Animals
Knock-in mice expressing HA-MOP (Oprm1em1Shlz, MGI:6117675) were generated by Applied StemCell (Menlo Park, USA). Mice were genotyped by PCR of genomic tail-biopsy DNA using the following primers: 5- TACCCATACGATGTTCCAGATTACGCT-3 and 5- GGAACTAGGTATTCAGAACATGCCTTACCTTAC-3, followed by RsaI restriction to detect presence of the HA-tagged mOprm1 gene. All HA-MOP mice were backcrossed to WT control mice JAX™ C57Bl/6J obtained from Charles River Laboratories (DE) which were also used for breeding of mutant strains and served as controls in all experiments. Animals were housed 2–5 per cage under a 12-hour light-dark cycle with ad libitum access to food and water. All animal experiments were performed in accordance with the Thuringian state authorities and complied with European Commission regulations for the care and use of laboratory animals. Our study is reported in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines16 In all experiments, male and female mice aged 8–16 weeks between 25 and 30 g body weight were used.
Drugs and routes of administration
All drugs were freshly prepared prior to use and were injected subcutaneously in lightly restrained, unanaesthetized mice at a volume of 10 µl g− 1 body weight. Drugs were diluted in saline for injections. Drugs were obtained and used as follows: morphine sulphate (30 mg kg− 1 for 30 min; Hameln Inc., Hameln, Germany), oxycodone hydrochloride (15 mg kg− 1 for 30 min; Mundipharma GmbH, Limburg, Germany), levomethadone hydrochloride (15 mg kg− 1 for 30 min; Sanofi-Aventis, Frankfurt, Germany), sufentanil (30 µg kg− 1 for 15 min; Hameln Inc., Hameln, Germany), fentanyl citrate (0.3 mg kg− 1 for 15 min; Rotexmedica, Trittau, Germany), etonitazene (30 µg kg− 1 for 30 min; Sigma Aldrich, Munich, Germany) and naltrexone hydrochloride (10 mg kg− 1 over night; Neuraxpharm, Langenfeld, Germany).
Reagents and antibodies
Pierce™ Anti-HA Magnetic Beads were obtained from Thermo Fisher Scientific (Schwerte, Germany), while the phosphorylation-independent antibodies were obtained as follows: rabbit monoclonal anti-HA antibody (Cell Signaling, Frankfurt, Germany), and anti-MOP antibody {UMB-3} (Epitomics, Burlingame, CA) and used as previously described17 The rabbit polyclonal phosphosite-specific µ-opioid receptor antibodies anti-pT370 {3195}, anti-pT376 {3722} and anti-pT379 {3686} were generated and extensively characterized as previously described18,19,20. The polyclonal phosphosite-specific anti-pS375 was obtained from Cell Signaling (Frankfurt, Germany). The polyclonal rabbit phosphorylation-independent-antibody for MOP1D was generated against the alternative C-terminal splice sequence NHQRNEEPSS (unpublished). This sequence corresponds to amino acids 384–393 of the mouse receptor. The antibodies were affinity-purified against their immunizing peptide using the SulfoLink kit (Thermo Scientific, Rockford, IL). In addition, the following commercially available secondary antibodies were used: polyclonal donkey anti-rabbit IgG Cy3 (Dianova, Hamburg, Germany) and goat anti-rabbit IgG, HRP-linked antibody (Cell Signaling, Frankfurt, Germany).
Immunoprecipitation of HA-MOP from brain lysates
Depending on the experiment, mice were either treated with agonists, antagonists, saline or received no treatment at all. Mice were anesthetized with isoflurane, killed by cervical dislocation, and brains were quickly dissected, excluding the cerebellum. The brain samples were immediately frozen in liquid nitrogen. Brains were transferred to ice-cold detergent buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), containing protease and phosphatase inhibitors), homogenized, and centrifuged at 14,000 × g for 30 min at 4 °C. The supernatant was then precipitated with HA-tagged magnetic beads (Thermo Fisher Scientific, Germany) for 60 min at 4 °C. Afterwards the receptor-beads-conjugates were separated from the supernatant using a special magnetic device (DynaMagTM-2, life technologies) and washed 3 times. Proteins were eluted from the beads with SDS-sample buffer for 25 min at 43 °C and then resolved on 8% SDS-polyacrylamide gels. After electroblotting, membranes were incubated with anti-pT370, anti-pS375, anti-pT376 or anti-pT379 antibody, followed by detection using a chemiluminescence detection system. Blots were subsequently stripped and incubated again with the phosphorylation-independent antibodies anti-HA and UMB-3 to confirm equal loading of the gels. Films exposed in the linear range were then densitized using ImageJ 1.37v.
