Characterization of Cannabinoid Receptors

Richard F. Cox1, Vincent Rash1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.26
DOI:  10.1002/0471141755.ph0126s63
Online Posting Date:  December, 2013
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Abstract

This unit describes the use of cannabinoid radioligands in competitive binding assays for determining affinity parameters (IC50, Ki) of unlabeled compounds at transfected CB1 and CB2 receptors expressed in cell lines. Curr. Protoc. Pharmacol. 63:1.26.1‐1.26.10. © 2013 by John Wiley & Sons, Inc.

Keywords: cannabinoids; receptor binding; GPR 55

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Membrane homogenates prepared from transfected cells or native tissues containing cannabinoid receptors (e.g., brain; use standard protocols to prepare membrane homogenates from cells or native tissues, see, e.g., units 1.4, 1.5, & 1.6)
  • Cannabinoid assay buffer (see recipe), room temperature and ice cold (adjust pH to 7.4 at each temperature)
  • Radioligand (e.g., [3H]rimonabant (SR141716), [3H]CP 55,940; see Table 1.26.3 and Introduction, above, for subtype selectivity; PerkinElmer NEN)
  • Unlabeled cannabinoid ligands for use as standards and to define nonspecific binding (Table 1.26.2; available, e.g., from Tocris or Sigma)
  • 100% dimethylsulfoxide (DMSO)
  • Test compounds
  • Scintillation fluid appropriate for microtiter plates (e.g., MicroScint from PerkinElmer NEN)
  • 96‐well microtiter plates (e.g., BD Falcon, cat. no. 35‐3075)
  • Opaque white and clear plate sealers
  • Platform shaker
  • 96‐well GF/C filter plates
  • 96‐well vacuum filtration membrane harvester (e.g., Packard Unifilter 196)
  • Scintillation counter capable of counting 96‐well microtiter plates (e.g., Packard TopCount)
  • Scientific graphing package (e.g., GraphPad Prism; http://www.graphpad.com)
  • Additional reagents and equipment for radioligand binding assays (units 1.2 & 1.3)
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Figures

Videos

Literature Cited

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  Baker, D., Pryce, G., Davies, W.L., and Hiley, C.R. 2006. In silico patent searching reveals a new cannabinoid receptor. Trends Pharmacol. Sci. 27:1‐4.
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  Hanus, L., Breuer, A., Tchilibon, S., Shiloah, S., Goldenberg, D., Horowitz, M., Pertwee, R.G., Ross, R.A., and Mechoulam, R. 1999. HU‐308: A specific agonist for CB2, a peripheral cannabinoid receptor. Proc. Natl. Acad. Sci. U.S.A. 96:14228‐14233.
  Howlett, A.C., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Devane, W.A., Felder, C.C., Herkenham, M., Mackie, K., Martin, B.R., Mechoulam, R., and Pertwee, R.G. 2002. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol. Rev. 54:161‐202.
  Iversen, L.L. 2000. The Science of Marijuana, Oxford University Press, Oxford, United Kingdom.
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  Matsuda, L.A., Lolait, S.J., Brownstein, M.J., Young, A.C., and Bonner, T.I. 1990. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 346:561‐564.
  McHugh, D., Hu, S.S., Rimmerman, N., Juknat, A., Vogel, Z., Walker, J.M., and Bradshaw, H.B. 2010. N‐arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor. BMC Neurosci. 11:44.
  McHugh, D., Page, J., Dunn, E., and Bradshaw, H.B. 2012. Δ(9)‐Tetrahydrocannabinol and N‐arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC‐1B cells. Br. J. Pharmacol. 165:2414‐2424.
  Munro, S., Thomas, K.L., and Abu‐shaar, M. 1993. Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61‐65.
  Pertwee, R.G. 1999. Pharmacology of cannabinoid receptor ligands. Curr. Med. Chem. 6:635‐664.
  Pertwee, R.G. 2010. Cannabinoid Receptor Ligands. Tocris Reviews No. 27. http://www.tocris.com/pdfs/pdf_downloads/Cannabinoid_Receptor_Ligands_Review.pdf.
  Pertwee, R.G., Howlett, A.C., Abood, M., Barth, F., Bonner, T.I., Cabral, G., Casellas, P., Cravatt, B.F., Devane, W.A., Elphick, M.R., Felder, C.C., Herkenham, M., Kunos, G., Mackie, K., Martin, B.R., and Mechoulam, R. 2011. Cannabinoid receptors, introductory chapter. IUPHAR database (IUPHAR‐DB). http://www.iuphar‐db.org/DATABASE/FamilyIntroductionForward ?familyId=13.
  Sharir, H. and Abood, M.E. 2010 Pharmacological characterization of GPR55, a putative cannabinoid receptor. Pharmacol. Therap. 126:301‐313.
  Thomas, B.F., Gilliam, A.F., Burch, D.F., Roche, M.J., and Seltzman, H.H. 1997. Comparative receptor binding analyses of cannabinoid agonists and antagonists. J. Pharm. Exp. Therap. 285:285‐292.
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Key References
  Pertwee, R.G. 2010. See above.
  Contains up to date summary tables of Kd and Ki values for cannabinoid ligands.
Internet Resources
  http://www.iuphar‐db.org/index.jsp
  IUPHAR Committee on Receptor Nomenclature and Drug Classification. IUPAR Database.
  http://www.graphpad.com
  A key resource on cannabinoid receptors.
  http://www.cannabinoidsociety.org
  Contains a data analysis resource center covering receptor binding theory and data analysis. The online radioactivity calculator is particularly useful.
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