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Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics

Acta Pharmacologica Sinica (2018) | Download Citation



Targeting peripheral CB1R is desirable for the treatment of metabolic syndromes without adverse neuropsychiatric effects. We previously reported a human hCB1b isoform that is selectively enriched in pancreatic beta-cells and hepatocytes, providing a potential peripheral therapeutic hCB1R target. It is unknown whether there are peripherally enriched mouse and rat CB1R (mCB1 and rCB1, respectively) isoforms. In this study, we found no evidence of peripherally enriched rodent CB1 isoforms; however, some mCB1R isoforms are absent in peripheral tissues. We show that the mouse Cnr1 gene contains six exons that are transcribed from a single promoter. We found that mCB1A is a spliced variant of extended exon 1 and protein-coding exon 6; mCB1B is a novel spliced variant containing unspliced exon 1, intron 1, and exon 2, which is then spliced to exon 6; and mCB1C is a spliced variant including all 6 exons. Using RNAscope in situ hybridization, we show that the isoforms mCB1A and mCB1B are expressed at a cellular level and colocalized in GABAergic neurons in the hippocampus and cortex. RT-qPCR reveals that mCB1A and mCB1B are enriched in the brain, while mCB1B is not expressed in the pancreas or the liver. Rat rCB1R isoforms are differentially expressed in primary cultured neurons, astrocytes, and microglia. We also investigated modulation of Cnr1 expression by insulin in vivo and carried out in silico modeling of CB1R with JD5037, a peripherally restricted CB1R inverse agonist, using the published crystal structure of hCB1R. The results provide models for future CB1R peripheral targeting.

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  1. 1.

    Mechoulam R, Parker LA. The endocannabinoid system and the brain. Annu Rev Psychol. 2013;64:21–47.

  2. 2.

    Nathan PJ, O’Neill BV, Napolitano A, Bullmore ET. Neuropsychiatric adverse effects of centrally acting antiobesity drugs. CNS Neurosci Ther. 2011;17:490–505.

  3. 3.

    Gonzalez-Mariscal I, Krzysik-Walker SM, Kim W, Rouse M, Egan JM. Blockade of cannabinoid 1 receptor improves GLP-1R mediated insulin secretion in mice. Mol Cell Endocrinol. 2016;423:1–10.

  4. 4.

    Perwitz N, Wenzel J, Wagner I, Buning J, Drenckhan M, Zarse K, et al. Cannabinoid type 1 receptor blockade induces transdifferentiation towards a brown fat phenotype in white adipocytes. Diabetes Obes Metab. 2010;12:158–66.

  5. 5.

    Udi S, Hinden L, Earley B, Drori A, Reuveni N, Hadar R, et al. Proximal tubular cannabinoid-1 receptor regulates obesity-induced CKD. J Am Soc Nephrol. 2017;28:3518–32.

  6. 6.

    Liu J, Zhou L, Xiong K, Godlewski G, Mukhopadhyay B, Tam J, et al. Hepatic cannabinoid receptor-1 mediates diet-induced insulin resistance via inhibition of insulin signaling and clearance in mice. Gastroenterology. 2012;142:1218–28 e1.

  7. 7.

    Ruiz de Azua I, Mancini G, Srivastava RK, Rey AA, Cardinal P, Tedesco L, et al. Adipocyte cannabinoid receptor CB1 regulates energy homeostasis and alternatively activated macrophages. J Clin Invest. 2017;127:4148–62.

  8. 8.

    Jalin AM, Rajasekaran M, Prather PL, Kwon JS, Gajulapati V, Choi Y, et al. Non-selective cannabinoid receptor antagonists, hinokiresinols reduce infiltration of microglia/macrophages into ischemic brain lesions in rat via modulating 2-arachidonolyglycerol-induced migration and mitochondrial activity. PLoS One. 2015;10:e0141600.

  9. 9.

    Mai P, Yang L, Tian L, Wang L, Jia S, Zhang Y, et al. Endocannabinoid system contributes to liver injury and inflammation by activation of bone marrow-derived monocytes/macrophages in a CB1-dependent manner. J Immunol. 2015;195:3390–401.

  10. 10.

    McPartland JM. Phylogenomic and chemotaxonomic analysis of the endocannabinoid system. Brain Res Brain Res Rev. 2004;45:18–29.