The same procedure was used for the MOP1D detection experiments. The membranes were incubated with either anti-MOP1D or UMB-3 antibody, followed by detection using a chemiluminescence detection system. Blots were subsequently stripped and incubated again with the phosphorylation-independent antibody anti-HA to confirm equal loading of the gels.
Immunodepletion of canonical HA-MOP
In order to enrich MOP variants that are alternatively-spliced at the C-terminus, we employed immunodepletion experiments. Brains were dissected from untreated HA-MOP mice, homogenized as described above and supernatants were pooled. Using the well-characterized antibody UMB-3, receptor proteins containing the canonical carboxyl-terminal 387LENLEAETAPLP398 motif were successively removed by immunoprecipitation using protein A-agarose beads. In theory, only MOP variants with non-canonical C-termini should thus remain in the lysate but should be detectable using their N-terminal HA-tag. A total of seven successive rounds of immunoprecipitation were performed and an aliquot was removed after each step. From each aliquot, remaining HA-MOP was precipitated using HA-beads as described and captured proteins were analyzed by Western blot probing for HA-epitopes. The quantitative capacity of anti-HA to precipitate HA-MOP was evaluated using the same immunodepletion strategy. Brain lysates were treated as described above and successively immunoprecipitated using HA-beads for 7 rounds. Aliquots from each step were analyzed by Western blot. Proteins were then loaded in a dilution series from 100–0.6% on 8% SDS-polyacrylamide gel. Staining intensities from these blots were used as a calibration standard to evaluate remaining HA-tagged proteins in the immunodepletion experiments.
Cell culture and transfection
HEK293 cells were obtained from the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany) and grown in Dulbecco´s modified Eagle´s medium supplemented with 10% fetal calf serum in a humidified atmosphere containing 5% CO2. Cells were transfected with plasmid encoding murine HA-tagged MOP or MOP1D using Lipofectamine according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Stable transfectants were selected in the presence of 1 µg/ml puromycin for HA-MOP or G-418 500 µg/ml for HA-MOP1D. HEK293 cells stably expressing MOP were characterized using radioligand-binding assays, Western blot analysis, immunocytochemistry, and cAMP assays as described previously6. For western blot analysis, cells were seeded onto poly-L-lysine-coated 60 mm dishes and grown to 90% confluence. Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0,5% sodium deoxycholate, 0,1% SDS) containing protease and phosphatase inhibitors (Complete mini and PhosSTOP; Roche Diagnostics, Mannheim, Germany). Pierce™ HA epitope tag Antibodies (Thermo Scientific, Rockford, USA) were used to enrich HA-tagged MOP. To elute proteins from the beads, the samples were incubated in SDS sample buffer for 25 min at 43 °C. Supernatants were separated from the beads, loaded on 8% SDS polyacrylamide gels and immunoblotted onto nitrocellulose afterwards. After blocking, membranes were incubated with either MOP1D antibody or UMB-3 antibody at 4 °C overnight. On the next day, membranes were incubated with peroxidase-conjugated secondary antibody followed by detection using a chemiluminescence system (90 mM p-coumaric-acid, 250 mM luminol, 30% hydrogen peroxide). Afterwards, blots were stripped and reprobed with anti-HA antibody to confirm equal loading of the gel. Protein bands on Western blots were exposed to X-ray films.
