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Cannabinoid Receptors CB1 and CB2 Form Functional Heteromers in Brain*

  • Lucía Callén
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • Estefanía Moreno
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • Pedro Barroso-Chinea
    Affiliations
    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain

    Centro de Investigación Médica Aplicada, Universidad de Navarra, 31008 Pamplona, Spain
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  • David Moreno-Delgado
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • Antoni Cortés
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • Josefa Mallol
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • Vicent Casadó
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • José Luis Lanciego
    Affiliations
    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain

    Centro de Investigación Médica Aplicada, Universidad de Navarra, 31008 Pamplona, Spain
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  • Rafael Franco
    Affiliations
    Centro de Investigación Médica Aplicada, Universidad de Navarra, 31008 Pamplona, Spain
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  • Carmen Lluis
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
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  • Enric I. Canela
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
    Search for articles by this author
  • Peter J. McCormick
    Correspondence
    A Ramón y Cajal Fellow. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 643 Avenida Diagonal, planta-2, Barcelona 08028, Spain. Tel.: 934039280; Fax: 934021559
    Footnotes
    Affiliations
    Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain

    Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Universidad de Navarra, 31008 Pamplona, Spain
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Spanish Ministerio de Economia and Competitividad Grants SAF2010-18472, SAF2008-03229-E, and SAF2008-03118-E within the framework of the Era-NET Neuron program) and a grant for collaborative projects (Grant PI2011/02-7) from the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED).
    1 A Formación de Profesorado Univesitario Fellow (Grant AP2007-00808).
    2 Both authors contributed equally to this work.
Open AccessPublished:April 24, 2012DOI:https://doi.org/10.1074/jbc.M111.335273
      Exploring the role of cannabinoid CB2 receptors in the brain, we present evidence of CB2 receptor molecular and functional interaction with cannabinoid CB1 receptors. Using biophysical and biochemical approaches, we discovered that CB2 receptors can form heteromers with CB1 receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB1-CB2 receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB1 and CB2 receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB1-CB2 receptor heteromers consists of both the ability of CB1 receptor antagonists to block the effect of CB2 receptor agonists and, conversely, the ability of CB2 receptor antagonists to block the effect of CB1 receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB2 receptors can negatively modulate CB1 receptor function.

      Introduction

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      Both CB1 and CB2 receptors are members of the GPCR
      The abbreviations used are:
      GPCR
      G protein-coupled receptor
      PLA
      proximity ligation assay(s)
      BRET
      bioluminescence resonance energy transfer
      ANOVA
      analysis of variance
      ACEA
      arachidonyl-2-chloroethylamide.
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      DISCUSSION

      The cannabinoid receptors CB1 and CB2 are increasingly becoming an important subject for investigation in a variety of neurological and immunological processes (
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      ). Although it has been described that both receptors can be co-expressed in the same brain areas (
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      • Song R.
      • Zhang H.Y.
      • Liu Q.R.
      • Yang H.J.
      • Bi G.H.
      • Li J.
      • Gardner E.L.
      Brain cannabinoid CB2 receptors modulate cocaine's actions in mice.
      ), the relationship between both receptors at the molecular level is not known. Among GPCRs, more and more evidence points to an important role of heteromer formation between GPCRs on receptor function modulation (
      • Ferré S.
      • Baler R.
      • Bouvier M.
      • Caron M.G.
      • Devi L.A.
      • Durroux T.
      • Fuxe K.
      • George S.R.
      • Javitch J.A.
      • Lohse M.J.
      • Mackie K.
      • Milligan G.
      • Pfleger K.D.
      • Pin J.P.
      • Volkow N.D.
      • Waldhoer M.
      • Woods A.S.
      • Franco R.
      Building a new conceptual framework for receptor heteromers.
      ,
      • Rozenfeld R.
      • Gupta A.
      • Gagnidze K.
      • Lim M.P.
      • Gomes I.
      • Lee-Ramos D.
      • Nieto N.
      • Devi L.A.
      AT1R-CBR heteromerization reveals a new mechanism for the pathogenic properties of angiotensin II.
      ,
      • Moreno E.
      • Vaz S.H.
      • Cai N.S.
