dynein light chain protein Tctex-1

Biochemical Pharmacology 99 (2016) 60–72

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Cannabinoid receptor 2 expression modulates Gb1g2 protein interaction with the activator of G protein signalling 2/dynein light chain protein Tctex-1 Marina Naglera,b , Lysann Palkowitschc , Sebastian Radinga,b , Barbara Moeppsb , Meliha Karsaka,b,* a Neuronal and Cellular Signal Transduction, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center Hamburg-Eppendorf (UKE), 20246 Hamburg, Germany b Institute of Pharmacology and Toxicology, Ulm University, 89081 Ulm, Germany c Institute of Physiological Chemistry, Ulm University, 89081 Ulm, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 August 2015 Accepted 22 September 2015 Available online 26 September 2015

The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gbg, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G proteincoupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gbg. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1–Gbg-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gbg interacts with Tctex-1, but competes with Tctex-1 in binding to Gbg. The Tctex-1–Gbg protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression “dosedependently” and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gbg–Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a noncanonical pathway. ã 2015 Elsevier Inc. All rights reserved.

Chemical compounds studied in this article: 2-AG (PubChem CID: 5282280) AM 630 (PubChem CID: 4302963) Ammonium chloride (PubChem CID: 25517) Bafilomycin A1 (PubChem CID: 25517) Cycloheximide (PubChem CID: 6197) JWH 133 (PubChem CID: 6918505) Leupeptin hemisulfate (PubChem CID: 2733491) MG132 (PubChem CID: 462382) Keywords: G protein-coupled receptor (GPCR) Cannabinoid receptor (CB2) Tctex-1 G protein subunit Protein degradation

1. Introduction The cannabinoid CB2 receptor belongs to the family of G protein-coupled receptors (GPCR) and is activated by lipophilic

substances like the phytocannabinoid delta-9-tetrahydrocannabinol (D9-THC) and the dietary cannabinoid beta-caryophyllene [1]. In addition, endogenously synthesized endocannabinoids such as arachidonoyl-glycerol derivatives stimulate CB2 receptors.

Abbreviations: AGS2, activator of G protein signalling; AM 630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone; 2-AG, 2-arachidonoyl-glycerol; CB1, cannabinoid CB1 receptor; CB2, cannabinoid CB2 receptor; co-IP, co-immunoprecipitation; DDM, n-dodecyl b-D-maltoside; DIC, dynein intermediate chain; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethylsulfoxide; Dynlt1, dynein light chain protein 1; Erk1/2, extracellular signal-regulated kinase 1/2; FCS, fetal calf serum; Gb, b subunit of GTP-binding protein; Gg, g subunit of GTP-binding protein; GEF-H1, rho guanine nucleotide exchange factor H1; GPCR, G proteincoupled receptor; GST, glutathione S-transferase; GTP, guanosine triphosphate; HEK293, human embryonic kidney cells; JWH 133, (6aR,10aR)-3-(1,1-dimethylbutyl)6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran; MG132, N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide; PTH1R, parathyroid hormone 1 receptor; RhoGEF-H1, rho guanine nucleotide exchange factor H1; SD, standard deviation; Tctex-1, T-complex testis-expressed protein 1; D9-THC, delta-9-tetrahydrocannabinol. * Corresponding author at: Neuronal and Cellular Signal Transduction, Center for Molecular Neurobiology Hamburg (ZMNH), University Medical Center HamburgEppendorf (UKE), Martinistr. 52, 20246 Hamburg, Germany. E-mail address: [email protected] (M. Karsak). http://dx.doi.org/10.1016/j.bcp.2015.09.017 0006-2952/ ã 2015 Elsevier Inc. All rights reserved.

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Increased CB2 receptor expression is associated with a wide range of human diseases and it has been shown to contribute to various pathological states in animal models of liver dysfunction, bone degeneration, atherosclerosis, and neurodegenerative diseases [2]. For example, whereas CB2 receptors are absent in healthy brain microglia [3], microglial expression levels of CB2 were found to be increased in Alzheimer’s brain tissue, amyotrophic lateral sclerosis, multiple sclerosis, as well as in models of neuropathic pain [4]. The increase in CB2 expression and/or activity possibly contributes to the observed increase in microglia activity in the course of the diseases [4]. Of note, a role of CB2 receptors in regulating neuronal progenitor proliferation and differentiation has been reported [5]. Higher CB2 expression is also observed in a number of tumours and cancer cells and the inhibitory effect of CB2 receptor activation on cell cycle progression and proliferation in different cancer types has been shown in a considerable number of studies [6]. In regard to these findings, modulation of CB2 receptor expression and/or activity has been proposed as an attractive potential therapeutic approach [2]. Due to the importance of CB2 receptors in pathological conditions, there is great interest in the biochemical characterization of CB2 receptor signalling and its regulation by e.g. intracellularly interacting proteins. Lack of specificity of commercially available CB2 antibodies [7] limits biochemical analysis in cells endogenously expressing CB2. Of note, studies raising concerns against specificity of antibodies increased generally for GPCRs [8–13]. Thus, we set out to identify and characterize receptor interacting proteins and/or receptor function regulating proteins in cultured model cells reconstituted with an epitopetagged CB2 receptor. As an activator of G protein signalling (AGS), Tctex-1 has been reported to interact with different GPCRs, like rhodopsin [14] parathyroid hormone 1 receptor (PTH1R) [15] and orexin 1 receptor [16]. Tctex-1 was identified as part of the cytoplasmic dynein motor complex, linking dynein cargos and adaptor proteins to dynein [17]. However, Tctex-1 also acts independently from the dynein complex by e.g. interacting with the b-subunit of GTP-binding protein (Gb) [18]. Accordingly, a Tctex-1–Gbg-complex was reported to be involved in the regulation of initial neurite sprouting, axonal specification and elongation of cultured neurons [19], and to be of relevance for cellular proliferation of neural progenitors [20]. For the dynein intermediate chain (DIC) and several Gb proteins an interaction with Tctex-1 via a putative binding domain K/R-K/R-X-X-K/R has been described [19,21]. Of interest, the CB2 receptor carries a putative Tctex-1 binding motif comprising the amino acid sequence K-K-C-V-R in its carboxyl terminal portion (Fig. 1A). Due to this fact, we analysed the putative interaction of Tctex-1 and CB2 receptor in detail. To this end, we investigated the potential interaction of both proteins by immunoprecipitation from lysates of HEK293 cells exogenously expressing Tctex-1 and CB2. We identified a correlation of the CB2 receptor expression level and the formation of Tctex-1-containing intracellular protein complexes, and studied if cannabinoids induce changes in these effects. Furthermore, results obtained by using protein degradation inhibitors indicate an induction of specific proteolytic pathways by a non-canonical CB2 receptor function. Our findings open future perspectives of CB2 receptor functions putatively relevant on various pathophysiological conditions where the receptor is up-regulated. 2. Methods 2.1. Chemicals The chemicals, used in this work, were purchased from following companies. DMEM (Dulbecco’s Modified Eagle Medium),

