Elsevier

Neuropharmacology

Volume 54, Issue 5, April 2008, Pages 815-823
Neuropharmacology

Antagonistic cannabinoid CB1/dopamine D2 receptor interactions in striatal CB1/D2 heteromers. A combined neurochemical and behavioral analysis

https://doi.org/10.1016/j.neuropharm.2007.12.011Get rights and content

Abstract

In vitro results show the ability of the CB1 receptor agonist CP 55,940 to reduce the affinity of D2 receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D2 receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB1–D2 receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB1/D2 receptor–receptor interactions may occur in CB1/D2 formed heteromers. Antagonistic CB1/D2 interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB1 receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB1 receptor antagonist rimonobant but also the specific A2A receptor antagonist MSX-3 blocked the inhibitory effect of CB1 receptor agonist on D2-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB1/D2 receptor–receptor interactions within CB1/D2 heteromers in which A2A receptors may also participate.

Introduction

It was proposed in 2003 that the anti-parkinsonian actions of cannabinoid (CB)1 receptor antagonists involve the counteraction of antagonistic CB1/dopamine (D)2 receptor interactions in CB1/D2 heteromeric complexes (Fuxe et al., 2003). Subsequent work also indicated the existence of CB1/D2 heteromers in HEK-293 cell lines (Kearn et al., 2005) based inter alia on co-immunoprecipitation experiments with the putative CB1/D2 heteromers becoming enhanced by concurrent agonist stimulation (Kearn et al., 2005). The cellular and sub-cellular localization of CB1 receptor and D2 receptors in the basal ganglia have been described in detail (Agnati et al., 2005, Egertova and Elphick, 2000, Khan et al., 1998, Matyas et al., 2006, Missale et al., 1998, Pickel et al., 2006). The evidence suggests a co-location of CB1 and D2 striatal receptors predominantly in the soma and dendrites of the ventral of striato-pallidal GABA neurons and also in cortico-striatal glutamate terminals where A2A receptors are also present (see Fuxe et al., 2005, Katona et al., 2006, Pickel et al., 2006, Uchigashima et al., 2007, Yin and Lovinger, 2006).

In the present study, the existence of CB1/D2 heteromers has been further established in cell lines using FRET-based analysis as well as demonstrating the existence of antagonistic CB1/D2 receptor interactions at the agonist recognition level in rat striatal membranes and by using receptor autoradiography in the rat nucleus accumbens. Through in vivo pharmacological analysis of D2 receptor agonist-induced hyperlocomotion, we have further demonstrated the existence of antagonistic CB1/D2 receptor interactions with an involvement of adenosine (A)2A receptor activation.

Section snippets

Autoradiography

Male specific free pathogen Sprague-Dawley rats (N = 8, 6 months old, weighted 275 ± 3 g) were provided by IFFA-CREDO (CHARLES RIVER, France). Animals were handled under standard conditions during 2 weeks. All animals were maintained in humidity and temperature controlled (20–22 °C) room under a 12:12-h light/dark cycle (lights on 08:00 and off 20:00 h). Food and water were available ad libitum. All experiments were performed between 09:00 and 13:00 in consideration of the animals' circadian rhythm.

Heteromerization of CB1 and D2 receptors in living cells

The formation of CB1–D2 receptor heteromers was demonstrated by using the FRET technique in cells transfected with fusion proteins consisting of each receptor and a fluorescent protein, either GFP2 or YFP. Expression of fusion proteins was assessed by Western blot and immunocytochemistry (data not shown). As seen in Fig. 1, a clear-cut positive FRET signal was observed in living cells co-transfected with the cDNA of vectors encoding for D2-GFP2 and CB1-YFP similar to the FRET signal obtained

Discussion

Previously, based on behavioral experiments, it has been proposed that CB1 and D2 receptors could antagonistically interact within CB1/D2 heteromeric complexes in striato-pallidal GABA neurons (Fuxe et al., 2003) and where co-immunoprecipitation assays have given further evidence for this proposal (Kearn et al., 2005). Here, strong evidence supporting direct interactions between CB1 and D2 receptors through CB1/D2 heteromerization is provided. Evidence indicating antagonistic CB1/D2 receptor

Acknowledgements

This study was supported by a Grant from the Swedish Research Council (K2006-04X-00715 to K. Fuxe), a Grant from the Spanish Ministry of Education and Science (SAF2005-00170 to C. Lluis and SAF2006-05481 to R. Franco) and by the statutory funds from the Institute of Pharmacology, Polish Academy of Sciences (M. Filip).

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      The direct physical interaction of the two receptors was further supported and confirmed by their functional interactions [155–158]. Although activation of the individual protomers with their respective agonists leads to signaling cascade via Gαi/o, the simultaneous co-activation of both receptors switches the signaling cascade from the Gαi/o to Gαs/olf, leading to altered signaling [63,155,156]. Moreover, recent studies revealed that this heterodimeric specific signaling requires the bidirectional allosteric interaction of the two receptors as lack of activation of either receptor abolishes the observed effect i.e. signal switching and co-internalization [155,156].

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