  11. 11.

    McPartland JM, Glass M, Pertwee RG. Meta-analysis of cannabinoid ligand binding affinity and receptor distribution: interspecies differences. Br J Pharmacol. 2007;152:583–93.

  12. 12.

    Gonzalez-Mariscal I, Krzysik-Walker SM, Doyle ME, Liu QR, Cimbro R, Santa-Cruz Calvo S, et al. Human CB1 receptor isoforms, present in hepatocytes and beta-cells, are involved in regulating metabolism. Sci Rep. 2016;6:33302.

  13. 13.

    Liu QR, Pan CH, Hishimoto A, Li CY, Xi ZX, Llorente-Berzal A, et al. Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands. Genes Brain Behav. 2009;8:519–30.

  14. 14.

    Zhang PW, Ishiguro H, Ohtsuki T, Hess J, Carillo F, Walther D, et al. Human cannabinoid receptor 1: 5′ exons, candidate regulatory regions, polymorphisms, haplotypes and association with polysubstance abuse. Mol Psychiatry. 2004;9:916–31.

  15. 15.

    Onaivi ES, Ishiguro H, Gu S, Liu QR. CNS effects of CB2 cannabinoid receptors: beyond neuro-immuno-cannabinoid activity. J Psychopharmacol. 2012;26:92–103.

  16. 16.

    Zhang HY, Bi GH, Li X, Li J, Qu H, Zhang SJ, et al. Species differences in cannabinoid receptor 2 and receptor responses to cocaine self-administration in mice and rats. Neuropsychopharmacology. 2015;40:1037–51.

  17. 17.

    Chiang YC, Lo YN, Chen JC. Crosstalk between dopamine D(2) receptors and cannabinoid CB(1) receptors regulates CNR1 promoter activity via ERK1/2 signaling. J Neurochem. 2013;127:163–76.

  18. 18.

    Iyer MR, Cinar R, Coffey NJ, Chorvat RJ, Kunos G. Synthesis of S-2-((S)-3-(4-chlorophenyl)-N’-((4-chlorophenyl)sulfonyl)-4-phenyl-4,5-dihydro-1H-pyrazole-1-carboximidamido)-3-(methyl-d3)butanamide-d5, octadeuterated JD5037. J Label Comp Radiopharm. 2017;60:460–5.

  19. 19.

    Hua T, Vemuri K, Nikas SP, Laprairie RB, Wu Y, Qu L, et al. Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature. 2017;547:468–71.

  20. 20.

    Hua T, Vemuri K, Pu M, Qu L, Han GW, Wu Y, et al. Crystal structure of the human cannabinoid receptor CB1. Cell. 2016;167:750–62 e14.

  21. 21.

    Liu QR, Rubio FJ, Bossert JM, Marchant NJ, Fanous S, Hou X, et al. Detection of molecular alterations in methamphetamine-activated Fos-expressing neurons from a single rat dorsal striatum using fluorescence-activated cell sorting (FACS). J Neurochem. 2014;128:173–85.

  22. 22.

    Harvey BK, Chou J, Shen H, Hoffer BJ, Wang Y. Diadenosine tetraphosphate reduces toxicity caused by high-dose methamphetamine administration. Neurotoxicology. 2009;30:436–44.

  23. 23.

    Zhang C, Deng Y, Dai H, Zhou W, Tian J, Bing G, et al. Effects of dimethyl sulfoxide on the morphology and viability of primary cultured neurons and astrocytes. Brain Res Bull. 2017;128:34–9.

  24. 24.

    Carninci P, Shibata Y, Hayatsu N, Sugahara Y, Shibata K, Itoh M, et al. Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res. 2000;10:1617–30.

  25. 25.

    Lee C, Huang CH. LASAGNA-Search: an integrated web tool for transcription factor binding site search and visualization. Biotechniques. 2013;54:141–53.

  26. 26.

    Khan A, Fornes O, Stigliani A, Gheorghe M, Castro-Mondragon JA, van der Lee R, et al. JASPAR 2018: update of the open-access database of transcription factor binding profiles and its web framework. Nucleic Acids Res. 2017;46:D1284.

  27. 27.

    Kozomara A, Griffiths-Jones S. miRBase: integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 2011;39:D152–7.

  28. 28.