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Penicillin-Streptomycin (10,000 U/ml), LipofectamineTM 2000 and Opti-MEM1 for cell culture were obtained from Life Technologies, Germany. FCS (fetal calf serum) with low endocannabinoid levels (#F7524, Lot: 030M3396, about 2 ng/ml anandamide and about 50 ng/ml 2-arachidonoyl-glycerol measured by the laboratory of Jürg Gertsch) was ordered from Sigma–Aldrich, Germany. PBS (phosphate buffered saline), DDM (n-dodecyl b-D-maltoside), EDTA (ethylenediaminotetraacetic acid), glycerol, sodium fluoride, bafilomycin A1, cycloheximide, GTP[gS] and anti-Flag1 M2 Affinity Gel were purchased from Sigma–Aldrich, Germany. Leupeptin hemisulfate, SDS pellets (sodium dodecylsulfate) and IPTG (isopropyl-b-D-1-thiogalactopyranoside) were obtained from Roth, Germany. Ammonium chloride, sodium chloride, magnesium chloride, HEPES (4-(2-hydroxyethyl)-1-piperazine-1-ethanesulfonic acid), PMSF (phenylmethanesulfonyl fluoride), b-glycerophosphate, Tris, DTT (2,3-dihydroxybutane-1,4-dithiol), skimmed milk powder, Tween1 20, Triton X-100 and aprotinin were purchased from AppliChem, Germany. Complete Mini protease inhibitor cocktail tablets and protein A agarose were obtained from Roche Applied Science, Germany. CB2 ligands JWH 133, AM 630, 2-AG as well as proteasome inhibitor MG132 were from Tocris, UK. Bromophenol blue was used from Merck, Germany. Glutathione sepharose beads were purchased from GE Healthcare, UK. 2.2. Primary and secondary antibodies Commercial antibodies were obtained from the following companies, rabbit anti-Flag (#F7425, dilution 1:750), mouse anti-Flag M2 (#F1804, dilution 1:750) and mouse anti-a-tubulin antibody (#T5168, dilution 1:1000) from Sigma–Aldrich, Germany; rabbit anti-Gb antibody (T-20, #sc-378, dilution 1:1000) from Santa Cruz Biotechnology, Inc., Germany; mouse anti-Myc (#2276, dilution 1:1000), mouse anti-Erk1/2 (#9107, dilution 1:2000), rabbit anti-phospho-Erk1/2 (#4370, dilution 1:1000) antibody from Cell Signaling Technology, USA; mouse anti-GST antibody (#34860, dilution 1:750) from Qiagen, Germany and rabbit antiTctex-1 antibody (#11954-1-AP, dilution 1:1000) from Proteintech, UK. Secondary anti-mouse and anti-rabbit horseradish peroxidaseconjugated antibodies (#sc-2030 and #sc-2005, dilution 1:5000) were purchased from Santa Cruz Biotechnology, Inc., Germany. Also anti-mouse and anti-rabbit IRDye1 680LT-conjugated secondary antibodies (#926-68022 and #926-68023, dilution 1:5000) or anti-mouse and anti-rabbit IRDye1 800CW-conjugated secondary antibodies (#926-32212 and #926-32213, dilution 1:5000) from LI-COR, Germany were used. Information about used pairs of primary and secondary antibodies for detection is given for each experiment in the corresponding figure legend. 2.3. Cell lines Human embryonic kidney cells HEK293 were purchased from CLS Cell Lines Service GmbH (Germany). The cells were maintained at 37
C in a humidified atmosphere of 5% CO2 in air in DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FCS (fetal calf serum) and 1% Penicillin-Streptomycin (10,000 U/ml). 2.4. DNA constructs The DNAs encoding for human proteins were cloned into TM pcDNA 3.1(+) expression vector (#V790-20, Life Technologies, Germany). Open reading frames of the human CB2 and PTH1R were cloned using primers attaching a DNA sequence encoding a Flagtag to the 50 ends of the coding regions. For cloning of CB2 receptor variant Q63–H316 human genomic DNA was used. The cDNA clone for human PTH1R was obtained from the ORFeome Collaboration and the Center for Cancer Systems Biology via Harvard PlasmID

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Fig. 1. Cannabinoid CB2 receptors do not interact with Tctex-1. (A) Schematic overview of the used constructs of CB2 receptor and Tctex-1 fusion proteins. The N-terminally Flag-tagged human CB2 receptor protein is illustrated with its seven transmembrane regions (TM1–TM7) and the localization of the putative binding motif of Tctex-1 in the intracellular C-terminal region of the receptor. This 60 aa of the C-terminus was also used as an N-terminally tagged GST-fusion protein (middle panel). In the lower panel the N-terminally Myc-tagged human Tctex-1 protein is depicted with its Gb-protein binding region at the C-terminus [19]. (B) Co-precipitation experiment using GST-CB2-C-terminal fusion proteins. GST protein alone or GST-CB2-Cterminus fusion protein bound to glutathione sepharose beads were incubated with cell lysate from Myc–Tctex-1 expressing HEK293 cells for 4 h at 4
C. Complexes of GST

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database. Human Tctex-1 DNA, obtained from HEK293 cDNA, was cloned using primers attaching a DNA sequence encoding a Myctag to the 50 ends of the coding regions. Gb1 and Gg2 DNA constructs, obtained from the Missouri S&T cDNA Resource Center, were used without any tag-encoding DNA sequences. DNA constructs used to express glutathione S-transferase (GST) and GST-fusion proteins were pGEX-4T-2 (#28-9545-50, GE Healthcare, Germany, GST expressing empty vector) and pGEX–CB2–CT. The latter contains cDNA encoding for the last 60 amino acids of human CB2 receptor. 2.5. Transfection of HEK293 cells For transfection, HEK293 cells were seeded in DMEM containing 10% FCS without antibiotics. About 24 h after seeding, when HEK293 cells were about 90% confluent, they were transfected with LipofectamineTM 2000 and Opti-MEM1 (Life Technologies, Germany) according to manufacturer’s protocol. The ratio of plasmid DNA to LipofectamineTM 2000 was 1:2. Transfected HEK293 cells were incubated at 37
C in a humidified atmosphere of 5% CO2 in air. About 24 h after transfection HEK293 cells were used for different experiments. 2.6. Blocking of protein synthesis in HEK293 cells Transiently transfected HEK293 cells were treated with 100 mM cycloheximide (Sigma–Aldrich, Germany) to block protein synthesis about 24 h after transfection for different time points. To this end, medium was aspirated and 500 ml of DMEM supplemented with 10% FCS and 100 mM cycloheximide were added. HEK293 cells were maintained at 37
C in a humidified atmosphere of 5% CO2 in air. Cell lysates were prepared after different time points of cycloheximide treatment. 2.7. Blocking of degradation pathways in HEK293 cells Transiently transfected HEK293 cells were treated with different inhibitors of cellular degradation pathways about 24 h after transfection. In brief, medium was aspirated and 500 ml of DMEM containing inhibitors or solvent control (0.1% EtOH or H2O), respectively, were added. Following compounds were used to inhibit cellular degradation pathways in HEK293 cells: 10 mM MG132 dissolved in absolute EtOH, 100 nM bafilomycin A1 dissolved in absolute EtOH and 200 mM leupeptin hemisulfate together with 20 mM ammonium chloride dissolved in H2O. HEK293 cells were maintained at 37
C in a humidified atmosphere of 5% CO2 in air for 3 h. Afterwards cell lysates were prepared. 2.8. Stimulation of HEK293 cells with CB2 receptor ligands Transiently transfected HEK293 cells were stimulated with different concentrations of CB2 ligands (CB2-selective agonist JWH 133 dissolved in absolute EtOH, endocannabinoid 2-AG dissolved