      • Ferrada C.
      • Quiroz C.
      • Barodia S.K.
      • Kabbani N.
      • Canela E.I.
      • McCormick P.J.
      • Lluis C.
      • Franco R.
      • Ribeiro J.A.
      • Sebastião A.M.
      • Ferré S.
      Dopamine-galanin receptor heteromers modulate cholinergic neurotransmission in the rat ventral hippocampus.
      ,
      • Fiorentini C.
      • Busi C.
      • Spano P.
      • Missale C.
      Dimerization of dopamine D1 and D3 receptors in the regulation of striatal function.
      ). However, determination of the heteromer functional characteristics and, mainly, identification of heteromers in tissue is often a challenge. Here, we present evidence of CB2 receptor molecular and functional interaction with CB1 receptors. Several conclusions can be drawn from the data. First, CB1 and CB2 receptors can form heteromers in transfected cells and in a variety of brain tissues, including pineal gland, nucleus accumbens, and globus pallidus. Second, one specific characteristic of CB1-CB2 receptor heteromers is that of bidirectional cross-antagonism (i.e. the ability of CB1 receptor antagonists to block the effect of CB2 receptor agonists and, conversely, the ability of CB2 receptor antagonists to block the effect of CB1 receptor agonists). Third, agonist co-activation of CB1-CB2 receptor heteromers results in a negative cross-talk in Akt phosphorylation and neurite outgrowth.
      Although it was known that CB1 receptors are highly expressed in the central nervous system, it was believed for a long time that CB2 receptors were restricted to the peripheral tissues (
      • Munro S.
      • Thomas K.L.
      • Abu-Shaar M.
      Molecular characterization of a peripheral receptor for cannabinoids.
      ). Recently, several studies have shown the expression of CB2 receptors in brain regions where CB1 receptors are also expressed (
      • Golech S.A.
      • McCarron R.M.
      • Chen Y.
      • Bembry J.
      • Lenz F.
      • Mechoulam R.
      • Shohami E.
      • Spatz M.
      Human brain endothelium. Coexpression and function of vanilloid and endocannabinoid receptors.
      ,
      • Van Sickle M.D.
      • Duncan M.
      • Kingsley P.J.
      • Mouihate A.
      • Urbani P.
      • Mackie K.
      • Stella N.
      • Makriyannis A.
      • Piomelli D.
      • Davison J.S.
      • Marnett L.J.
      • Di Marzo V.
      • Pittman Q.J.
      • Patel K.D.
      • Sharkey K.A.
      Identification and functional characterization of brainstem cannabinoid CB2 receptors.
      ,
      • Onaivi E.S.
      • Ishiguro H.
      • Gong J.P.
      • Patel S.
      • Perchuk A.
      • Meozzi P.A.
      • Myers L.
      • Mora Z.
      • Tagliaferro P.
      • Gardner E.
      • Brusco A.
      • Akinshola B.E.
      • Liu Q.R.
      • Hope B.
      • Iwasaki S.
      • Arinami T.
      • Teasenfitz L.
      • Uhl G.R.
      Discovery of the presence and functional expression of cannabinoid CB2 receptors in brain.
      ,
      • Brusco A.
      • Tagliaferro P.
      • Saez T.
      • Onaivi E.S.
      Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus.
      ,
      • Núñez E.
      • Benito C.
      • Pazos M.R.
      • Barbachano A.
      • Fajardo O.
      • González S.
      • Tolón R.M.
      • Romero J.
      Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain. An immunohistochemical study.
      ,
      • Koch M.
      • Habazettl I.
      • Dehghani F.
      • Korf H.W.
      The rat pineal gland comprises an endocannabinoid system.
      ,
      • Suárez J.
      • Llorente R.
      • Romero-Zerbo S.Y.
      • Mateos B.
      • Bermúdez-Silva F.J.
      • de Fonseca F.R.
      • Viveros M.P.
      Early maternal deprivation induces gender-dependent changes on the expression of hippocampal CB1 and CB2 cannabinoid receptors of neonatal rats.
      ,
      • Xi Z.X.
      • Peng X.Q.
      • Li X.
      • Song R.
      • Zhang H.Y.
      • Liu Q.R.
      • Yang H.J.
