase, whereas antagonism decrease overt attention to the information-rich eye region. The expected linear effect of MOR manipulation on visual attention to the stimuli was observed, such that MOR agonism increased while antagonism decreased visual exploration of faces and overt attention to the eyes. The observed effects suggest that the human MOR system promotes overt visual attention to socially significant cues, in line with theories linking reward value to gaze control and target selection. Enhanced attention to others' faces and eyes represents a putative behavioral mechanism through which the human MOR system promotes social interest.

| S-EPMC5141966 | biostudies-literature

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