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in DMSO and CB2-selective inverse agonist AM 630 dissolved in DMSO were used) about 24 h after transfection. To this end, medium was aspirated and 500 ml of DMEM containing 10% FCS and cannabinoids or solvent control (0.1% EtOH for JWH 133 or 0.1% DMSO for 2-AG and AM630, respectively), were added and cells were incubated at 37
C in a humidified atmosphere of 5% CO2 in air for additional 2 h. Afterwards cell lysates were prepared. To obtain a time–response curve, transiently transfected HEK293 cells were stimulated with 1 mM JWH 133 dissolved in absolute EtOH about 24 h after transfection. To this end, medium was aspirated and 500 ml of DMEM containing 10% FCS and 1 mM JWH 133 or solvent control (0.1% EtOH), respectively, were added and cells were incubated at 37
C in a humidified atmosphere of 5% CO2 in air for additional 15 min, 45 min or 120 min, respectively. Afterwards cell lysates were prepared. 2.9. Preparation of HEK293 cell lysates To obtain cell lysates from HEK293 cells, medium was aspirated and cells were washed once with ice-cold phosphate buffered saline (PBS). HEK293 cells were lysed with DDM lysis buffer containing 0.2% DDM, 150 mM NaCl, 5 mM HEPES, 1 mM EDTA (pH 8.0), 10% glycerol, 2 mM leupeptin hemisulfate, 1 mM PMSF (in EtOH), 20 mM NaF, 20 mM b-glycerophosphate supplemented with protease inhibitor cocktail (Roche Applied Science, Germany) in dH2O directly in the cell culture plate for 30 min at 4
C under moderate rocking. Cell lysates were transferred to 1.5 ml reaction vessel and cell debris was removed by centrifugation at 16,200  g and 4
C for 15 min. 2.10. Western blotting analysis Cell lysates were mixed with 6 Laemmli buffer containing 100 mM Tris–HCl (pH 6.8), 4% SDS, 60% glycerol, 0.2% bromophenol blue and 10 mM DTT in dH2O. Proteins of the cell lysate were separated by SDS-PAGE with constant voltage of 100 V on 12% gels for 90 min and transferred to Amersham Hybond ECL nitrocellulose membrane (#RPN2032D, GE Healthcare, Germany). After blocking with 5% skimmed milk powder in TBS-T (150 mM NaCl, 10 mM Tris and 0.025% Tween1 20 in dH2O), the membrane was incubated with specific primary antibodies in TBS-T or blocking buffer over night at 4
C. After three washing steps with TBS-T the membrane was incubated with horseradish peroxidase-coupled secondary antibodies for 1 h at room temperature. The membrane was washed for three times in TBS-T and immunoreactive proteins were visualized with the Pierce1 ECL Western Blotting Substrate (#32106, Thermo Scientific, Germany). For detection of additional proteins on the membranes also the LI-COR Odyssey1 imaging system was used, therefore membranes were incubated with fluorescent dye-conjugated antibodies for 1 h at room temperature. Immunoreactive proteins were detected with the twochannel IR direct detection imaging system from Odyssey1 LI-COR, Germany.

fusion proteins and glutathione sepharose were washed with cell lysis buffer, SDS loading buffer was added, and the proteins were separated by 12% SDS-PAGE. Protein expression was analysed by immunoblotting using anti-GST (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody) and anti-Tctex-1 antibodies (detected with anti-rabbit horseradish peroxidase-conjugated secondary antibody), respectively. Lane 1 shows HEK293 cell lysates containing Myc–Tctex-1 used for co-precipitation experiments. Lanes 2 and 3 show GST alone or GST-CB2-C-terminus fusion protein, respectively, bound to glutathione sepharose beads after incubation with Myc–Tctex-1containing HEK293 cell lysates. The protein contents of the lysates remaining after co-precipitations are given for GST alone (lane 4) or GST-CB2-C-terminus fusion protein (lane 5). Lanes run on different parts of the same gel are shown. (C) Immunoprecipitation of solubilized CB2 receptor. HEK293 cells were seeded in 6-well plates and transfected with 1000 ng pcDNA3.1–Myc–Tctex-1 plasmid, 500 ng pcDNA3.1-Gb1 plasmid, 500 ng pcDNA3.1–Gg2 plasmid and 500 ng pcDNA3.1–Flag–CB2 plasmid or 500 ng pcDNA3.1–Flag–PTH1R as indicated in the figure. In total 2500 ng plasmid DNA were transfected per well, if necessary samples were filled up to 2500 ng plasmid DNA with empty vector pcDNA3.1(+). For control HEK293 cells were transfected with empty vector pcDNA3.1(+) alone (control). About 24 h after transfection cells were harvested and cell extracts were prepared by adding 30 mM GTP[gS] and 5 mM MgCl2 according to the protocol described in Pavlos et al. [22]. For the immunoprecipitation experiments cell lysates were incubated with anti-Flag1 M2 Affinity Gel for 1 h at 4
C. Western blots from cell lysates and immunoprecipitates were probed with anti-Flag and anti-Myc antibodies, respectively. Both primary antibodies were detected with anti-mouse horseradish peroxidase-conjugated secondary antibody. One representative blot from three independent experiments (n = 3) is shown. Lanes run on different parts of the same gel are shown.

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2.11. GST-pulldown The carboxyl terminal portion of CB2 receptor (CB2–CT) cloned into the bacterial expression vector pGEX-4T2 was expressed as glutathione S-transferase fusion protein in Escherichia coli BL21. GST protein alone was expressed as control. Bacteria were lysed by addition of bacteria lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, 5 mM MgCl2, 1 mM DTT, 1 mM PMSF, 10 mg/ml aprotinin, 2 mM leupeptin hemisulfate in dH2O and moderate sonification. After removal of bacterial cell debris by centrifugation the bacterial lysates were used for GST pulldown experiments. To bind GST fusion proteins to glutathione sepharose beads (GE Healthcare, UK) 50 ml of beads-containing slurry were incubated by end-over-end rotation overnight with the bacterial lysate at 4
C. Complexes of GST fusion proteins and glutathione sepharose were washed three times with washing buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 5 mM MgCl2, 1 mM DTT, 0,1 mM PMSF, 10 mg/ml aprotinin, 2 mM leupeptin hemisulfate in dH2O and once with cell lysis buffer. HEK293 cells were transfected with pcDNA3.1–Myc–Tctex-1 DNA and lysed about 48 h after transfection with cell lysis buffer containing 50 mM Tris (pH 7.5), 0.5 M EDTA, 0.5 mM DTT, 0.1 mM PMSF, 10 mg/ml aprotinin, 2 mM leupeptin hemisulfate in dH2O. Cell debris was removed via centrifugation for 10 min at 16,000  g and 4
C. The soluble fraction was added to GST fusion proteins bound to glutathione sepharose beads and incubated by end-over-

end rotation for 4 h at 4
C. Complexes were washed three times with cell lysis buffer, 6 Laemmli buffer was added to the complexes and the samples were incubated at 95
C for 5 min. Complexes of GST fusion proteins and glutathione were separated by 12% SDS-PAGE and precipitated proteins were identified by immunoblotting, respectively. 2.12. Immunoprecipitation of Gb proteins and Flag-tagged receptors For immunoprecipitation of Gb proteins or Flag-tagged proteins transfected HEK293 cells were washed once with ice-cold PBS and scraped with 200 ml lysis buffer containing 150 mM NaCl, 50 mM HEPES-KOH (pH 7.2), 1 mM MgCl2, 1 mM PMSF (in EtOH), 1 mg/ml aprotinin, 2 mM leupeptin hemisulfate and Complete Mini protease inhibitor cocktail in dH2O. Cell lysates for co-immunoprecipitation experiments were produced in the presence of 30 mM GTP[gS] and 5 mM MgCl2 according to the published protocol [22]. Gb proteins were immunoprecipitated with monoclonal anti-Gb antibody (#sc-378, T-20, Santa Cruz Biotechnology, Germany) overnight at 4
C, and immunocomplexes were captured by adding 8 ml Protein A Agarose slurry (Roche Applied Science, Germany) and by end-to-end rotation for 2 h at 4
C. Immunoprecipitates coupled to Protein A Agarose were washed three times with lysis buffer containing 0.2% Triton1 X-100. To immunoprecipitate Flag-tagged GPCRs (Flag–CB2 and Flag–PTH1R) 8 ml of mouse anti-Flag1 M2 Affinity Gel slurry (Sigma–Aldrich, Germany) were added to cell lysate and incubated

