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Heteromultimerization of Cannabinoid CB1 Receptor and Orexin OX1 Receptor Generates a Unique Complex in Which Both Protomers Are Regulated by Orexin A*

  • Richard J. Ward
    Affiliations
    Molecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • John D. Pediani
    Affiliations
    Molecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Graeme Milligan
    Correspondence
    To whom correspondence should be addressed: Wolfson Link Bldg. 253, University of Glasgow, Glasgow G12 8QQ, Scotland, UK. Tel.: 44-141-330-5557; Fax: 44-141-330-5481
    Affiliations
    Molecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
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  • Author Footnotes
    * This work was supported by Medical Research Council Grant G0900050.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.
Open AccessPublished:September 09, 2011DOI:https://doi.org/10.1074/jbc.M111.287649
      Agonist-induced internalization was observed for both inducible and constitutively expressed forms of the cannabinoid CB1 receptor. These were also internalized by the peptide orexin A, which has no direct affinity for the cannabinoid CB1 receptor, but only when the orexin OX1 receptor was co-expressed along with the cannabinoid CB1 receptor. This effect of orexin A was concentration-dependent and blocked by OX1 receptor antagonists. Moreover, the ability of orexin A to internalize the CB1 receptor was also blocked by CB1 receptor antagonists. Remarkably, orexin A was substantially more potent in producing internalization of the CB1 receptor than in causing internalization of the bulk OX1 receptor population, and this was true in cells in which the CB1 receptor was maintained at a constant level, whereas levels of OX1 could be varied and vice versa. Both co-immunoprecipitation and cell surface, homogenous time-resolved fluorescence resonance energy transfer based on covalent labeling of N-terminal “SNAP” and “CLIP” tags present in the extracellular N-terminal domain of the receptors confirmed the capacity of these two receptors to heteromultimerize. These studies confirm the capacity of the CB1 and OX1 receptors to interact directly and demonstrate that this complex has unique regulatory characteristics. The higher potency of the agonist orexin A to regulate the CB1-OX1 heteromer compared with the OX1-OX1 homomer present in the same cells and the effects of CB1 receptor antagonists on the function of orexin A suggest an interplay between these two systems that may modulate appetite, feeding, and wakefulness.

      Introduction

      It is now widely recognized that many and indeed perhaps all members of the G protein-coupled receptor (GPCR)
      The abbreviations used are: GPCR
      G protein-coupled receptor
      htrFRET
      homogenous time-resolved FRET
      VSV-G
      vesicular stomatitis virus G
      5-HT
      5hydroxytryptamine
      Bis-Tris
      2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
      mGluR
      metabotropic glutamate receptor.
      superfamily are able to form homodimers or homomultimers (
      • Milligan G.
      • Canals M.
      • Pediani J.D.
      • Ellis J.
      • Lopez-Gimenez J.F.
      ,
      • Milligan G.
      ,
      • Dalrymple M.B.
      • Pfleger K.D.
      • Eidne K.A.
      ,
      • Pin J.P.
      • Comps-Agrar L.
      • Maurel D.
      • Monnier C.
      • Rives M.L.
      • Trinquet E.
      • Kniazeff J.
      • Rondard P.
      • Prézeau L.
      ). There are also a substantial number of reports of heterointeractions between co-expressed pairs of GPCRs (
      • Milligan G.
      ,
      • Birdsall N.J.
      ,
      • Pin J.P.
      • Neubig R.
      • Bouvier M.
      • Devi L.
      • Filizola M.
      • Javitch J.A.
      • Lohse M.J.
      • Milligan G.
      • Palczewski K.
      • Parmentier M.
      • Spedding M.
      ,
      • Rozenfeld R.
      • Devi L.A.
      ,
      • Ferré S.
      • Navarro G.
      • Casadó V.
      • Cortés A.
      • Mallol J.
      • Canela E.I.
      • Lluís C.
      • Franco R.
      ). Although the functional significance and consequences of a number of such pairings, including those between dopamine D1 and D2 (
      • Hasbi A.
