Elsevier

Cellular Signalling

Volume 10, Issue 9, October 1998, Pages 667-674
Cellular Signalling

Differential Receptor–G-Protein Coupling Evoked by Dissimilar Cannabinoid Receptor Agonists

https://doi.org/10.1016/S0898-6568(98)00013-8Get rights and content

Abstract

Multiple affinity states are revealed by agonist competition for radioantagonist [3H]SR141716A binding to rat brain CB1 cannabinoid receptors. Desacetyllevonantradol (DALN), a tricyclic cannabinoid, and WIN55212-2, an aminoalkylindole, both bound in two discrete affinity states (30% high affinity), but the ratios of the IC50 revealed distinct differences. Other affinity-state differences between the agonists were: Na+ reduced the affinity for the membrane-bound receptor by 10-fold for DALN but minimally for WIN55212-2; a non-hydrolysable GTP analogue decreased the fraction of high-affinity WIN55212-2 binding but not that of DALN unless Na+ was also present. Detergent solubilisation increased the fraction of high-affinity binding for both agonists but eliminated any effect of Na+ on the agonist affinities. In detergent solution, the GTP analogue reduced the WIN55212-2 high-affinity fraction but not that of DALN, even though the IC50 values increased for both DALN and WIN55212-2. The differential modulation of CB1 receptor–G-protein coupling by Na+ and guanine nucleotides is dependent upon the cannabimimetic agonist bound.

Introduction

The biologically active compound in marihuana, Δ9-tetrahydrocannabinol, promotes specific central nervous system effects in humans. These actions are conveyed through specific membrane CB1 cannabinoid receptors found in the brain and result in the well-known psychoactive effects of marihuana such as sedation, euphoria and loss of short-term memory as well as analgesic, antiemetic and anticonvulsant actions (reviewed in 1, 2). At the cellular level, cannabimimetic agonists are known to inhibit the activity of adenylate cyclase and to alter the activity of potassium and calcium ion channels (see [3] for review). These interactions are transduced through the guanine nucleotide binding proteins Go and Gi. However, it is unclear which subtypes of these proteins (Go1, Go2, Gαi1, Gαi2, Gαi3) are implicated in effector activation.

Most of the data regarding CB1 cannabinoid receptor interactions with G proteins have been generated by using agonist radioligand binding assays. The high-affinity synthetic cannabinoid agonist [3H]CP55940 has been used to characterise the CB1 cannabinoid receptor 4, 5. The aminoalkylindole agonists belong to a second class of compounds having a cannabimimetic profile identical with that of the cannabinoid ligands in vivo and in vitro—reviewed in [6]. [3H]WIN55212-2 is the agonist radioligand developed from this class of compounds [7]. [3H]CP55940 binding to rat brain membranes revealed that, like many other G-protein-coupled receptors, cannabinoid agonist binding could be affected by the presence of Na+ or guanine nucleotides, indicating that different states of binding affinity exist 4, 5. These binding states are indicative of the degree of receptor interaction with G proteins. The interaction of CB1 receptors with G proteins is thought to be tight, because high- affinity agonist binding is maintained after detergent solubilisation from membranes and is diminished by 75% with the addition of guanine nucleotides [5]. Unlike some receptor systems, complete inhibition of radioagonist binding was not achieved, even in the presence of high (100 μM) concentrations of the non-hydrolysable GTP analogue guanosine 5′-O-(3-thio)triphosphate (GTPγS) [5].

Recently, the characterisation of SR141716A, a CB1 selective antagonist, was reported [8]. The tritium labelling of SR141716A provides a new tool for the analysis of cannabinoid receptor signal transduction. The present study examines the displacement of [3H]SR141716A binding to CB1 cannabinoid receptors in rat brain membranes and detergent-solubilised receptor preparations by cannabinoid desacetyllevonantradol (DALN) and aminoalkylindole WIN55212-2. The effects of Na+ and GTPγS in modulating the affinity of these two structurally distinct cannabimimetic agonists are compared.

Section snippets

Preparation of Membranes and Solubilization

Membranes were prepared from frozen rat brains (Pel-Freez) as previously described [4] with the exception that 100 μM aminoethylbenzenesulphonylfluoride (AEBSF) (CalBiochem) was included in the initial homogenisation. Aliquots were stored in TME buffer comprising 20 mM Tris-HCl, pH 7.5, 5 mM MgCl and 1 mM ethylenediaminetetraacetic acid (EDTA). Protein concentrations were determined by the Bio-Rad DC assay. Detergent solubilisation of cannabinoid receptors was accomplished by adjusting

Results

Figure 1 is typical of the binding profile observed for competitive displacement of the radiolabelled cannabinoid agonist [3H]CP55940. DALN and WIN55212-2 represent two structurally different classes of cannabimimetic agonists, known as tricyclic cannabinoids and aminoalkylindoles, respectively. Both agonists bind CB1 cannabinoid receptors with high affinity; however, WIN55212-2 is less potent than DALN.

Characterisation of SR141716A as a CB1-selective antagonist revealed the compound to be

Discussion

A basic tenet of molecular pharmacology is that agonists, but not antagonists, stabilise an active conformation of the receptor, which in turn promotes effector activation. The ternary complex model for G-protein-coupled receptors equates active receptor with a complex of agonist (A), receptor (R) and G protein (G) and explains multiple agonist–receptor affinity states by the conversion of high-affinity receptors (RG) into low-affinity receptors (R) in the presence of guanine nucleotides [18].