      • Bi G.H.
      • Li J.
      • Gardner E.L.
      Brain cannabinoid CB2 receptors modulate cocaine's actions in mice.
      ), including the pineal gland (
      • Koch M.
      • Habazettl I.
      • Dehghani F.
      • Korf H.W.
      The rat pineal gland comprises an endocannabinoid system.
      ), the nucleus accumbens (
      • Xi Z.X.
      • Peng X.Q.
      • Li X.
      • Song R.
      • Zhang H.Y.
      • Liu Q.R.
      • Yang H.J.
      • Bi G.H.
      • Li J.
      • Gardner E.L.
      Brain cannabinoid CB2 receptors modulate cocaine's actions in mice.
      ), and the globus pallidus (
      • Lanciego J.L.
      • Barroso-Chinea P.
      • Rico A.J.
      • Conte-Perales L.
      • Callén L.
      • Roda E.
      • Gómez-Bautista V.
      • López I.P.
      • Lluis C.
      • Labandeira-García J.L.
      • Franco R.
      Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis.
      ). Because the physiological role of the expression of two different receptors for the same endogenous ligands in the same brain regions is not obvious, we explored if both receptors are modulating each other and, for this reason, we first examined the possibility of direct receptor-receptor interaction by heteromerization. The identification of CB1-CB2 receptor heteromers was first performed via BRET, a biophysical technique, in co-transfected cells and by immunofluorescence using the PLA in a neuronal cell model. The definition of a receptor heteromer is that the heteromer is a macromolecular complex composed of at least two functional receptor units with biochemical properties that are demonstrably different from those of its individual receptors (
      • Ferré S.
      • Baler R.
      • Bouvier M.
      • Caron M.G.
      • Devi L.A.
      • Durroux T.
      • Fuxe K.
      • George S.R.
      • Javitch J.A.
      • Lohse M.J.
      • Mackie K.
      • Milligan G.
      • Pfleger K.D.
      • Pin J.P.
      • Volkow N.D.
      • Waldhoer M.
      • Woods A.S.
      • Franco R.
      Building a new conceptual framework for receptor heteromers.
      ). Thus, we focused on the determination of the functional characteristics of CB1-CB2 receptor heteromers expressed in neuroblastoma cells. Both cannabinoid receptors have been shown to signal through the MAPK and Akt/PKB pathways (
      • Molina-Holgado E.
      • Vela J.M.
      • Arévalo-Martín A.
      • Almazán G.
      • Molina-Holgado F.
      • Borrell J.
      • Guaza C.
      Cannabinoids promote oligodendrocyte progenitor survival. Involvement of cannabinoid receptors and phosphatidylinositol-3 kinase/Akt signaling.
      ,
      • Sánchez M.G.
      • Ruiz-Llorente L.
      • Sánchez A.M.
      • Díaz-Laviada I.
      Activation of phosphoinositide 3-kinase/PKB pathway by CB1 and CB2 cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction.
      ); therefore, we explored the implication of heteromer formation on these pathways. We first investigated whether there were changes in Akt/PKB (Ser-473 Akt phosphorylation) signaling when heteromers were both co-stimulated with agonists or blocked with antagonists. When neuroblastoma cells were co-stimulated with both receptor agonists, a negative cross-talk was observed between CB1 and CB2 receptors in Akt/PKB phosphorylation signaling. It has been described that activation of PI3K/Akt signaling is involved in neural differentiation of SH-SY5Y cells (
      • López-Carballo G.
      • Moreno L.
      • Masiá S.
      • Pérez P.
      • Barettino D.
      Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells.
      ); according to this, we observed that CB1 and CB2 receptor agonists promoted neuritogenesis in our SH-SY5Y neuronal cell model, and, interestingly, we also observed a negative cross-talk in neuritogenesis when cells were co-stimulated with both receptor agonists. Thus, both CB1 and CB2 receptors might be negatively modulating each other in signaling pathways where endocannabinoids are involved, such as brain development and neural cell differentiation (
      • Fernández-Ruiz J.
      • Gómez M.
      • Hernández M.
      • de Miguel R.
      • Ramos J.A.
      Cannabinoids and gene expression during brain development.
      ,
      • Harkany T.