Fig. 2. Heterologous expression of CB2 receptor leads to dose-dependent decrease of Myc–Tctex-1 protein. (A) HEK293 cells were seeded in 24-well plates and transfected with different amounts of pcDNA3.1–Flag–CB2 and pcDNA3.1–Myc–Tctex-1 plasmids as indicated in the figure. In total 1000 ng plasmid DNA were transfected per well, if necessary samples were filled up to 1000 ng plasmid DNA with empty vector pcDNA3.1(+). For control HEK293 cells were transfected with empty vector pcDNA3.1(+) alone (control). About 24 h after transfection cell lysates were prepared with DDM lysis buffer. Protein expression was analysed by Western blotting using anti-Flag and anti-Myc antibodies, respectively. Detection of a-tubulin using anti-a-tubulin antibody served as control for equal loading. All primary antibodies were detected with anti-mouse horseradish peroxidase-conjugated secondary antibody. One representative blot from three independent experiments (n = 3) is shown. (B) HEK293 cells were seeded in 24-well plates and transfected with 400 ng pcDNA3.1–Flag–CB2 plasmid and 400 ng pcDNA3.1–Myc–Tctex-1 plasmid. In total 800 ng plasmid DNA were transfected per well, if necessary samples were filled up to 800 ng plasmid DNA with empty vector pcDNA3.1(+). About 24 h after transfection cells were treated with 100 mM cycloheximide for the indicated time points. Cells were harvested and cell lysates prepared using DDM lysis buffer. Protein expression was analysed by Western blotting using antibodies anti-Flag (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody) and anti-Myc (detected with anti-mouse IRDye1 800CW-conjugated secondary antibody), respectively. Detection of a-tubulin using anti-a-tubulin antibody (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody) served as control for equal loading. One representative blot from three independent experiments (n = 3) is shown.

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for 1 h at 4
C. Immunoprecipitates bound to agarose were washed three times with lysis buffer and one time with PBS. After washing procedure 18 ml of 6 Laemmli buffer were added to the immunoprecipitates and samples were incubated at 70
C for 10 min. As a control for the proteins applied to immunoprecipitation 20 ml of cell lysates were directly mixed with 5 ml 6 Laemmli buffer and stored on ice or at 80
C, respectively, until SDS-PAGE was performed. Gb as well as Flag-tagged receptor immunoprecipitates and lysate controls were analysed by SDS-PAGE on 4–12% SDS gradient precast gels (Expedeon, Germany) and immunoblotting. 2.13. Statistical analysis All data are presented as mean  SD. Comparisons among the groups were performed by 2-way ANOVA test. The comparison between two groups was performed using 2-tailed Student t tests. p  0.05 was considered statistically significant. GraphPad Prism 5 (GraphPad software, USA) was used for data analysis and figure preparation. 3. Results 3.1. CB2 receptors do not – neither directly nor indirectly via Gbg – interact with Tctex-1 As Tctex-1 is known to interact with various GPCRs, we set out to investigate a possible Tctex-1 and CB2 receptor interaction and/ or function in detail. First, we addressed the question whether Tctex-1 directly interacts with the CB2 receptor. To this end, pull down studies were performed using glutathione S-transferase fusion proteins of the carboxyl terminal portion of CB2 receptor (GST–CB2–CT) and HEK293 cell lysates containing Myc-tagged Tctex-1 (schematic overview Fig. 1A). The sixty amino acid long carboxyl terminal portion of CB2 fused to GST contained the putative Tctex-1 binding motif K-K-C-V-R. As shown in Fig. 1, using GST–CB2–CT as a bait no Myc–Tctex-1 protein was co-precipitated (Fig. 1B, lane 3), although the corresponding Myc-tagged Tctex-1 protein was shown to be present in the cell lysate used in these pull down studies (Fig. 1B, lane 1, 4 and 5). These findings indicate that Tctex-1 does not directly interact with the GST-fusion protein of the carboxyl terminal portion of CB2. Assuming that more than the carboxyl terminal part of CB2 might be necessary for the interaction with Tctex-1 we performed co-immunoprecipitation (co-IP) experiments using an amino terminally Flag-tagged full length CB2 receptor and Myc-tagged Tctex-1 co-expressed in HEK293 cells. Since Tctex-1 had been shown to interact with parathyroid hormone receptors 1 (PTH1R) we included this receptor in our co-immunoprecipitation experiments as a positive control [15]. Co-expression of Gb1g2 subunits was included in this set of experiments to additionally determine whether interaction of Tctex-1 with CB2 receptor may indirectly be achieved via Gbg proteins. An interaction of Tctex-1 with Gb proteins is well documented [18,19]. Since Gb1 Gb2, Gb3, Gb5, Gb5L isoforms have been shown to interact with Tctex-1 and because the co-expression of Gg1 or Gg2 together with Gb1 had no influence on the Gb1– Tctex-1-interaction [19], we decided to use the combination of Gb1 g2 co-transfection in our set of experiments in addition to the endogenously expressed G protein subunits. Although only very low amounts of PTH1R protein were detected in the input samples used for immunoprecipitation – corresponding in particular to dimeric receptor protein – PTH1 receptors co-precipitated with Myc–Tctex-1 as expected (Fig. 1C). Both monomeric and dimeric PTH receptor protein was detected after immunoprecipitation (Fig. 1C). In contrast to the PTH1 receptor precipitation, Myc–Tctex-1 failed to co-precipitate with

