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Research ArticleArticle

The Third Transmembrane Helix of the Cannabinoid Receptor Plays a Role in the Selectivity of Aminoalkylindoles for CB2, Peripheral Cannabinoid Receptor

Chen-ni Chin, James W. Murphy, John W. Huffman and Debra A. Kendall
Journal of Pharmacology and Experimental Therapeutics November 1999, 291 (2) 837-844;
Chen-ni Chin
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James W. Murphy
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John W. Huffman
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Debra A. Kendall
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Abstract

Two subtypes of the human cannabinoid receptor have been identified. The CB1 receptor is primarily distributed in the central nervous system, whereas the CB2 receptor is associated with peripheral tissue, including the spleen. These two subtypes are also distinguished by their ligand-binding profiles. The goal of this study was to identify critical residues in transmembrane region III (TM3) of the receptors that contribute to subtype specificity in ligand binding. For this purpose, a chimeric cannabinoid receptor [CB1/2(TM3)] was generated in which the TM3 of CB1 was replaced with the corresponding region of CB2. These receptors were stably expressed in Chinese hamster ovary cells for evaluation. The binding affinities of CB1/2(TM3) and the wild-type CB1 receptor to several prototype ligands were similar with one notable exception: the chimeric receptor exhibited a 4-fold enhancement in binding affinity to WIN 55,212-2 (Kd = 4.8 nM) relative to that observed with CB1 (Kd = 21.7 nM). Two additional aminoalkylindoles, JWH 015 and JWH 018, also bound the chimeric receptor (Ki = 1.0 μM and 1.4 nM, respectively) with higher affinity compared with the wild-type CB1 (Ki = 5.2 μM and 9.8 nM, respectively). Furthermore, the increase in binding affinities of the aminoalkylindoles were reflected in the EC50 values for the ligand-induced inhibition of intracellular cAMP levels mediated by the chimeric receptor. This pattern mirrors the selectivity of WIN 55,212-2 binding to CB2 compared with CB1. Site-specific mutagenesis of the most notable amino acid changes in the chimeric receptor, Gly195 to Ser and Ala198 to Met, revealed that the enhancement in WIN 55,212-2 binding is contributed to by the Ser but not by the Met residue. The data indicate that the amino acid differences in TM3 between CB1 and CB2 play a critical role in subtype selectivity for this class of compounds.

Footnotes

  • Send reprint requests to: Dr. Debra A. Kendall, Department of Molecular and Cell Biology, Box U-44, 75 North Eagleville Rd., University of Connecticut, Storrs, CT 06269-3044. E-mail:kendall{at}uconnvm.uconn.edu

  • ↵1 This work was supported in part by the Critical Technologies Program of Connecticut Innovations, Inc., and grants from the National Institute on Drug Abuse (DA09158) and General Medical Science (GM37639) (all to D.A.K.) and by a grant from the National Institute on Drug Abuse (DA03590) (to J.W.H.).

  • ↵2 Present address: Department of Biochemistry, Stockholm University, 106 91 Stockholm, Sweden.

  • Abbreviations:
    THC
    Δ9-tetrahydrocannabinol
    TM
    transmembrane region
    AAI
    aminoalkyindole
    CB1
    central cannabinoid receptor
    CB2
    peripheral cannabinoid receptor
    CP 55,940
    (−)-cis-3[2-hydroxy-4-(1′,1′-dimethylheptyl)phenyl]trans-4-(3-hydroxypropyl)cyclohexanol
    WIN 55,212-2
    (R)-(+)-[2.3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthalenyl)methanone monomethanesulfonate
    SR 141716A
    N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride
    JWH 015
    1-propyl-2-methyl-3-(1-naphthoyl)indole
    JWH 018
    1-pentyl-3-(1-naphthoyl)indole
    (R)-methanandamide
    (R)-(+)-arachidonyl-1′-hydroxy-2′-propylamide
    MEM
    minimal essential medium
    CHO
    Chinese hamster ovary
    • Received March 15, 1999.
    • Accepted July 8, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 291 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 291, Issue 2
1 Nov 1999
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Research ArticleArticle

The Third Transmembrane Helix of the Cannabinoid Receptor Plays a Role in the Selectivity of Aminoalkylindoles for CB2, Peripheral Cannabinoid Receptor

Chen-ni Chin, James W. Murphy, John W. Huffman and Debra A. Kendall
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 837-844;

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Research ArticleArticle

The Third Transmembrane Helix of the Cannabinoid Receptor Plays a Role in the Selectivity of Aminoalkylindoles for CB2, Peripheral Cannabinoid Receptor

Chen-ni Chin, James W. Murphy, John W. Huffman and Debra A. Kendall
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 837-844;
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