      • Guzmán M.
      • Galve-Roperh I.
      • Berghuis P.
      • Devi L.A.
      • Mackie K.
      The emerging functions of endocannabinoid signaling during CNS development.
      ,
      • Mulder J.
      • Aguado T.
      • Keimpema E.
      • Barabás K.
      • Ballester Rosado C.J.
      • Nguyen L.
      • Monory K.
      • Marsicano G.
      • Di Marzo V.
      • Hurd Y.L.
      • Guillemot F.
      • Mackie K.
      • Lutz B.
      • Guzmán M.
      • Lu H.C.
      • Galve-Roperh I.
      • Harkany T.
      Endocannabinoid signaling controls pyramidal cell specification and long-range axon patterning.
      ), Although these data, coupled with the BRET and PLA experiments, implicate heteromers in this receptor modulation, they could also be explained via simple signaling cross-talk rather than via physical interaction between receptors. However, the results obtained with the antagonists clearly show that CB1-CB2 receptor heteromers are the signaling units. Antagonists, by definition, do not signal; thus, the fact that a CB1 receptor-specific antagonist can block CB2 receptor signaling strongly argues against a cross-talk at the intracellular signaling level. A more likely explanation is that binding of the antagonist to CB1-CB2 receptor heteromers leads to a conformational change that reduces CB2 receptor-induced Akt/PKB phosphorylation. We found this cross-antagonism to be bidirectional in Akt/PKB phosphorylation signaling and in neurite growth.
      A question arising from our findings is the following. Are the CB1-CB2 receptor heteromers indeed expressed in the brain? We explored this possibility using different approaches. The cross-antagonism discussed above can be considered as a heteromer fingerprint and can be exploited to demonstrate the expression of CB1-CB2 receptor heteromers in globus pallidus, a brain region that expresses a high amount of CB2 receptors (
      • Lanciego J.L.
      • Barroso-Chinea P.
      • Rico A.J.
      • Conte-Perales L.
      • Callén L.
      • Roda E.
      • Gómez-Bautista V.
      • López I.P.
      • Lluis C.
      • Labandeira-García J.L.
      • Franco R.
      Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis.
      ). CB1 and CB2 receptors were poorly coupled to Akt/PKB phosphorylation in the rat globus pallidus, but the activation of both receptors increased ERK1/2 phosphorylation in rat globus pallidus slices. A clear cross-antagonism was observed for this signaling pathway, indicating that CB1-CB2 receptor heteromers are expressed in the globus pallidus. A second question arising from our findings is the following. Can CB1-CB2 receptor heteromers explain some of the reported results in the brain? Recently it has been described that both CB1 and CB2 receptors are co-expressed in the pineal gland, where they may be involved in the control of pineal physiology (
      • Koch M.
      • Habazettl I.
      • Dehghani F.
      • Korf H.W.
      The rat pineal gland comprises an endocannabinoid system.
      ). In fact, CB1 and CB2 receptors and the enzymes catalyzing endocannabinoid biosynthesis and degradation were expressed in pinealocytes, and immunosignals for the CB2 receptor did not vary under a 12-h light/12-h dark cycle, whereas CB1 receptor immunoreaction was significantly reduced at the end of the light phase, when the expression of both receptors is more balanced (
      • Koch M.
      • Habazettl I.
      • Dehghani F.
      • Korf H.W.
      The rat pineal gland comprises an endocannabinoid system.
      ). We isolated pinealocytes from rat pineal glands extracted at the end of the light period, and by taking advantage of the PLA, we demonstrated the expression of CB1-CB2 receptor heteromers in pinealocytes. In the pineal gland, the rhythm in melatonin biosynthesis is under control of norepinephrine-mediated regulation of arylalkylamine N-acetyltransferase, the penultimate enzyme of melatonin biosynthesis (
      • Klein D.C.
      Arylalkylamine N-acetyltransferase. “The Timezyme”.
      ), and it has been described that phytocannabinoids like tetrahydrocannabinol reduce arylalkylamine N-acetyltransferase activity and attenuate melatonin biosynthesis in rat pineal glands (
      • Koch M.
      • Dehghani F.
      • Habazettl I.
      • Schomerus C.
      • Korf H.W.