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the CB2 receptor, indicating that CB2 receptors do not interact with Tctex-1 even not indirectly via Gb1g2 (Fig. 1C). These findings confirmed afore made assumption that CB2 and Tctex-1 do not directly interact. However, we consistently noticed that CB2 receptor co-expression substantially reduced Tctex-1 protein levels when co-expressed in HEK293 cells. 3.2. CB2 receptor co-expression leads to dose-dependent decrease of Tctex-1 protein levels To confirm that CB2 expression influences Tctex-1 protein levels, increasing amounts of CB2 receptor were co-expressed with Tctex-1 in HEK293 cells and Tctex-1 expression was analysed. Fig. 2A shows that Tctex-1 protein levels inversely correlated with CB2 protein levels. Already little amounts of CB2 receptor protein were sufficient to strongly reduce Tctex-1 protein and increasing amounts of CB2 diminished Tctex-1 to roughly estimated 10% of the control Tctex-1 levels. These findings indicate a very sensitive balance between both proteins. Of note, this phenomenon was independent of the promotor used to transiently express the proteins and gave similar results with both CMV and EF1 promotors (data not shown). Also of interest, co-expression of Gb1g2 had the opposite effect on Tctex-1 protein levels (Fig. 1C, input). 3.3. CB2 receptor expression increase Tctex-1 protein degradation and turnover in a ligand-independent manner To further explore the modulatory effect of CB2 on Tctex-1 protein levels and to unravel the molecular mechanism behind we asked the question whether CB2 receptor co-expression induced a change in the half-life time of Tctex-1 protein. Therefore, we studied the effect of the protein biosynthesis inhibitor cycloheximide (100 mM) on CB2 and Tctex-1 expressing HEK293 cells 24 h after transfection and analysed the protein amounts of Tctex-1 after blocking protein translation in a time dependent manner. In absence of CB2 receptors, we noticed a strong expression of Myc–Tctex-1 protein at the beginning of the experiment with first slight reduction after one hour followed by a drastic degradation three to five hours after addition of the protein biosynthesis blocker. The expression of Tctex-1 remained low up to the end of the experiment (after 24 h) (Fig. 2B). In presence of CB2 receptors cells already started with a very low expression level of Tctex-1 24 h after transfection, showing that the major portion of Tctex-1 already was removed. The protein level did not change in the further time course of cycloheximide treatment, indicating that a main part of Tctex-1 protein has a half-life time in the range of few hours and that a smaller part of the protein is more stable. Next, we aimed to analyse the influence of the CB2 receptor activity on this effect. We therefore applied the CB2 synthetic agonist JWH 133 on CB2 expressing HEK293 cells and analysed the amount of co-expressed Tctex-1 protein. First, a time–response study for incubation with 1 mM JWH 133 was performed (Fig. 3). Despite the CB2 receptor-dependent decrease of Tctex-1 we did not observe obvious changes in Tctex-1 protein levels after JWH 133 stimulation for 0 min, 15 min, 45 min, and 120 min, as deduced by Western blotting (Fig. 3A). The results given in Fig. 3B are derived from an experiment that has been performed two times in triplicates, and has been densitometrically normalized to the expression of a-tubulin. Then, the normalized Tctex-1 level in the solvent control group at 0 min was set to 100% and was used to calculate the percentage relative expression values of normalized Myc–Tctex-1 levels depicted as mean  SD. The statistical analysis by 2-way ANOVA revealed no significant agonist-induced changes of Myc–Tctex-1 expression throughout the time points analysed; identifying the CB2-induced Tctex-1 reduction as ligand-independent (Fig. 3B). As a

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control, the CB2-induced phosphorylation of extracellular signalregulated kinase 1/2 (Erk1/2) in the presence and absence of JWH 133 was analysed. As shown in Fig. 3A and C expression of CB2 constitutively induced Erk1/2 phosphorylation that could be slightly further stimulated by JWH 133, indicating that the latter was active on CB2. These findings correspond to data described by Correa et al. [23]. Of note, after 15 min a slight increase in Erk1/2 phosphorylation was observed in the presence of the solvent and in the absence of CB2 (Fig. 3A lower panel and C). Second, to exclude that the amounts of JWH 133 applied were insufficient to induce receptor-mediated effects, a dose–response study was performed. To this end, the cells were incubated for 120 min with increasing concentrations of JWH 133 (up to 3 mM). To see any putative receptor independent effects of JWH 133 we used cells exogenously expressing Tctex-1 as control. As shown in a representative blot in Fig. 4A we observed a similar strong reduction of Tctex-1 in the presence of increasing concentrations of JWH 133 that was dependent on CB2 receptor presence but independent of its stimulation. The statistical analysis of the experiments that has been performed three times in duplicates revealed no significant effects of JWH 133 treatment (Fig. 4B). To test whether the endocannabinoid 2-arachidonoyl-glycerol (2-AG) rather than synthetic JWH 133 was able to regulate the CB2 effect

on Tctex-1, cells were incubated with increasing concentration of 2-AG (Fig. 4C). As shown in Fig. 4D, the CB2 receptor presence caused a statistical significant decrease in Tctex-1 protein levels independent of the amount of 2-AG present. These findings indicate that downregulation of Tctex-1 by the CB2 receptor is independent of both the synthetic ligand JWH 133 and the endocannbinoid 2-AG. Again, the constitutive and/or ligand-stimulated CB2 receptor activity was controlled by analysing the receptor-induced phosphorylation of Erk1/2. As shown in Fig. 4E, in cells treated with increasing concentrations of JWH 133 the mean phospho-Erk1/2 levels increased (n = 6), whereas the 2-AG treatment did result in little if any changes in Erk1/2 phosphorylation (p = ns; n = 4; Fig. 4F). Last, to address the question whether CB2 receptor-mediated downregulation of Tctex-1 protein is due to the known constitutive activity of CB2 [24], the analysis were repeated in the presence of increasing concentrations of the inverse agonist AM 630 (Fig. 5A). As shown, similar to the former findings also the inverse agonist had no statistical significant impact on Tctex-1 protein amount in the presence of CB2 supporting the idea of a receptor activityindependent effect of CB2 receptors (Fig. 5B). Of note, there was no statistically significant increase in phosphorylation of Erk1/2 kinases induced by the constitutively active CB2 receptor indicating

Fig. 3. Selective CB2 receptor agonist JWH 133 has no time-dependent influence on CB2-mediated downregulation of Myc–Tctex-1. (A–C) HEK293 cells were seeded in 24-well plates and transfected with 300 ng pcDNA3.1–Flag–CB2 and 300 ng pcDNA3.1–Myc–Tctex-1 plasmid as indicated in the figure. In total 600 ng plasmid DNA were transfected per well, if necessary samples were filled up to 600 ng plasmid DNA with empty vector pcDNA3.1(+). About 24 h after transfection cells were treated with 1 mM JWH 133 for different time points as indicated in the figure. Incubation of cells with 0.1% EtOH for different time points served as solvent control. Cells were harvested using DDM lysis puffer. Protein expression was analyzed by Western blotting using antibodies anti-Flag (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody), anti-Myc (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody) and anti-phospho-Erk1/2 (detected with anti-rabbit IRDye1 800CW-conjugated secondary antibody), respectively. Detection of a-tubulin using anti-a-tubulin antibody (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody) served as control for equal loading. The experiment was performed for two independent times in biological triplicates (n = 6). (A) One representative blot is shown. Signals of (B) Myc–Tctex-1 and (C) phosphorylated Erk1/2 proteins were quantified using ImageJ software, normalized to the presence of a-tubulin and set as a percentage relative to the “Myc–Tctex-1” group. The data were analysed using GraphPad Prism 5 software. Data are presented as means  SD. Comparisons among the groups were performed by 2-way ANOVA test. *p  0.05, **p  0.01, ***p  0.001, ns = not significant.