Immunohistochemistry
Mice were anesthetized with isoflurane and transcardially perfused with Tyrode’s solution followed by Zamboni’s fixative (4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4). Brains were rapidly dissected and postfixed in the same solution for 2 hours. Then the tissue was cryoprotected by immersion in 10% sucrose, followed by 30% sucrose for 48 hours at 4 °C before sectioning using a freezing microtome. Free-floating sections (40 µm) were washed multiple times, blocked and incubated with anti-HA antibody (Cell Signaling, Frankfurt, Germany) overnight. On the following day, Cy3-conjugated anti-rabbit antibody (Dianova, Hamburg, Germany) was used for detection. Cy3 was imaged with excitation at 568 nm using a Zeiss LSM510 META laser scanning confocal microscope.
In-gel tryptic digestion, nanoLC-MS/MS analysis and database searches
For mass spectrometry (MS) analysis, HA-immunoprecipitated samples were reduced for 30 min at 37 °C by adding 1 x SDS sample buffer containing 30 mM DTT, and then alkylated in 90 mM iodoacetamide for 30 min in the dark at room temperature. 60 µl of reduced/alkylated protein samples were separated by SDS-PAGE on 10% polyacrylamide gels followed by gel staining with colloidal Coomassie blue. At the expected molecular weight of HA-MOP receptor, a band was excised and subjected to in-gel tryptic digestion using modified porcine trypsin (Promega, France) at 20 ng/µl. Tryptic peptides were extracted and analyzed in triplicate by on-line nanoLC using an Ultimate 3000 system (Dionex, Amsterdam, The Netherlands) coupled to an ETD-enabled LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) as previously described21,22. Survey scan MS was performed in the Orbitrap over a 300–2000 m/z mass range with resolution set to a value of 60,000 at m/z 400. The 20 most intense ions per survey scan were selected for subsequent CID/ETD fragmentation, and the resulting fragments were analyzed in the linear trap (LTQ). The settings for the data-dependent decision tree-based CID/ETD method were as follows: ETD was performed instead of CID if charge state was 3 and m/z less than 650, or if the charge state was 4 and the m/z less than 900, or if the charge state was 5 and the m/z less than 950. ETD was performed for all precursor ions with charge states > 5. The normalized collision energy was set to 35% for CID. The reaction time was set to 100 ms and supplemental activation was enabled for ETD. Dynamic exclusion was employed within 30 seconds to prevent repetitive selection of the same peptide. For internal calibration the 445.120025 ion was used as lock mass. 3–4 technical replicates were performed for each condition. All raw mass spectrometry files were processed with Proteome Discoverer software (version 2.1, Thermo Fisher Scientific) for database search with the Mascot search engine (version 2.6.0, Matrix Science, London, UK) combined with the Percolator algorithm (version 2.05) for PSM search optimization and the phosphoRS algorithm (version 3.1,23) for phosphorylation site localization. For both fragmentation techniques, the parameters set for creation of the peak lists were: parent ions in the mass range 300–5000 Da and no grouping of MS/MS scans. The non-fragmented filter was used to simplify ETD spectra with the following settings: the precursor peak was removed within a 4 Da window, charged reduced precursors were removed within a 2 Da window, and neutral losses from charged reduced precursors were removed within a 2 Da window (the maximum neutral loss mass was set to 120 Da). Peak lists were searched against SwissProt database with taxonomy Mus musculus (16761 sequences) implemented with the mouse HA-tagged MOP receptor sequence and the sequences of 19 predicted MOP isoforms produced by alternative splicing already described in the UniProt entry. Enzyme specificity was set to trypsin/P and a maximum of three missed cleavages were allowed. Carbamidomethylation of cysteine was set as fixed modification whereas oxidation of methionine and phosphorylation of serine, threonine and tyrosine were set as variable modifications. Mass tolerances in MS and MS/MS were set to 10 ppm and 0.6 Da, respectively. Mascot results were validated by the target-decoy approach using a reverse database at the same size. The Percolator algorithm was used to calculate a q-value for each peptide-spectrum match (PSM), peptides and PSM were validated based on Percolator q-values at a False Discovery Rate (FDR) set to 5%. Then, peptide identifications were grouped into proteins according to the law of parsimony and filtered to 5% FDR.
Data Analysis
Data were analyzed using ImageJ and GraphPad Prism 4.0 software.