      Cannabinoids attenuate norepinephrine-induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N-acetyltransferase activity without involvement of cannabinoid receptors.
      ). Thus, our results favor the hypothesis that through the negative cross-talk in CB1-CB2 receptor heteromers, CB2 receptor, mainly at the end of the light period, can negatively modulate the CB1 receptor-mediated tetrahydrocannabinol effect on arylalkylamine N-acetyltransferase activity and thus modulate melatonin synthesis. Another brain region where the CB2 receptor expression was reported is the nucleus accumbens, where CB2 receptor may be directly involved in many of the neurochemical and motivational properties of cocaine that are responsible for addiction (
      • Xi Z.X.
      • Peng X.Q.
      • Li X.
      • Song R.
      • Zhang H.Y.
      • Liu Q.R.
      • Yang H.J.
      • Bi G.H.
      • Li J.
      • Gardner E.L.
      Brain cannabinoid CB2 receptors modulate cocaine's actions in mice.
      ). In fact, Xi et al. (
      • Xi Z.X.
      • Peng X.Q.
      • Li X.
      • Song R.
      • Zhang H.Y.
      • Liu Q.R.
      • Yang H.J.
      • Bi G.H.
      • Li J.
      • Gardner E.L.
      Brain cannabinoid CB2 receptors modulate cocaine's actions in mice.
      ) found that CB2 receptor agonists directly infused into the nucleus accumbens decreased the cocaine intake, CB2 receptors regulate the psychomotor stimulant properties of cocaine, and CB2 receptors are endogenously activated by endocannabinoids in nucleus accumbens, where they control locomotor activity. Because CB1 receptors are also expressed in the striatum (
      • Pickel V.M.
      • Chan J.
      • Kearn C.S.
      • Mackie K.
      Targeting dopamine D2 and cannabinoid-1 (CB1) receptors in rat nucleus accumbens.
      ,
      • Martín A.B.
      • Fernandez-Espejo E.
      • Ferrer B.
      • Gorriti M.A.
      • Bilbao A.
      • Navarro M.
      • Rodriguez de Fonseca F.
      • Moratalla R.
      Expression and function of CB1 receptor in the rat striatum. Localization and effects on D1 and D2 dopamine receptor-mediated motor behaviors.
      ) and are reported to be involved in the locomotor activity control (
      • Martín A.B.
      • Fernandez-Espejo E.
      • Ferrer B.
      • Gorriti M.A.
      • Bilbao A.
      • Navarro M.
      • Rodriguez de Fonseca F.
      • Moratalla R.
      Expression and function of CB1 receptor in the rat striatum. Localization and effects on D1 and D2 dopamine receptor-mediated motor behaviors.
      ), we determined the expression of CB1-CB2 receptor heteromers in rat nucleus accumbens slices by PLA. We found CB1-CB2 receptor heteromer expression in this brain region. Although the CB2 receptor-mediated effects on locomotor activity are also seen in CB1−/− mice (
      • Xi Z.X.
      • Peng X.Q.
      • Li X.
      • Song R.
      • Zhang H.Y.
      • Liu Q.R.
      • Yang H.J.
      • Bi G.H.
      • Li J.
      • Gardner E.L.
      Brain cannabinoid CB2 receptors modulate cocaine's actions in mice.
      ), our results provide a new perspective by which CB1 and CB2 receptors might be modulating each other to control locomotion through CB1-CB2 receptor heteromers, using one partner as a “brake” for the other partner's action when both are co-expressed in the same neuron. CB1 receptors are extremely abundant, but CB2 receptors, at least in neurons, are thought to be much less abundant. Altering the expression of CB2 receptors may provide a possible level of regulation of CB1 receptor function by changing the amount of CB1-CB2 receptor heteromers. Further studies will have to be performed to investigate this possibility.
      Finally, our data may provide explanations for several previously controversial points concerning CB1 and CB2 receptors. A confounding problem with the cannabinoid receptors has been the expression levels of the two receptors in the brain. Specifically, there have been varying reports as to the amount of CB2 receptor in the brain and whether those levels change in pathological settings as well as speculation on the role of the neuronal CB2 receptor (
      • Fernández-Ruiz J.
      • Romero J.