    Zhou Y, Zeng P, Li YH, Zhang Z, Cui Q. SRAMP: prediction of mammalian N6-methyladenosine (m6A) sites based on sequence-derived features. Nucleic Acids Res. 2016;44:e91.

  29. 29.

    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem. 2004;25:1605–12.

  30. 30.

    Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;30:2785–91.

  31. 31.

    Abood ME, Ditto KE, Noel MA, Showalter VM, Tao Q. Isolation and expression of a mouse CB1 cannabinoid receptor gene. Comparison of binding properties with those of native CB1 receptors in mouse brain and N18TG2 neuroblastoma cells. Biochem Pharmacol. 1997;53:207–14.

  32. 32.

    McCaw EA, Hu H, Gomez GT, Hebb AL, Kelly ME, Denovan-Wright EM. Structure, expression and regulation of the cannabinoid receptor gene (CB1) in Huntington’s disease transgenic mice. Eur J Biochem. 2004;271:4909–20.

  33. 33.

    Pruitt KD, Tatusova T, Maglott DR. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2005;33:D501–4.

  34. 34.

    Bonaldo MF, Lennon G, Soares MB. Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res. 1996;6:791–806.

  35. 35.

    Ruehle S, Wager-Miller J, Straiker A, Farnsworth J, Murphy MN, Loch S, et al. Discovery and characterization of two novel CB1 receptor splice variants with modified N-termini in mouse. J Neurochem. 2017;142:521–33.

  36. 36.

    Gonzalez-Mariscal I, Montoro RA, Doyle ME, Liu QR, Rouse M, O’Connell JF, et al. Absence of cannabinoid 1 receptor in beta cells protects against high-fat/high-sugar diet-induced beta cell dysfunction and inflammation in murine islets. Diabetologia. 2018;61:1470–83.

  37. 37.

    Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990;346:561–4.

  38. 38.

    McLaughlin CR, Martin BR, Compton DR, Abood ME. Cannabinoid receptors in developing rats: detection of mRNA and receptor binding. Drug Alcohol Depend. 1994;36:27–31.

  39. 39.

    Shao Z, Yin J, Chapman K, Grzemska M, Clark L, Wang J, et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature. 2016;540:602–6.

  40. 40.

    Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J Mol Biol. 1987;196:261–82.

  41. 41.

    Zheng GX, Ravi A, Gould GM, Burge CB, Sharp PA. Genome-wide impact of a recently expanded microRNA cluster in mouse. Proc Natl Acad Sci U S A. 2011;108:15804–9.

  42. 42.

    Belarbi Y, Mejhert N, Gao H, Arner P, Ryden M, Kulyte A. MicroRNAs-361-5p and miR-574-5p associate with human adipose morphology and regulate EBF1 expression in white adipose tissue. Mol Cell Endocrinol. 2017;472:50–6.

  43. 43.

    Olivares AM, Jelcick AS, Reinecke J, Leehy B, Haider A, Morrison MA, et al. Multimodal regulation orchestrates normal and complex disease states in the retina. Sci Rep. 2017;7:690.

  44. 44.

    Allen SE, Toro CP, Andrade A, Lopez-Soto EJ, Denome S, Lipscombe D. Cell-specific RNA binding protein Rbfox2 regulates CaV2.2 mRNA exon composition and CaV2.2 current size.eNeuro.2017;4:pii: ENEURO.0332-16.2017

  45. 45.

    Iijima T, Iijima Y, Witte H, Scheiffele P. Neuronal cell type-specific alternative splicing is regulated by the KH domain protein SLM1. J Cell Biol. 2014;204:331–42.

  46. 46.

    Adhikari S, Xiao W, Zhao YL, Yang YG. m(6)A: signaling for mRNA splicing. RNA Biol. 2016;13:756–9.

  47. 47.

    Patil DP, Pickering BF, Jaffrey SR. Reading m(6)A in the transcriptome: m(6)A-binding proteins. Trends Cell Biol. 2018;28:113–27.

  48. 48.

    Liu QR, Lu L, Zhu XG, Gong JP, Shaham Y, Uhl GR. Rodent BDNF genes, novel promoters, novel splice variants, and regulation by cocaine. Brain Res. 2006;1067:1–12.

  49. 49.

