Molecular and Cellular Pharmacology
Cannabinoid CB1 receptor ligand binding and function examined through mutagenesis studies of F200 and S383

https://doi.org/10.1016/j.ejphar.2010.10.056Get rights and content

Abstract

The cannabinoid CB1 G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB1 receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB1 receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB1 receptor ligands.

Introduction

The cannabinoid CB1 receptor is a seven transmembrane spanning G protein-coupled receptor that is highly expressed in the brain (Howlett et al., 2002). The natural ligands of the cannabinoid CB1 receptor are the endogenous cannabinoids, anandamide and 2-arachidonylglycerol (2-AG). This receptor subtype has been shown to mediate the orexigenic actions of endogenous cannabinoids (Wiley et al., 2005). The inverse agonist rimonabant (SR141716A, 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide) has been demonstrated to reduce body weight and waist circumference in clinical trials and, until recently, was on the market as an anti-obesity agent in Europe (Van Gaal et al., 2005). Based on these observations, the cannabinoid CB1 receptor is considered to be an important regulator of energy homeostasis in humans.

Cannabinoid CB1 receptor mutagenesis studies have been used to refine homology models for both active and inactive forms of the receptor, and to relate three-dimensional models to receptor function and ligand binding (Abood, 2005, D'Antona et al., 2006, Hurst et al., 2002, Kapur et al., 2007, Kapur et al., 2008, McAllister et al., 2004, Nebane et al., 2006, Picone et al., 2005). Previous reports have shown that a human cannabinoid CB1 receptor F200 mutation to alanine (position 3.36 using the Ballasteros numbering system (Ballesteros, 1995)) results in a constitutively active receptor and leads to changes in receptor affinity for SR141716A and the agonist WIN55,212-2 ((3R)-5-methyl-3-(morpholinomethyl)-2,3,41,9a-tetrahydro-[1,4]oxazino[2,3,4-hi]indol-6-yl)(naphthalen-1-yl)methanone, but no change for the agonist CP55,940 2-((1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl)-5-(2-methyloctan-2-yl)phenol (McAllister et al., 2004, McAllister et al., 2003, Shen et al., 2006). Structurally, F200 along with several other aromatic residues is proposed to line a pocket where SR141716A and WIN55,212-2 bind via aromatic stacking (McAllister et al., 2003), and it is hypothesized that a rotameric toggle involving F200 and W356 is key to cannabinoid CB1 receptor activation (McAllister et al., 2004). Previous publications on mutant S383A (position 7.39) found no effect on constitutive activity (Kapur et al., 2007), an impact on binding of small molecule agonist CP55,940 and inverse agonist taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide), but no impact on that of the agonist WIN55,212-2 or the inverse agonist SR141716A (Kapur et al., 2007, Lin et al., 2008). S383 is believed to reside closer to the extracellular compartment in the cannabinoid CB1 receptor binding site relative to F200, based on homology models.

To further examine the roles of these residues in binding of cannabinoid CB1 receptor ligands and in cannabinoid CB1 receptor function, we have developed human cannabinoid CB1 receptors with an F200A, F200L or S383A mutation — the substitutions at F200 may allow separation of hydrophobic from pi–pi aromatic interactions involving this residue, enabling assessment of the importance of aromatic stacking to function and binding in this region. We report the agonist-induced and constitutive functional activity of the mutant receptors, and the effects of mutations on binding of several well-studied literature cannabinoid CB1 receptor ligands as well as compounds from an agonist series exemplified by compound 1 (quinolin-8-yl 4-methyl-3-(morpholinosulfonyl)benzoate, Fig. 1) (Lambeng et al., 2007) and a recently disclosed tetrahydroquinoline-based inverse agonist series exemplified by compound 4 ((R)-N-(1-benzyl-6-(trifluoromethyl)-1,2,3,4-tetrahydroquinolin-3-yl)benzenesulfonamide, Fig. 1) (Sun et al., 2006).

Section snippets

Materials

[3H]-CP55,940, [3H]-SR141716A and [35S]GTPγS were purchased from PerkinElmer Life Sciences (Boston, MA). Anandamide, 2-Arachidonylglycerol, WIN55,212-2 and CP55,940 were purchased from Tocris Bioscience (Ellisville, MO). GDP was purchased from Sigma-Aldrich (St. Louis, MO). GTPγS, Anti-c-myc and Fugen 6 were obtained from Roche Applied Science (Indianapolis, IN). Dulbecco's modified Eagle's medium (DMEM), geneticin, l-glutamine, fetal bovine serum (FBS), penicillin/streptomycin, and Dynabeads

Characterization of c-myc tagged wild-type and mutant cannabinoid CB1 receptor constructs in CHO cells

The cannabinoid CB1 membrane protein preparations were characterized by saturation binding analysis to determine Kd and Bmax values (Fig. 2 and Table 1). The Kd and Bmax values for [3H]-CP55,940 binding to F200A and F200L are essentially similar to the wild-type cannabinoid CB1 receptor values. The Kd and Bmax values of [3H]-SR141716A binding to S383A are also comparable to the wild-type receptor values. The binding characteristics of c-myc tagged wild-type cannabinoid CB1 receptor to the

Discussion

Mutation of human cannabinoid CB1 receptor residues F200 and S383 affects binding affinities of the agonists and inverse agonists examined here in diverse but distinctive patterns. As has been found previously, WIN55212-2 and SR141716A/AM251 show a large decrease in binding affinity in only the F200A mutant, whereas CP55,940 loses significant affinity in only the S383A mutant (Kapur et al., 2007, McAllister et al., 2003, Shen et al., 2006). Anandamide binding remains unchanged in both F200A and

Acknowledgements

We thank Brian Murphy for assistance with statistical analyses of the data and Stanley Krystek for valuable advice on cannabinoid CB1 receptor and general GPCR modeling.

References (23)

  • A.C. Howlett et al.

    International Union of Pharmacology. XXVII. Classification of cannabinoid receptors

    Pharmacol. Rev.

    (2002)
  • Cited by (7)

    View all citing articles on Scopus
    1

    Present address: 2115 S. Tan Ct., Chicago, IL 60616, USA.

    2

    Present address: Legal Regulatory/Commercialization, Bristol-Myers Squibb Company, 777 Scudders Mill Road, Plainsboro, NJ 08536, USA.

    3

    Present address: Anchor Therapeutics, Inc., 67 Rogers Street, Cambridge, MA 02142, USA.

    View full text