      • Velasco G.
      • Tolón R.M.
      • Ramos J.A.
      • Guzmán M.
      Cannabinoid CB2 receptor. A new target for controlling neural cell survival?.
      ,
      • Brusco A.
      • Tagliaferro P.A.
      • Saez T.
      • Onaivi E.S.
      Ultrastructural localization of neuronal brain CB2 cannabinoid receptors.
      ,
      • Van Sickle M.D.
      • Duncan M.
      • Kingsley P.J.
      • Mouihate A.
      • Urbani P.
      • Mackie K.
      • Stella N.
      • Makriyannis A.
      • Piomelli D.
      • Davison J.S.
      • Marnett L.J.
      • Di Marzo V.
      • Pittman Q.J.
      • Patel K.D.
      • Sharkey K.A.
      Identification and functional characterization of brainstem cannabinoid CB2 receptors.
      ,
      • Gong J.P.
      • Onaivi E.S.
      • Ishiguro H.
      • Liu Q.R.
      • Tagliaferro P.A.
      • Brusco A.
      • Uhl G.R.
      Cannabinoid CB2 receptors. Immunohistochemical localization in rat brain.
      ,
      • Brusco A.
      • Tagliaferro P.
      • Saez T.
      • Onaivi E.S.
      Postsynaptic localization of CB2 cannabinoid receptors in the rat hippocampus.
      ,
      • Lanciego J.L.
      • Barroso-Chinea P.
      • Rico A.J.
      • Conte-Perales L.
      • Callén L.
      • Roda E.
      • Gómez-Bautista V.
      • López I.P.
      • Lluis C.
      • Labandeira-García J.L.
      • Franco R.
      Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis.
      ,
      • Benito C.
      • Núñez E.
      • Tolón R.M.
      • Carrier E.J.
      • Rábano A.
      • Hillard C.J.
      • Romero J.
      Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains.
      ,
      • Ehrhart J.
      • Obregon D.
      • Mori T.
      • Hou H.
      • Sun N.
      • Bai Y.
      • Klein T.
      • Fernandez F.
      • Tan J.
      • Shytle R.D.
      Stimulation of cannabinoid receptor 2 (CB2) suppresses microglial activation.
      ,
      • Onaivi E.S.
      • Ishiguro H.
      • Gong J.P.
      • Patel S.
      • Meozzi P.A.
      • Myers L.
      • Perchuk A.
      • Mora Z.
      • Tagliaferro P.A.
      • Gardner E.
      • Brusco A.
      • Akinshola B.E.
      • Liu Q.R.
      • Chirwa S.S.
      • Hope B.
      • Lujilde J.
      • Inada T.
      • Iwasaki S.
      • Macharia D.
      • Teasenfitz L.
      • Arinami T.
      • Uhl G.R.
      Functional expression of brain neuronal CB2 cannabinoid receptors are involved in the effects of drugs of abuse and in depression.
      ,
      • Sagredo O.
      • González S.
      • Aroyo I.
      • Pazos M.R.
      • Benito C.
      • Lastres-Becker I.
      • Romero J.P.
      • Tolón R.M.
      • Mechoulam R.
      • Brouillet E.
      • Romero J.
      • Fernández-Ruiz J.
      Cannabinoid CB2 receptor agonists protect the striatum against malonate toxicity. Relevance for Huntington's disease.
      ,
      • García-Gutiérrez M.S.
      • Manzanares J.
      Overexpression of CB2 cannabinoid receptors decreased vulnerability to anxiety and impaired anxiolytic action of alprazolam in mice.
      ,
      • Onaivi E.S.
      • Ishiguro H.
      • Gu S.
      • Liu Q.R.
      CNS effects of CB2 cannabinoid receptors. Beyond neuro-immuno-cannabinoid activity.
      ). Our results suggest that, even at low expression levels, CB2 receptors could have a significant effect on signaling from CB1 receptor by reducing the cellular response through CB1 receptor. This modulation could be up-regulated upon injury or disease, supporting the thesis proposed recently by Onaivi (
      • Onaivi E.S.
      Commentary. Functional neuronal CB2 cannabinoid receptors in the CNS.