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Fig. 4. Selective CB2 receptor agonist JWH 133 and the endocannabinoid 2-AG have no dose-dependent influence on CB2-mediated downregulation of Myc–Tctex-1. (A–F) HEK293 cells were seeded in 24-well plates and transfected with 300 ng pcDNA3.1–Flag–CB2 and 300 ng pcDNA3.1–Myc–Tctex-1 plasmid as indicated in the figure. In total 600 ng plasmid DNA were transfected per well, if necessary samples were filled up to 600 ng plasmid DNA with empty vector pcDNA3.1(+). About 24 h after transfection cells were treated with different concentrations, as indicated in the figure, of selective CB2 agonist JWH 133 (A, B, E) or endocannabinoid 2-AG (C, D, F) for 2 h. Incubation of cells with 0.1% EtOH (A, B, E) or 0.1% DMSO (C, D, F), respectively, served as solvent control. (A–F) Cells were harvested using DDM lysis puffer. Protein expression was analyzed by Western blotting using antibodies anti-Flag (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody), anti-Myc (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody) and anti-phospho-Erk1/2 (detected with anti-rabbit IRDye1 800CW-conjugated secondary antibody), respectively. Detection of a-tubulin using anti-a-tubulin antibody (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody) served as control for equal loading. (A, B, E) The experiments were performed for three independent times in biological duplicates (n = 6). (C, D, F) The experiment was performed for two independent times in biological duplicates (n = 4). (A, C) One representative blot is shown. Signals of (B, D) Myc–Tctex-1 and (E, F) phosphorylated Erk1/2 proteins were quantified using ImageJ software, normalized to the presence of a-tubulin and set as a percentage relative to the “Myc–Tctex-1” group. The data were analysed using GraphPad Prism 5 software. Data are presented as means  SD. Comparisons among the groups were performed by 2-way ANOVA test. *p  0.05, **p  0.01, ***p  0.001, ns = not significant.

that AM 630 treatment achieved an inhibition of CB2 constitutive activity (Fig. 5A), as shown by quantification analysis given in Fig. 5C. Again, a small increase in Erk1/2 phosphorylation and a reduction in CB2 receptor expression was observed in the presence of the solvents ethanol and DMSO (Figs. 3A, 4A and C, 5A). 3.4. Tctex-1 is degraded by the proteasome To analyse how expression of CB2 receptors influences protein stability of Tctex-1 in transiently expressing HEK293 cells, pharmacologic protein degradation inhibitors were used to investigate different degradation mechanisms. In brief, we studied the effect of MG132, a potent inhibitor of the proteasome, on cells co-expressing CB2 and Tctex-1 proteins. In parallel we performed

experiments using ammonium chloride with leupeptin or bafilomycin to inhibit proteases, lysosomes and the maturation of autophagic vacuoles, respectively. For the quantification analysis the expression levels of Myc–Tctex-1 protein has been normalized to total Erk1/2 amounts and then set as a percentage relative to the Myc–Tctex-1 group. Interestingly, we observed that CB2 receptorinduced degradation of Tctex-1 was predominantly inhibited by MG132 (Fig. 6A and B). This blocking effect was observed in control cells solely expressing Tctex-1 but was significantly elevated in CB2 and Tctex-1 co-expressing cells. The quantification showed that vehicle treated control cells led to a CB2-induced degradation of 66%  10% (Fig. 6B). In comparison MG132 treatment was able to inhibit the CB2 induced degradation to only 22%  10%. This effect was significantly different (p = 0.002, n = 3). A milder effect

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Fig. 5. Selective CB2 receptor inverse agonist AM 630 has no dose-dependent influence on CB2-mediated downregulation of Myc–Tctex-1. (A–C) HEK293 cells were seeded in 24-well plates and transfected with 300 ng pcDNA3.1–Flag–CB2 and 300 ng pcDNA3.1–Myc–Tctex-1 plasmid as indicated in the figure. In total 600 ng plasmid DNA were transfected per well, if necessary samples were filled up to 600 ng plasmid DNA with empty vector pcDNA3.1(+). About 24 h after transfection cells were treated with different concentrations, as indicated in the figure, of selective CB2 receptor inverse agonist AM 630 for 2 h. Incubation of cells with 0.1% DMSO served as solvent control. Cells were harvested using DDM lysis puffer. Protein expression was analyzed by Western blotting using antibodies anti-Flag (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody), anti-Myc (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody) and anti-phosphoErk1/2 (detected with anti-rabbit IRDye1 800CW-conjugated secondary antibody), respectively. Detection of a-tubulin using anti-a-tubulin antibody (detected with antimouse IRDye1 680LT-conjugated secondary antibody) served as control for equal loading. The experiments were performed for three independent times in biological duplicates (n = 6). (A) One representative blot is shown. signals of (B) Myc–Tctex-1 and (C) phosphorylated Erk1/2 proteins were quantified using ImageJ software, normalized to the presence of a-tubulin and set as a percentage relative to the “Myc–Tctex-1” group. The data were analysed using GraphPad Prism 5 software. Data are presented as means  SD. Comparisons among the groups were performed by 2-way ANOVA test. *p  0.05, **p  0.01, ***p  0.001, ns = not significant.

(p = 0.039, n = 7) was seen in cells treated with NH4Cl/leupeptin resulting in an inhibition from 65%  17% in solvent control treated cells to 45%  27% reduction of Tctex-1 protein degradation by CB2 receptor-induction (Fig. 6C and D). In contrast, bafilomycin treatment showed no changes in CB2 dependent degradation (69%  16%) in comparison to solvent control (64%  18%) (Fig. 6E and F, n = 3). In conclusion, these experiments indicate that CB2 receptor presence mainly induced proteasomal and partly lysosomal degradation of Tctex-1 in HEK293 cells. 3.5. CB2 receptors suppress Tctex-1 interaction with Gb proteins As Gb and Tctex-1 are known interaction partners [18] and as we could observe an increase of Tctex-1 proteins by co-expression of Gb1g2 (Fig. 1C), we set out to analyse if Gbg stabilized Tctex-1 proteins and whether CB2 receptors may interfere or modulate the

interaction of Gbg and Tctex-1. To test this hypothesis, we overexpressed the proteins of interest, precipitated Gb proteins, and studied the co-precipitated Tctex-1 protein amount in the presence and absence of CB2 receptor (Fig. 7). Of note, the low amount of Gb protein detected in lysates of cells not expressing exogenous Gbg reflects endogenous Gb protein (Fig. 7A left panel, lanes 1 and 3, and right panel, lanes 1 and 3). The quantification of Tctex-1 was performed in experimental triplicates, normalized to Gb protein and related to the value with highest Tctex-1 levels, which was set to 100% (Fig. 7B). This analysis show that CB2-induced reduction of Tctex-1 was significant changed (p = 0.0341, t-test) in the presence of Gb1g2 reaching 66%  11%, mean  SD (corresponding to Fig. 7A, lane 5 left panel). Of importance, the amount of Tctex-1 protein that was co-precipitated with Gb was significantly reduced to 12%  4% (p = 0.0006, t-test) in the presence of CB2 (Fig. 7B), although the protein levels of Tctex-1 in the sample applied for

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Fig. 6. Tctex-1 protein levels are predominantly decreased by proteasomal and lysosomal degradation. (A–F) HEK293 cells were seeded in 24-well plates and transfected with 300 ng pcDNA3.1–Flag–CB2 and 300 ng pcDNA3.1–Myc–Tctex-1 as indicated in the figure. In total 900 ng plasmid DNA were transfected per well, if necessary samples were filled up to 900 ng plasmid DNA with empty vector pcDNA3.1(+). About 24 h after transfection cells were incubated with 10 mM MG132 (A, B), 20 mM ammonium chloride and 200 mM leupeptin (C, D) or 100 nM bafilomycin (E, F). Cells incubated with 0.1% EtOH (A, E) or water (C) served as solvent control. After 3 h of incubation cells were harvested using DDM lysis puffer. Protein expression was analysed by Western blotting using antibodies anti-Flag (detected with anti-mouse IRDye1 800CW-conjugated secondary antibody) and anti-Myc (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody), respectively. Detection of Erk1/2 using anti-Erk1/2 antibody (detected with anti-mouse IRDye1 680LT-conjugated secondary antibody) served as control for equal loading. The experiments were performed for several independent times (n = 3 for (B) and (F) and n = 7 for D). In (A), (C) and (E) one representative blot with lanes run on different parts of the same gel is shown, respectively. In (B), (D), and (F) Myc–Tctex-1 signals were quantified with ImageJ software, normalized to the presence of Erk1/2 and set as a percentage relative to the “Myc–Tctex-1” group. The data were analysed using GraphPad Prism 5 software. Normalized Myc–Tctex-1 levels with and without Flag–CB2 overexpression are shown. Data are presented as means  SD. The differences between Tctex-1 levels with and without Flag–CB2 overexpression are depicted. *p  0.05, **p  0.01, ns = not significant.