    Maynard KR, Hobbs JW, Sukumar M, Kardian AS, Jimenez DV, Schloesser RJ, et al. Bdnf mRNA splice variants differentially impact CA1 and CA3 dendrite complexity and spine morphology in the hippocampus. Brain Struct Funct. 2017;222:3295–307.

  50. 50.

    Elphick MR. Evolution of cannabinoid receptors in vertebrates: identification of a CB(2) gene in the puffer fish Fugu rubripes. Biol Bull. 2002;202:104–7.

  51. 51.

    Shire D, Calandra B, Rinaldi-Carmona M, Oustric D, Pessegue B, Bonnin-Cabanne O, et al. Molecular cloning, expression and function of the murine CB2 peripheral cannabinoid receptor. Biochim Biophys Acta. 1996;1307:132–6.

  52. 52.

    Vijayakumar S, Depreux FF, Jodelka FM, Lentz JJ, Rigo F, Jones TA, et al. Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide. Hum Mol Genet. 2017;26:3482–94.

  53. 53.

    Tam J, Liu J, Mukhopadhyay B, Cinar R, Godlewski G, Kunos G. Endocannabinoids in liver disease. Hepatology. 2011;53:346–55.

  54. 54.

    Zhang HY, Gao M, Liu QR, Bi GH, Li X, Yang HJ, et al. Cannabinoid CB2 receptors modulate midbrain dopamine neuronal activity and dopamine-related behavior in mice. Proc Natl Acad Sci U S A. 2014;111:E5007–15.

  55. 55.

    Liu QR, Canseco-Alba A, Zhang HY, Tagliaferro P, Chung M, Dennis E, et al. Cannabinoid type 2 receptors in dopamine neurons inhibits psychomotor behaviors, alters anxiety, depression and alcohol preference. Sci Rep. 2017;7:17410.

  56. 56.

    Tam J, Szanda G, Drori A, Liu Z, Cinar R, Kashiwaya Y, et al. Peripheral cannabinoid-1 receptor blockade restores hypothalamic leptin signaling. Mol Metab. 2017;6:1113–25.

  57. 57.

    Lin X, Dhopeshwarkar AS, Huibregtse M, Mackie K, Hohmann AG. Slowly signaling G protein-biased CB2 cannabinoid receptor agonist LY2828360 suppresses neuropathic pain with sustained efficacy and attenuates morphine tolerance and dependence. Mol Pharmacol. 2018;93:49–62.

  58. 58.

    Rowley S, Sun X, Lima IV, Tavenier A, de Oliveira ACP, Dey SK, et al. Cannabinoid receptor 1/2 double-knockout mice develop epilepsy. Epilepsia. 2017;58:e162–e6.

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JME, Q-RL, NSH, IG-M, JFO, and SS-C-C are supported by the Intramural Research Program of the National Institute on Aging; Z-XX by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health; HQ by the National Science Foundation of China (31671375); ESO by NIH grant DA032890; and YW by the National Health Research Institutes and a Central Government S & T Grant, Taiwan (106-1901-01-10-02).

Author contributions

Q-RL, ESO, and JME conceptualized the study and wrote the manuscript. Q-RL, JME, JFO, IG-M, MD, and SS-C-C performed S961 treatment and mouse and rat tissue dissections. Q-RL and HQ analyzed the gene structures and protein modeling, respectively, using the bioinformatics tools. Q-RL, NSH, and Z-XX performed RT-qPCR, RNAscope ISH, imaging, and data analysis. Primary cell culture and isolation of neurons, astrocytes, and microglia were conducted by YW. All authors reviewed and approved the publication of this manuscript.

Author information


  1. Laboratory of Clinical Investigation, National Institute on Aging, NIH, Baltimore, MD, USA

    • Qing-Rong Liu
    • , Nicholas S. Huang
    • , Jennifer F. O’Connell
    • , Isabel Gonzalez-Mariscal
    • , Sara Santa-Cruz-Calvo
    • , Maire E. Doyle
    •  & Josephine M. Egan
  2. Center for Bioinformatics, State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China

    • Hong Qu
  3. Addiction Biology Unit, Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse, NIH, Baltimore, MD, USA

    • Zheng-Xiong. Xi
  4. Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan

    • Yun Wang
  5. Department of Biology, William Paterson University, Wayne, NJ, USA

    • Emmanuel. S. Onaivi


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The authors declare no competing interests.

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Correspondence to Qing-Rong Liu.

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