      • Fan T.
      • Alijaniaram M.
      • Nguyen T.
      • Perreault M.L.
      • O'Dowd B.F.
      • George S.R.
      ,
      • Rashid A.J.
      • So C.H.
      • Kong M.M.
      • Furtak T.
      • El-Ghundi M.
      • Cheng R.
      • O'Dowd B.F.
      • George S.R.
      ,
      • Pei L.
      • Li S.
      • Wang M.
      • Diwan M.
      • Anisman H.
      • Fletcher P.J.
      • Nobrega J.N.
      • Liu F.
      ) and μ- and δ-opioid (
      • van Rijn R.M.
      • Whistler J.L.
      • Waldhoer M.
      ,
      • Milan-Lobo L.
      • Whistler J.L.
      ,
      • He S.Q.
      • Zhang Z.N.
      • Guan J.S.
      • Liu H.R.
      • Zhao B.
      • Wang H.B.
      • Li Q.
      • Yang H.
      • Luo J.
      • Li Z.Y.
      • Wang Q.
      • Lu Y.J.
      • Bao L.
      • Zhang X.
      ,
      • Gomes I.
      • Ijzerman A.P.
      • Ye K.
      • Maillet E.L.
      • Devi L.A.
      ) receptor subtypes, have been explored, the relevance of other pairings has been studied less extensively. With notable exceptions such as interactions between adenosine and dopamine receptor subtypes (
      • Borroto-Escuela D.O.
      • Romero-Fernandez W.
      • Tarakanov A.O.
      • Ciruela F.
      • Agnati L.F.
      • Fuxe K.
      ,
      • Soriano A.
      • Ventura R.
      • Molero A.
      • Hoen R.
      • Casadó V.
      • Cortés A.
      • Fanelli F.
      • Albericio F.
      • Lluís C.
      • Franco R.
      • Royo M.
      ), this is particularly true of pairings between GPCRs for which the endogenous agonist ligands are distinct. Despite this, a number of commentators have discussed the potential for such heteromers to respond to ligands in unique ways and to offer the potential as novel sets of drug targets (
      • Dalrymple M.B.
      • Pfleger K.D.
      • Eidne K.A.
      ,
      • Casadó V.
      • Cortés A.
      • Mallol J.
      • Pérez-Capote K.
      • Ferré S.
      • Lluis C.
      • Franco R.
      • Canela E.I.
      ,
      • Milligan G.
      ,
      • Panetta R.
      • Greenwood M.T.
      ,
      • Filizola M.
      ). In substantial part, this reflects a growing understanding of the mechanisms of G protein activation via GPCR dimers (
      • Rovira X.
      • Pin J.P.
      • Giraldo J.
      ) and the appreciation that interactions between protomeric elements of GPCR homomers and heteromers must cause allosteric effects upon one another (
      • Rovira X.
      • Pin J.P.
      • Giraldo J.
      ,
      • Smith N.J.
      • Milligan G.
      ,
      • Springael J.Y.
      • Urizar E.
      • Costagliola S.
      • Vassart G.
      • Parmentier M.
      ,
      • Milligan G.
      • Smith N.J.
      ). The consequences of such allosteric effects are likely to be unique for different GPCR pairs and indeed potentially for different ligands. One means to explore such effects is via ligand binding studies (
      • Birdsall N.J.
      ,
      • Rovira X.
      • Vivó M.
      • Serra J.
      • Roche D.
      • Strange P.G.
      • Giraldo J.
      ). Although potentially powerful, in a number of cases, this may be limited by the availability of suitable, particularly agonist radiolabeled probes. As such, measures of ligand function at GPCR multimers have been more widely explored. For example, the potency of the hallucinogenic serotonin 5-HT2A agonist 2,5-dimethoxy-4-iodoamphetamine to activate Gi family G proteins is increased more than 100-fold when the 5-HT2A receptor is co-expressed alongside and interacts with the metabotropic glutamate receptor 2 (mGluR2), and this is reversed upon co-addition of an mGluR2 agonist (
      • González-Maeso J.