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Fig. 7. CB2 receptor interferes with Gb-Tctex-1-interaction. (A) HEK293 cells were seeded in 6-well plates and transfected with 1000 ng pcDNA3.1–Myc–Tctex-1 plasmid, 500 ng pcDNA3.1-Gb1 plasmid, 500 ng pcDNA3.1-Gg2 plasmid and 500 ng pcDNA3.1–Flag–CB2 or pcDNA3.1–Flag–PTH1R as indicated in the figure. In total 2500 ng plasmid DNA were transfected per well, if necessary samples were filled up to 2500 ng plasmid DNA with empty vector pcDNA3.1(+). For control HEK293 cells were transfected with empty vector pcDNA3.1(+) alone (control). About 24 h after transfection cells were harvested and cell extracts were prepared by adding 30 mM GTP[gS] and 5 mM MgCl2 according to the protocol described in Pavlos et al. [22]. For the immunoprecipitation experiments cell lysates were incubated with anti-Gb-antibody overnight at 4
C. Protein–antibody-complexes were captured by incubating with protein A agarose beads for 2 h at 4
C. Western blots from cell lysates and immunoprecipitates were probed with anti-Flag (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody), anti-Myc (detected with anti-mouse horseradish peroxidase-conjugated secondary antibody) and anti-Gb antibodies (detected with anti-rabbit IRDye1 800CW-conjugated secondary antibody). The experiment was performed for three independent times (n = 3). In (A) one representative blot is shown. (B) Myc–Tctex-1 signals were quantified with ImageJ software, normalized to the presence of Gb proteins and set as a percentage relative to the “Myc–Tctex-1 + Gb1g2” group. The data were analysed using GraphPad Prism 5 software. Data are presented as means  SD. *p  0.05, ***p  0.001.

immunoprecipitation were strongly increased in the presence of exogenous Gb1g2 (Fig. 7A lower panel). The CB2-induced Tctex-1 decrease that reached 60–70% in the absence of exogenous Gb1g2 (see i.e. Fig. 4B and D) was reduced to 30–40% in the presence of Gb1g2 (Fig. 7B, input) supporting the idea of a Gbg stabilizing effect on Tctex-1. Two major conclusions were drawn from these experiments. First, Tctex-1 protein amounts were increased by coexpression of Gb1g2 subunits. Second, Gb almost completely failed to co-precipitate its interaction partner Tctex-1 in the presence of CB2 receptors. As a control, the effect of PTH1 receptor expression on GbTctex-1-complex formation was analysed. In contrast to CB2,

PTH1R expression had only little effect on Gb-Tctex-1 interaction. Interestingly, Gb was able to co-precipitate both, PTH1R and Tctex1 proteins. In addition to formerly published results of Sugai et al. [15] showing that PTH1 receptors directly interact with Tctex-1, our findings revealed that Gb subunits are also present in the receptor-Tctex-1-protein complex. Taken together, our results show that expression of CB2 interferes with the complex formation of Tctex-1 and Gbg proteins. The finding that CB2 receptors co-precipitated with Gb proteins (Fig. 7) indicate that CB2 receptors are able to disturb the interaction of Gbg proteins with Tctex-1 possibly in a competitive manner. The results support the concept of a stabilizing impact of

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Gbg proteins and introduced a degrading effect of cannabinoid CB2 receptor on Tctex-1 proteins. The degrading effect of CB2 receptors is probably due to the disruption of the Tctex-1–Gbg-complex by the CB2 receptor conveying Tctex-1 to the degradation by the proteasome and partly by lysosomes. 4. Discussion & conclusion Due to their immunmodulatory and neuroprotective functions and their role in modulating analgesic responses, CB2 receptors are attractive therapeutic targets. Accordingly, CB2 receptor overexpression and/or increased signalling was found to be correlated with various pathophysiological conditions including Alzheimer’s disease [2]. Therefore the identification and understanding of intracellular interaction and signalling complexes of CB2 receptors is of importance to invent new pharmacological applications. In the present study we aimed to identify and characterize new CB2 receptor interaction partners and/or intracellular protein platforms regulated by CB2 receptor. The activator of G protein signalling AGS2 (also known as Dynlt1 or Tctex-1) received our attention since it directly interacts with several GPCRs such as the carboxyl terminal portion of rhodopsin [14], the parathyroid hormone 1 receptor (PTH1R) [15] and orexin 1 receptor [16]. Former studies revealed that Tctex-1 as a dynein motor complex component is involved in regulating receptor trafficking, e.g. of rhodopsin [14]. Furthermore Tctex-1 was shown to directly bind to the Gb subunits of Gbg proteins and to control cellular functions as a Gbg–Tctex-1 complex, e.g. in neurite outgrowth [19], cell cycle progression [20], and activation of the microtubule-associated RhoA exchange factor GEF-H1 [25]. The results presented in this study indicate that Tctex-1 does not directly interact with CB2 receptors, although it carries a putative Tctex-1 binding motif K-K-C-V-R in its carboxyl terminal portion. In both, GST-pull down experiments using a carboxyl terminal fragment of CB2 and immunoprecipitation experiments using full-length receptors in the absence (data not shown) and presence of exogenous Gb1g2 we failed to identify co-precipitated Tctex-1. Of note, comparable extraction and solubilisation procedures/conditions used in these experiments had been successfully applied for other Tctex-1 interacting receptors, e.g. PTH1 receptor (present work), Gb and Rab3D [19,22]. However, we observed a CB2 receptor correlated decrease in Tctex-1 protein expression which partly was rescued in the presence of exogenously expressed Gb1g2. Furthermore, as shown herein, CB2 receptor expression was found to compete with Tctex-1 in binding to Gbg and to induce a release of Tctex-1 from its binding partner Gbg, followed by proteasomal degradation of Tctex-1 proteins. Additionally, a part of the CB2 receptor-induced degradation of Tctex-1 seemed to take place in lysosomes. Interestingly, Sachdev et al. observed a similar competition between the dynein intermediate chain (DIC) and Gbg in binding to Tctex-1 [19]. Very recently, a collaboration of Ga and Gbg subunits in separating the protein complex formed by Tctex-1, by the RhoGEF-H1 and by DIC was reported [25]. Binding of Gbg subunits to Tctex-1 was found to disrupt the Tctex-1 interaction with DIC, while binding of Ga to GEF-H1 was reported to separate the RhoGEF-H1 from the complex and to induce its GEF activity [25]. In regards to this findings the results reported herein indicate that the CB2 receptor disrupt the Gbg–Tctex-1-complex to build up a CB2-Gbg-complex. We suggest that the Tctex-1 pool generated by the disturbance of Gbg–Tctex-1 protein–protein interaction represents a free pool of Tctex-1 which is disposable for protein degradation, and is eliminated in particular by the proteasomal/lysosomal pathways. The basal levels of “stable” Tctex-1 proteins observed in cells expressing CB2 in the presence and absence of the protein biosynthesis inhibitor cycloheximide

