Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes

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Highlights

  • D2R-5-HT2A heteroreceptor complexes exist in the ventral and dorsal striatum.

  • LSD and DOI but not TCB2 increased the recognition of the D2R protomer.

  • DOI and LSD but not TCB2 enhanced agonist induced increase of D2R protomer signaling.

  • The increases of D2R protomer function by LSD and DOI involves an allosteric mechanisms.

  • The psychotic actions of LSD and DOI involves biased actions at the 5-HT2AR protomer.

Abstract

Dopamine D2LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor–receptor interactions. In the current study the existence of D2L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist 3H-raclopride binding sites and significantly reduced the pKiH values of the high affinity D2R agonist binding sites in 3H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists.

Introduction

Biophysical methods demonstrated dopamine D2LR-serotonin 5-HT2AR heteromers in cellular models after cotransfection of the two receptors with bidirectional receptor–receptor interactions [1], [2]. The 5-HT2AR-mediated phospholipase C activation by 5-HT was synergistically enhanced by the concomitant activation of the D2LR protomer by the D2R agonist quinpirole as shown in a NFAT-luciferase reporter gene assay. A specific and significant elevation of the intracellular calcium levels was also observed when both receptor protomers were simultaneously activated. Conversely, when the D2LR-mediated adenylyl cyclase inhibition by the D2R agonist quinpirole was assayed coagonist stimulation of D2LR and 5-HT2AR protomers by quinpirole and 5-HT, respectively, led to a reduction of the D2LR protomer signaling. These results suggested the existence of a 5-HT2AR-mediated D2LR trans-inhibition phenomenon [1]. The D2LR-5-HT2AR heteromer represents a novel target for antipsychotic drugs since such drugs are well known to exert their therapeutic actions at least with regard to positive symptoms via blockade of D2Rs [3].

Albizu et al. [4] recently observed that activation of D2Rs by quinpirole increases the affinity of the hallucinogenic 5-HT2AR agonist DOI ((±)-2,5-dimethoxy-4-iodoamphetamine) for the 5-HT2AR and reduces the DOI induced Gq/11 coupling to the 5-HT2AR as seen from the diminished production of inositol phosphate by DOI. This is different from the case when the endogenous ligand 5-HT was used to activate the 5-HT2AR [1]. These findings have led us to study if the observed ability of 5-HT to produce an allosteric counteraction of D2R agonist induced D2R signaling via the 5-HT2AR protomer [1] is altered when using the known hallucinogenic high affinity 5-HT2AR agonists lysergic acid diethylamide (LSD) and DOI [5], [6]. The 5-HT receptor agonist activity of LSD in the CNS was first described in 1968 [7]. Such an alteration if enhancing D2R protomer function may contribute to their psychotic actions. The current study tested this hypothesis in HEK293 cells and ventral striatum studying 5-HT2AR-D2R interactions using LSD and DOI and a standard 5-HT2AR agonist TCB2 [8] to modulate D2R binding and signaling. Indications were obtained for the existence of D2R-5-HT2AR heteroreceptor complexes in ventral and dorsal striatum with proximity ligation assays (PLA) [9].

Section snippets

Materials and methods

Detailed descriptions are available in Supplementary Material and Methods on: chemicals, reagents and drugs; antibodies; cell culture and transfection; membrane preparation and immunofluorescence microscopy.

PLA and immunofluorescence experiments

PLA demonstrated the presence of red clusters (blobs) of D2R and 5-HT2AR, representing D2-5-HT2A heteroreceptor complexes in discrete regions of the neuropil of the dorsal striatum and nucleus accumbens core but not in the corpus callosum and the anterior limb of the anterior commissure (Fig. 1). Also no PLA positive clusters were observed in the cortical regions analized at the Bregma level −1.7. These results were validated in HEK293 cells transiently cotransfected with human D2LR and 5-HT2AR

Discussion

Experimental evidence was obtained that the hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 in the nanomolar range produced an enhancement of the D2R agonist induced D2R protomer recognition and signaling. In contrast, the endogenous ligand 5-HT was previously shown to exert an allosteric antagonistic action on D2R signalling in D2LR-5-HT2AR cotransfected HEK293 cells [1]. TCB2 in the range of 10–100 nM which can fully activate the 5-HT2A signaling pathways

Acknowledgments

This work has been supported by the Swedish Medical Research Council (04X-715), Telethon TV3’s La Marató Foundation 2008 and Hjärnfonden to KF; by Grants from the Swedish Royal Academy of Sciences (Stiftelsen B. von Beskows Fond and Stiftelsen Hierta-Retzius stipendiefond) and Karolinska Institutets Forskningsstiftelser 2011 and 2012 to D.O.B-E. D.O.B-E. and W.R-F. belong to “Academia de Biólogos Cubanos” group. George Milicevic is acknowledged for his practical help.

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