      • Ang R.L.
      • Yuen T.
      • Chan P.
      • Weisstaub N.V.
      • López-Giménez J.F.
      • Zhou M.
      • Okawa Y.
      • Callado L.F.
      • Milligan G.
      • Gingrich J.A.
      • Filizola M.
      • Meana J.J.
      • Sealfon S.C.
      ). A further pairing in which alterations in potency of agonist ligands has been observed following co-expression is the cannabinoid CB1 receptor and the orexin OX1 receptor (
      • Hilairet S.
      • Bouaboula M.
      • Carrière D.
      • Le Fur G.
      • Casellas P.
      ,
      • Ellis J.
      • Pediani J.D.
      • Canals M.
      • Milasta S.
      • Milligan G.
      ). Here, Hilairet et al. (
      • Hilairet S.
      • Bouaboula M.
      • Carrière D.
      • Le Fur G.
      • Casellas P.
      ) observed that the potency of the peptide agonist orexin A to activate the mitogen-activated protein kinase pathway via the OX1 receptor was increased 100-fold in the presence of the CB1 receptor, whereas Ellis et al. (
      • Ellis J.
      • Pediani J.D.
      • Canals M.
      • Milasta S.
      • Milligan G.
      ) noted that the CB1 receptor antagonist/inverse agonist SR141716A caused a decrease in potency of orexin A to activate the mitogen-activated protein kinases ERK1/2 only in cells co-expressing the two receptors. There have also been a number of reports of coordinated trafficking of pairs of GPCRs by individual ligands that are expected only to occupy and activate one of the pair (
      • Milligan G.
      ). These studies are at least consistent with the concept of GPCR heteromultimerization (
      • Milligan G.
      ,
      • Birdsall N.J.
      ,
      • Pin J.P.
      • Neubig R.
      • Bouvier M.
      • Devi L.
      • Filizola M.
      • Javitch J.A.
      • Lohse M.J.
      • Milligan G.
      • Palczewski K.
      • Parmentier M.
      • Spedding M.
      ,
      • Rozenfeld R.
      • Devi L.A.
      ,
      • Ferré S.
      • Navarro G.
      • Casadó V.
      • Cortés A.
      • Mallol J.
      • Canela E.I.
      • Lluís C.
      • Franco R.
      ).
      In recent times, the ability to monitor cell surface multimerization of GPCRs in intact cells has been enhanced greatly by the development of SNAP and CLIP tag variants of the enzyme O6-alkylguanine-DNA-alkyltransferase. These allow the covalent attachment of a variety of fluorophores and other probes to such modified receptors (
      • Maurel D.
      • Comps-Agrar L.
      • Brock C.
      • Rives M.L.
      • Bourrier E.
      • Ayoub M.A.
      • Bazin H.
      • Tinel N.
      • Durroux T.
      • Prézeau L.
      • Trinquet E.
      • Pin J.P.
      ,
      • Alvarez-Curto E.
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ,
      • Albizu L.
      • Cottet M.
      • Kralikova M.
      • Stoev S.
      • Seyer R.
      • Brabet I.
      • Roux T.
      • Bazin H.
      • Bourrier E.
      • Lamarque L.
      • Breton C.
      • Rives M.L.
      • Newman A.
      • Javitch J.
      • Trinquet E.
      • Manning M.
      • Pin J.P.
      • Mouillac B.
      • Durroux T.
      ,
      • Monnier C.
      • Tu H.
      • Bourrier E.
      • Vol C.
      • Lamarque L.
      • Trinquet E.
      • Pin J.P.
      • Rondard P.
      ,
      • Xu T.R.
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ). This tagging approach has also proved valuable in monitoring ligand-induced receptor trafficking (
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ). Herein, we used a range of such approaches to confirm heteromultimerization between the CB1 receptor and the orexin OX1 receptor and show a substantially greater potency of orexin A to promote internalization of this heteromer compared with the OX1 receptor homomer. The concept that an endogenously produced peptide ligand has higher affinity/potency at a receptor heteromer than at the supposed primary monomeric/homomeric target is both novel and fascinating and provides new insights into both physiological signaling processes and the prospects for drug discovery.