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might represent Tctex-1 proteins bound in complexes with either DIC or Gb, respectively, which are thereby protected against CB2induced degradation. This idea would be in line with the results obtained by increased Gb1g2 expression where Tctex-1 protein levels seemed to be stabilized even in the presence of CB2 receptor and therefore are higher than in experiments without coexpressed Gb1g2. Our findings thus indicate an afore unknown non-canonical regulatory role of CB2 receptor for downstream signalling, in particular for signalling controlled by Tctex-1. Of note, this CB2 receptor function is independent of ligand-induced CB2 signalling, since addition of the synthetic agonist JWH 133 or the endocannabinoid 2-AG had no effect. In brief, no ligand-dependent effect was observed at all concentrations and all time points analysed, excluding that we missed the ligand induced effect. By the use of inverse agonist treatment AM 630 up to a concentration of 3 mM we could also rule out, that the CB2 receptor constitutive activity or its activation by traces of endocannabinoids present in the fetal calf serum added to cell culture media [26] is responsible for this effect. Of note, AM 630 was effective in inhibiting the CB2-mediated constitutive phosphorylation of Erk1/2. However, since the CB2 receptor expression levels were slightly decreased by solvents (ethanol/DMSO) we cannot completely rule out that the solvents influence/masked any agonist or inverse agonist effects. It also has to be addressed in future experiments whether Gai subunits play a role in the formation of Gb-Tctex-1-complex in the presence of CB2, and whether CB2 receptor interaction with b-arrestin and its internalization is of importance for the observed CB2 receptor-mediated downregulation of Tctex-1. CB2 receptors are known to couple to Gai proteins [27,28], and a role of b-arrestin in agonist-mediated internalization has been reported [29]. Despite the fact that only studies with over-expressed proteins have been performed in the present work, our findings may have a number of interesting and potentially important physiologic and pathophysiologic implications. For example, Tctex-1 is known to regulate cell cycle progression and cell proliferation by its interaction with Gbg proteins in neuronal progenitor cells, preventing neuronal differentiation [20,30]. In particular, phosphorylation of Tctex-1 at amino acid 94 of the encoded protein was shown to be involved in the control of cell cycle progression of these cells [30]. A role of CB2 receptors in regulating proliferation and cell fate decisions in neuronal progenitor cells also has been described [5,31,32]. However, whether the latter is associated with the disruption of Gb-Tctex-1-complexes by CB2 followed by Tctex1 degradation remains to be elucidated. A role of cannabinoid receptors in the control of survival, proliferation, and differentiation in a variety of cancer cells has been described [6]. For instance, the activation of endogenous CB2 receptor via D9-THC reduces human breast cancer cell proliferation [33], and stimulation of different human leukemia and lymphoma cell lines with D9-THC or the arachidonoyl-glycerol derivative anandamide results in increased apoptosis of the cells [34]. The reduced cell proliferation in breast cancer was correlated with CB2 receptor expression and/or function and was attributed to the inhibition of cell cycle progression, probably via downregulation of Cdc2 [33]. Whether the findings reported in our manuscript provide an additional mechanistic explanation for changes in cell cycle progression in the presence of increased CB2 expression await further investigation. In our opinion influencing the expression level of CB2 receptors may represent an additional treatment strategy to control CB2-related pathological states. Future experimentation using cells with endogenously expressing CB2 receptors and Tctex-1, e.g. cultured macrophages, or cancer cells, and knockdown of Tctex-1 and/or CB2 by the application of siRNAs in these cells will help to address these questions.

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Conflict of interest The authors declare that they have no conflict of interest. Author contributions MN, LP, SR performed the research. MK designed the research study. BM participated in the study design and contributed essential reagents. MN, LP, SR and MK analysed the data. MN, BM and MK wrote the paper. Acknowledgements The project was granted by the “Juniorprofessoren Programm” of the Baden-Württemberg Stiftung to MK (Az. 31-7635-52/Karsak /1). MK was also supported by a grant from the DFG (KA 2306/3-1). For providing cDNA clones and plasmids we thank Harvard PlasmID databank (plasmid.med.harvard.edu) and the Missouri S&T cDNA Resource Center (www.cdna.org). We thank the Institute of Physiological Chemistry at Ulm University, Prof. Dr. Thomas Wirth and his colleagues, for supporting this work. We also wish to thank Prof. Dr. Jürg Gertsch and his lab members at the Institute of Biochemistry and Molecular Medicine at the University of Bern for analysing endocannabinoid levels in fetal calf serum samples. We also thank our colleagues Dr. Nevena Djogo and Leonore Mensching at the ZMNH for their help. References [1] J. Gertsch, M. Leonti, S. Raduner, I. Racz, J.-Z. Chen, X.-Q. Xie, et al., Betacaryophyllene is a dietary cannabinoid, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 9099–9104. [2] P. Pacher, R. Mechoulam, Is lipid signaling through cannabinoid 2 receptors part of a protective system? Prog. Lipid Res. 50 (2011) 193–211. [3] N. Stella, Cannabinoid signaling in glial cells, Glia 48 (2004) 267–277. [4] B.K. Atwood, K. Mackie, CB2: a cannabinoid receptor with an identity crisis, Br. J. Pharmacol. 160 (2010) 467–479. [5] I. Galve-Roperh, V. Chiurchiù, J. Díaz-Alonso, M. Bari, M. Guzmán, M. Maccarrone, Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation, Prog. Lipid Res. 52 (2013) 633–650. [6] G. Velasco, C. Sánchez, M. Guzmán, Towards the use of cannabinoids as antitumour agents, Nat. Rev. Cancer 12 (2012) 436–444. [7] B. Cécyre, S. Thomas, M. Ptito, C. Casanova, J.F. Bouchard, Evaluation of the specificity of antibodies raised against cannabinoid receptor type 2 in the mouse retina, Naunyn Schmiedebergs Arch. Pharmacol. 387 (2014) 175–184. [8] N.L. Grimsey, C.E. Goodfellow, E.L. Scotter, M.J. Dowie, M. Glass, E.S. Graham, Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal!, J. Neurosci. Methods 171 (2008) 78–86. [9] B.C. Jensen, P.M. Swigart, P.C. Simpson, Ten commercial antibodies for alpha-1adrenergic receptor subtypes are nonspecific, Naunyn Schmiedebergs Arch. Pharmacol. 379 (2009) 409–412. [10] N. Hamdani, J. van der Velden, Lack of specificity of antibodies directed against human beta-adrenergic receptors, Naunyn Schmiedebergs Arch. Pharmacol. 379 (2009) 403–407. [11] S. Beermann, R. Seifert, D. Neumann, Commercially available antibodies against human and murine histamine H(4)-receptor lack specificity, Naunyn Schmiedebergs Arch. Pharmacol. 385 (2012) 125–135. [12] H. Cernecka, P. Ochodnicky, W.H. Lamers, M.C. Michel, Specificity evaluation of antibodies against human b3-adrenoceptors, Naunyn Schmiedebergs Arch. Pharmacol. 385 (2012) 875–882.

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