Characterization of Cannabinoid Receptors

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

This unit describes the use of cannabinoid radioligands in competitive binding assays for determining affinity parameters (IC₅₀ , K _i ) 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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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., [³ H]rimonabant (SR141716), [³ H]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

Figure 1.26.1 Competition binding curves demonstrating inhibition of [³ H]CP 55,940 binding to the human CB1 receptor by competition with unlabeled WIN 55,212‐2, HU‐210, and CP 55,940. Data from Table were fit using GraphPad Prism software.

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Literature Cited

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	Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A., and Mechoulan, R. 1992. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258:1946‐1949.
	Fattore, L. and Fratta, W. 2011. Beyond THC: The new generation of cannabinoid designer drugs. Front. Behav. Neurosci. 5:60.
	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.
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	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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	Yao B.B., Mukherjee, S., Fan, Y., Garrison, T.R., Daza, A.V., Grayson, H.K., Hooker, B.A., Dart, M.J., Sullivan, J.P., and Meyer, M.D. 2006 In vitro pharmacological characterization of AM1241: A protean agonist at the cannabinoid CB2 receptor? Br. J. Pharmacol. 149:145‐154.
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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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Characterization of Cannabinoid Receptors

Abstract

Table of Contents

Materials

Basic Protocol 1:

Figures

Videos

Literature Cited