      DISCUSSION

      Previous studies have either inferred physical interactions between CB1 and OX1 receptors based on alterations in function of receptor selective agonists in cells transfected to co-express this pair of GPCRs (
      • Hilairet S.
      • Bouaboula M.
      • Carrière D.
      • Le Fur G.
      • Casellas P.
      ) or used FRET between C-terminally cyan fluorescent protein- and YFP-tagged variants to detect such interactions in intracellular structures that may represent recycling endocytic vesicles (
      • Ellis J.
      • Pediani J.D.
      • Canals M.
      • Milasta S.
      • Milligan G.
      ). A further set of observations consistent with the presence of CB1-OX1 heteromers is that selective antagonists for either receptor are able to traffic both receptors to the cell surface from an intracellular location (
      • Ellis J.
      • Pediani J.D.
      • Canals M.
      • Milasta S.
      • Milligan G.
      ). However, although consistent with such an interaction, much of the evidence for the existence of a CB1-OX1 heteromer has been indirect and/or has not addressed whether such a complex is present at the surface of cells. In recent times, the introduction of SNAP or CLIP tags into the N-terminal domain of GPCRs has become an effective way of detecting cell surface receptors because the N-terminal region is exposed to the extracellular environment, and the SNAP and CLIP tags can be labeled covalently with a variety of cell-impermeant fluorophores or other reagents (
      • Maurel D.
      • Comps-Agrar L.
      • Brock C.
      • Rives M.L.
      • Bourrier E.
      • Ayoub M.A.
      • Bazin H.
      • Tinel N.
      • Durroux T.
      • Prézeau L.
      • Trinquet E.
      • Pin J.P.
      ,
      • Alvarez-Curto E.
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ,
      • Albizu L.
      • Cottet M.
      • Kralikova M.
      • Stoev S.
      • Seyer R.
      • Brabet I.
      • Roux T.
      • Bazin H.
      • Bourrier E.
      • Lamarque L.
      • Breton C.
      • Rives M.L.
      • Newman A.
      • Javitch J.
      • Trinquet E.
      • Manning M.
      • Pin J.P.
      • Mouillac B.
      • Durroux T.
      ,
      • Monnier C.
      • Tu H.
      • Bourrier E.
      • Vol C.
      • Lamarque L.
      • Trinquet E.
      • Pin J.P.
      • Rondard P.
      ,
      • Xu T.R.
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ,
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ). As such, cell surface receptors can be labeled and imaged, and the effects of ligands on the cellular location of the receptor can be monitored by following the location of the fluorophore because the ligand binding site of the receptor is not altered by this procedure (
      • Alvarez-Curto E.
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ,
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ) unlike when using irreversible receptor ligands or those with high affinity and very slow dissociation rates (
      • Hern J.A.
      • Baig A.H.
      • Mashanov G.I.
      • Birdsall B.
      • Corrie J.E.
      • Lazareno S.
      • Molloy J.E.
      • Birdsall N.J.
      ). Although we have previously used this approach to monitor individually cell surface location and agonist-induced internalization of both the CB1 receptor and the OX1 receptor (
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ), the most extensive use of SNAP tagging and related technologies has been to covalently label cell surface receptors with pairs of htrFRET-competent donors and acceptors to detect protein-protein interactions at the cell surface (
      • Maurel D.
      • Comps-Agrar L.
      • Brock C.
      • Rives M.L.
      • Bourrier E.
      • Ayoub M.A.
      • Bazin H.
      • Tinel N.
      • Durroux T.
      • Prézeau L.
      • Trinquet E.
      • Pin J.P.
      ,
      • Alvarez-Curto E.
      • Ward R.J.
      • Pediani J.D.
      • Milligan G.
      ). In studies on heteromeric interactions, separate SNAP and CLIP tagging of a pair of proteins allows the concurrent addition of distinct FRET acceptors/donors that label the two proteins differentially and the subsequent detection of htrFRET if the proteins are located within FRET-compliant distances of one another (
      • Maurel D.