Acknowledgements

This work was supported by NIH grants R01-DA03690, R01-DA06312 and P01-DA09158.

References (39)

  • M.E. Abood et al.

    Trends Pharmacol. Sci.

    (1992)
  • M. Rinaldi-Carmona et al.

    FEBS Lett.

    (1994)
  • S. Swillens et al.

    Trends Pharmacol. Sci.

    (1995)
  • D.A. Horstman et al.

    J. Biol. Chem.

    (1990)
  • A. De Lean et al.

    J. Biol. Chem.

    (1980)
  • P. Samama et al.

    J. Biol. Chem.

    (1993)
  • R.J. Lefkowitz et al.

    Trends Pharmacol. Sci.

    (1993)
  • B.P. Ceresa et al.

    J. Biol. Chem.

    (1994)
  • K. Befort et al.

    J. Biol. Chem.

    (1996)
  • H.L. Wiener et al.

    Eur. J. Pharmacol.

    (1991)
  • B. Frances et al.

    Eur. J. Pharmacol.

    (1985)
  • W.L. Dewey

    Pharmacol. Rev.

    (1986)
  • A.C. Howlett

    Cannabinoid Compounds and Signal Transduction Mechanisms

  • W.A. Devane et al.

    Mol. Pharmacol.

    (1988)
  • D.B. Houston et al.

    Mol. Pharmacol.

    (1993)
  • A.C. Howlett et al.

    Curr. Pharm. Design

    (1995)
  • J.E. Kuster et al.

    J. Pharmacol. Exp. Ther.

    (1993)
  • C.B. Pert et al.

    Mol. Pharmacol.

    (1974)
  • L.E. Limbird et al.

    Mol. Pharmacol.

    (1982)
  • Cited by (53)

    • Analysis of the pharmacological properties of JWH-122 isomers and THJ-2201, RCS-4 and AB-CHMINACA in HEK293T cells and hippocampal neurons

      2018, European Journal of Pharmacology
      Citation Excerpt :

      It is becoming increasingly evident that CB1-mediated signaling is not well described by classical pharmacological models. Specifically, several studies have reported that CB1 signaling displays ligand bias or differential G-protein coupling (Glass and Northup, 1999; Houston and Howlett, 1998; Kenakin, 2007), as well as inefficient G-protein coupling (Breivogel et al., 1997; Howlett et al., 2004; Sim et al., 1996). Furthermore, CB1 is known to exert pleiotropic effects by virtue of its ability to activate multiple G-proteins (Gi/o, Gs, Gq) in the same cell.

    • Driving the need to feed: Insight into the collaborative interaction between ghrelin and endocannabinoid systems in modulating brain reward systems

      2016, Neuroscience and Biobehavioral Reviews
      Citation Excerpt :

      The signaling cascades initiated following CB-1R activation depend largely on the G-protein type coupled to CB-1Rs (Bosier et al., 2010). Most predominately, CB-1Rs associate with the Gi/o family of G proteins, ultimately leading to the activation of inwardly rectifying K+ currents and MAPK cascades and the inhibition of voltage-gated Ca2+ channels and adenylyl cyclase, following receptor activation (Bosier et al., 2010; Houston and Howlett, 1998; Howlett, 1985; Howlett et al., 2002; Kano et al., 2009; Pertwee, 1997). These CB-1R mediated effects are not consistent with those known to activate AMPK (Hardie, 2013; Lim et al., 2010; Omar et al., 2009).

    • Pharmacological characterization of emerging synthetic cannabinoids in HEK293T cells and hippocampal neurons

      2016, European Journal of Pharmacology
      Citation Excerpt :

      Furthermore, CB1 exerts pleiotropic effects through multiple G-proteins (Gi/o, Gs, Gq) and activation of signaling pathways is agonist-specific (Bosier et al., 2010; Glass and Northup, 1999; Laprairie et al., 2014; Lauckner et al., 2005; Mukhopadhyay and Howlett, 2005). Cannabinoid receptor signaling is notably complex, displaying ligand bias, differential G-protein coupling (Glass and Northup, 1999; Houston and Howlett, 1998; Kenakin, 2007), and inefficient G-protein coupling (Breivogel et al., 1997; Howlett et al., 2004). It has been reported that certain compounds activate different G-protein cohorts in endogenous versus heterologous CB1 receptors (Breivogel et al., 1998; Landsman et al., 1997).

    • Cannabinoid receptors: Nomenclature and pharmacological principles

      2012, Progress in Neuro-Psychopharmacology and Biological Psychiatry
      Citation Excerpt :

      Generally, constitutively active receptors are also constitutively phosphorylated and desensitized, providing support for a model where a single active state conformation is the target for phosphorylation, internalization and desensitization (Leurs et al., 1998). However, a study on the angiotensin II receptor and a series of studies on the CB1 receptor suggest that GPCRs may possess several transition states, each associated with conformationally distinguishable states of receptor activation and regulation (Houston and Howlett, 1998; Hsieh et al., 1999; Jin et al., 1999; Roche et al., 1999; Thomas et al., 2000). Nie and Lewis (2001) found that the C-terminal domain contributes to constitutive activity of CB1.

    View all citing articles on Scopus

    Present address: National Enzyme Company, Forsyth, MO 65653, USA.

    View full text