      ) that CB1 and CB2 receptors can work independently and/or cooperatively in differing neuronal populations. This interdependence in neurons expressing both receptors could be through heteromers. At the ligand level, low and high levels of cannabinoid receptor ligands have given different results (
      • Bambico F.R.
      • Katz N.
      • Debonnel G.
      • Gobbi G.
      Cannabinoids elicit antidepressant-like behavior and activate serotonergic neurons through the medial prefrontal cortex.
      ,
      • Bellocchio L.
      • Lafenêtre P.
      • Cannich A.
      • Cota D.
      • Puente N.
      • Grandes P.
      • Chaouloff F.
      • Piazza P.V.
      • Marsicano G.
      Bimodal control of stimulated food intake by the endocannabinoid system.
      ), which were interpreted as affecting different populations of neurons, or by the presence of another cannabinoid ligand-mediated receptor, such as TRPV1 or GPR55, in the same neurons, but an alternative explanation could also be the presence of receptor heteromers. Conflicting results have also been observed on the serotonin system at high and low doses of the nonspecific cannabinoid receptor agonist WIN55212,2. At low doses, there was an increase in neuronal excitation that decreased at higher concentrations. The authors argued these effects must be through CB1 receptor because they could be blocked by the CB1 receptor-specific antagonist, rimonabant. However, we show here that a CB1 receptor antagonist could also block CB2 receptor-mediated signaling via receptor heteromers. It would be interesting to revisit these experiments using a CB2 receptor-specific antagonist as well. Heteromers could also help explain seemingly opposite effects seen with the ligands 2-arachidonoylglycerol and anandamide, which have been reported to have seemingly opposite effects on striatal spiny neurons and sensory neurons of the dorsal root ganglion (reviewed by di Marzo (
      • Di Marzo V.
      Endocannabinoid signaling in the brain. Biosynthetic mechanisms in the limelight.
      )). Part of these effects could also be through differential signaling via CB1-CB2 receptor heteromers. More studies will be required to elucidate how these heteromers behave in different tissues at different concentrations of these two endogenous agonists. Another complication has been the discovery that multiple isoforms of the CB2 receptors are expressed. Perhaps the different isoforms of the CB2 receptor can differentially modulate CB1 receptor and/or vice versa, depending on the tissue environment. Further studies may provide a clue as to how, at the mechanistic level, CB2 receptor is altering CB1 receptor signaling or vice versa and help clarify if any differences in isoforms exist. Finally, a third family of the cannabinoid receptors has recently been proposed, the GPR55 receptor (
      • Pertwee R.G.
      GPR55. A new member of the cannabinoid receptor clan?.
      ,
      • Ryberg E.
      • Larsson N.
      • Sjögren S.
      • Hjorth S.
      • Hermansson N.O.
      • Leonova J.
      • Elebring T.
      • Nilsson K.
      • Drmota T.
      • Greasley P.J.
      The orphan receptor GPR55 is a novel cannabinoid receptor.
      ), with which a functional interaction with CB2 receptor has also been described (
      • Balenga N.A.
      • Aflaki E.
      • Kargl J.
      • Platzer W.
      • Schröder R.
      • Blättermann S.
      • Kostenis E.
      • Brown A.J.
      • Heinemann A.
      • Waldhoer M.
      GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils.
      ,
      • Irving A.
      New blood brothers. The GPR55 and CB2 partnership.
      ). It will be interesting to further characterize its role, if any, in the brain and whether it too can form heteromers with the other members of the family. In conclusion, we report the presence of CB1-CB2 receptor heteromers in a variety of brain regions. These heteromers may have a profound impact on CNS function in a variety of neurological and immunological systems, and our data suggest that these heteromers must be taken into account when designing therapeutic approaches toward alterations involving the endocannabinoid system.

      Note Added in Proof

      An article by Kleyer et al. (
      • Kleyer J.
      • Nicolussi S.
      • Taylor P.
      • Simonelli D.
      • Furger E.
      • Anderle P.
      • Gertsch J.
      Cannabinoid receptor trafficking in peripheral cells is dynamically regulated by a binary biochemical switch.
      ) showing similar findings has recently been published.

      Acknowledgment

      We thank Jasmina Jiménez (University of Barcelona) for technical help.

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