      • Comps-Agrar L.
      • Brock C.
      • Rives M.L.
      • Bourrier E.
      • Ayoub M.A.
      • Bazin H.
      • Tinel N.
      • Durroux T.
      • Prézeau L.
      • Trinquet E.
      • Pin J.P.
      ). To assess the presence of CB1-OX1 heteromers, we generated sets of cell lines in which either a SNAP-tagged CB1 or OX1 receptor was expressed constitutively, whereas the complementary CLIP-tagged receptor was harbored at a doxycycline-inducible locus. In cells in which the SNAP-tagged receptor was at the inducible locus, no binding of the FRET donor SNAP-Lumi4Tb was detected until the receptor was induced. However, when induced and in the presence of the FRET acceptor CLIP-Red, the htrFRET signal reported the presence of the CB1-OX1 heteromer at the cell surface. As a more conventional approach, we also took advantage of the presence of HA and VSV-G epitope tags that were also engineered into the N-terminal domain of each receptor to show that no matter which receptor was SNAP- or CLIP-tagged or which receptor was at the inducible locus co-immunoprecipitation was achieved after addition of doxycycline to cells to cause receptor co-expression.
      A further use of the SNAP/CLIP tags is to monitor receptor internalization. Only a cell surface receptor is available to be labeled with SNAP/CLIP-Lumi4Tb, and in cells expressing only SNAP-CB1, CB1 agonists substantially reduced levels of SNAP-Lumi4Tb binding over short time periods. As this construct also contained the VSV-G epitope tag, a similar effect of CB1 agonists was observed in intact cell ELISA studies using a VSV-G antibody. However, the signal to background ratio in studies using SNAP-Lumi4Tb binding was far superior to ELISA; therefore, SNAP-Lumi4Tb was used routinely in these studies. Although initially unexpected, in cells co-expressing both CB1 and OX1, maximally effective concentrations of orexin A were as effective at producing internalization of the CB1 receptor as CB1 receptor agonists. This is consistent with the CB1-OX1 heteromer being a stable complex and binding of only orexin A causing the entire complex to be internalized. Importantly, when exploring the potency of orexin A to produce this effect, we noted in different cell lines that orexin A was some 10-fold more potent in producing internalization of the CB1 receptor than in causing internalization of the co-expressed OX1 receptor. This may seem contrary to the concept of internalization of a stable CB1-OX1 heteromer, but it may be anticipated that only a proportion of the expressed OX1 receptor is within such a heteromeric complex. As such, the most obvious but also most interesting interpretation is that orexin A has substantially higher affinity for the CB1-OX1 heteromer than for the OX1-OX1 homomer (or OX1 monomer). This would be consistent with the earlier observation that orexin A is substantially more potent in promoting ERK mitogen-activated protein kinase phosphorylation when the OX1 receptor is co-expressed with the CB1 receptor (
      • Hilairet S.
      • Bouaboula M.
      • Carrière D.
      • Le Fur G.
      • Casellas P.
      ) and the concept that receptor heteromers are unique species.
      To assess whether OX1-OX1 homomers were also present in SNAP-CB1/CLIP-OX1-co-expressing cells, we added a combination of CLIP-Lumi4Tb and CLIP-Red, which can label only the OX1 receptor population, and again were able to detect htrFRET consistent with such an OX1-OX1 interaction. Equivalent studies using the equivalent pair of SNAP labels also identified CB1-CB1 interactions. Although certainly consistent with mixtures of homodimers and heterodimers, these results are also potentially consistent with the CB1 and OX1 receptors co-existing in larger complexes such as tetramers, and there is growing evidence for such oligomeric GPCR complexes (
      • Lopez-Gimenez J.F.
      • Canals M.
      • Pediani J.D.
      • Milligan G.
      ,
      • Klco J.M.
      • Lassere T.B.
      • Baranski T.J.
      ,
      • Guo W.
      • Urizar E.
      • Kralikova M.
      • Mobarec J.C.
      • Shi L.
      • Filizola M.
      • Javitch J.A.
      ), including tetramers (
      • Pisterzi L.F.
      • Jansma D.B.
      • Georgiou J.
      • Woodside M.J.
      • Chou J.T.
      • Angers S.
      • Raicu V.
      • Wells J.W.
      ,
      • Raicu V.
      • Stoneman M.R.
      • Fung R.
      • Melnichuk M.
      • Jansma D.B.
      • Pisterzi LF
      • Rath S.
      • Fox M.
      • Wells J.W.
      • Saldin D.K.
      ,
      • Fung J.J.
      • Deupi X.
      • Pardo L.
      • Yao X.J.
      • Velez-Ruiz G.A.
      • Devree B.T.
      • Sunahara R.K.
      • Kobilka B.K.
      ,
      • Comps-Agrar L.
      • Kniazeff J.
      • N⊘rskov-Lauritsen L.
      • Maurel D.
      • Gassmann M.
      • Gregor N.
      • Prézeau L.
      • Bettler B.
      • Durroux T.
      • Trinquet E.
      • Pin J.P.
      ). Although requiring further analysis, this would also be consistent with the ability of orexin A to internalize similar proportions of the CB1 receptor as the cannabinoid agonists.
      Co-internalization of the CB1 receptor along with the OX1 receptor in response to orexin A was also observed in cell surface biotinylation protection experiments that were designed to provide a biochemical correlate of the htrFRET studies. Equally, by labeling the receptors with cell-impermeant fluorescent SNAP and CLIP tag substrates, we were able to image the co-internalization of both OX1 and CB1 receptors in response to addition of orexin A. This approach also demonstrated, as shown previously using a pair of receptors C-terminally tagged with autofluorescent proteins (
      • Ellis J.
      • Pediani J.D.
      • Canals M.
      • Milasta S.
      • Milligan G.
      ), that the simple presence of the CB1 receptor alters directly the cellular location profile of the OX1 receptor even in the absence of receptor ligands. Co-internalization of co-expressed receptors has been observed in a substantial number of cases. For example, the μ-opioid receptor agonist [d-Ala2,N-MePhe4,Gly-ol]enkephalin is able to cause internalization of the mGluR5 as well as the μ-opioid receptor when the two receptors are co-expressed, whereas the non-competitive mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine limits [d-Ala2,N-MePhe4,Gly-ol]enkephalin-induced internalization of the μ-opioid receptor (
      • Schröder H.
      • Wu D.F.
      • Seifert A.
      • Rankovic M.
      • Schulz S.
      • Höllt V.
      • Koch T.
      ). Similarly, interactions between purinergic P2Y11 and P2Y1 receptors are reported to allow agonist internalization of P2Y11, although this receptor is not generally able to be internalized in response to agonist when expressed in isolation (
      • Ecke D.
      • Hanck T.
      • Tulapurkar M.E.
      • Schäfer R.
      • Kassack M.
      • Stricker R.
      • Reiser G.
      ).
      It is noteworthy that the CB1 receptor appears to be able to form heteromers with distinct properties with a variety of other receptors (
      • Hudson B.D.
      • Hébert T.E.
      • Kelly M.E.
      ). For example, interactions with the angiotensin AT1 receptor (
      • Rozenfeld R.
      • Gupta A.
      • Gagnidze K.
      • Lim M.P.
      • Gomes I.
      • Lee-Ramos D.
      • Nieto N.
      • Devi L.A.
      ) results in CB1 blockers limiting mitogenic signaling by the AT1 receptor, and although not observed in all studies (
      • Canals M.
      • Milligan G.
      ), interactions of the CB1 receptor with the μ-opioid receptor (
      • Rios C.
      • Gomes I.
      • Devi L.A.
      ,
      • Hojo M.
      • Sudo Y.
      • Ando Y.
      • Minami K.
      • Takada M.
      • Matsubara T.
      • Kanaide M.
      • Taniyama K.
      • Sumikawa K.
      • Uezono Y.
      ) have also been reported to modulate function. Further interactions with the dopamine D2 receptor (
      • Marcellino D.
      • Carriba P.
      • Filip M.
      • Borgkvist A.
      • Frankowska M.
      • Bellido I.
      • Tanganelli S.
      • Müller C.E.
      • Fisone G.
      • Lluis C.
      • Agnati L.F.
      • Franco R.
      • Fuxe K.
      ,
      • Przybyla J. A
      • Watts V.J.
      ) and with the β2-adrenoreceptor (
      • Hudson B.D.
      • Hébert T.E.
      • Kelly M.E.
      ) have also been reported to have functional sequelae. By contrast, there is little information on other heteromers that incorporate the OX1 receptor. However, this may simply reflect the much more extensive literature on cannabinoid receptors than on the OX1 receptor rather than a more limited propensity to make such interactions.
      Although initial focus on the OX1 receptor centered on potential roles in appetite and feeding, much recent work has concentrated on the capacity of orexin receptor antagonists potentially targeting the orexin OX2 receptor as well as the OX1 receptor to limit wakefulness and hence treat insomnia (
      • Scammell T.E.
      • Winrow C.J.
      ,
      • Coleman P.J.
      • Cox C.D.
      • Roecker A.J.
      ). By contrast, until recently, there have been very limited efforts to identify small molecule orexin receptor agonists, and because of this, the current studies have been limited to using the native peptide agonist orexin A as an activator of the OX1 receptor. This may change with indications that agonists at the OX1 receptor could be effective in the treatment of colon cancer (

      Laburthe, M., Voisin, T. (2011) Br. J. Pharmacol., in press.

      ,
      • Voisin T.
      • El Firar A.
      • Fasseu M.
      • Rouyer-Fessard C.
      • Descatoire V.
      • Walker F.
      • Paradis V.
      • Bedossa P.
      • Henin D.
      • Lehy T.
      • Laburthe M.
      ). The CB1 receptor has been a target of great interest not least because of the psychotropic effects of the active ingredient of cannabis produced via activation of this receptor. The association of increased feeding with cannabis use resulted in the development of CB1 receptor antagonists/inverse agonists for weight loss and the approval (later retracted because of the potential for psychiatric side effects) of the ligand rimonabant (
      • Christopoulou F.D.
      • Kiortsis D.N.
      ). Despite this, the contribution of CB1 receptors to energy balance and fat storage in the periphery has suggested that peripherally restricted CB1 receptor antagonists/inverse agonists might well have clinical potency without the central nervous system liabilities (
      • Christopoulou F.D.
      • Kiortsis D.N.
      ,
      • Wu Y.K.
      • Yeh C.F.
      • Ly T.W.
      • Hung M.S.
      ,
      • Tam J.
      • Vemuri V.K.
      • Liu J.
      • Bátkai S.
      • Mukhopadhyay B.
      • Godlewski G.
      • Osei-Hyiaman D.
      • Ohnuma S.
      • Ambudkar S.V.
      • Pickel J.
      • Makriyannis A.
      • Kunos G.
      ). Peripherally restricted CB1 agonists have also been promoted as potential therapeutic agents in both inflammatory and neuropathic pain (
      • Yu X.H.
      • Cao C.Q.
      • Martino G.
      • Puma C.
      • Morinville A.
      • St-Onge S.
      • Lessard E.
      • Perkins M.N.
      • Laird J.M.
      ). The effects of heteromeric interactions between the CB1 and OX1 receptors on such end points remain uncertain, but the substantial effects on ligand potency reported previously (
      • Hilairet S.
      • Bouaboula M.
      • Carrière D.
      • Le Fur G.
      • Casellas P.
      ,
      • Ellis J.
      • Pediani J.D.
      • Canals M.
      • Milasta S.
      • Milligan G.
      ) and the effects of orexin A and presumably synthetic OX1 receptor agonists as they are developed on CB1 receptor trafficking and function will need to be explored.

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