Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction is a common property of clinically effective antipsychotics - PubMed (original) (raw)

Antagonism of dopamine D2 receptor/beta-arrestin 2 interaction is a common property of clinically effective antipsychotics

Bernard Masri et al. Proc Natl Acad Sci U S A. 2008.

Abstract

Since the unexpected discovery of the antipsychotic activity of chlorpromazine, a variety of therapeutic agents have been developed for the treatment of schizophrenia. Despite differences in their activities at various neurotransmitter systems, all clinically effective antipsychotics share the ability to interact with D2 class dopamine receptors (D2R). D2R mediate their physiological effects via both G protein-dependent and independent (beta-arrestin 2-dependent) signaling, but the role of these D2R-mediated signaling events in the actions of antipsychotics remains unclear. We demonstrate here that while different classes of antipsychotics have complex pharmacological profiles at G protein-dependent D2R long isoform (D2(L)R) signaling, they share the common property of antagonizing dopamine-mediated interaction of D2(L)R with beta-arrestin 2. Using two cellular assays based on a bioluminescence resonance energy transfer (BRET) approach, we demonstrate that a series of antipsychotics including haloperidol, clozapine, aripiprazole, chlorpromazine, quetiapine, olanzapine, risperidone, and ziprasidone all potently antagonize the beta-arrestin 2 recruitment to D2(L)R induced by quinpirole. However, these antipsychotics have various effects on D2(L)R mediated G(i/o) protein activation ranging from inverse to partial agonists and antagonists with highly variable efficacies and potencies at quinpirole-induced cAMP inhibition. These results suggest that the different classes of clinically effective antipsychotics share a common molecular mechanism involving inhibition of D2(L)R/beta-arrestin 2 mediated signaling. Thus, selective targeting of D2(L)R/beta-arrestin 2 interaction and related signaling pathways may provide new opportunities for antipsychotic development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

BRET measurements of cAMP levels in living cells to monitor dopamine D2L receptor activation. (A and B) Variations of cAMP levels in HEK293 cells stably expressing the EPAC biosensor and D2LR. (A) Kinetics of cAMP variations after treatment of the cells at time 0 with forskolin (25 μM) in the absence (▵) or presence (■) of quinpirole 1 μM as indicated and the emission ratios (_R_luc/YFP) were measured. Cells incubated in PBS (○) were used as a negative control. (B) Dose-response curve of quinpirole for inhibiting forskolin-stimulated cAMP accumulation in HEK cells. cAMP production was normalized to the percentage of forskolin-stimulated cAMP accumulation (set at 100%).

Fig. 2.

Fig. 2.

BRET measurements of β-arrestin 2 recruitment to dopamine D2L receptor in living cells. (A) BRET titration curve for the β-arrestin 2 recruitment to D2LR. BRET was measured in cells expressing a fixed amount of _R_luc-tagged D2LR and increasing amounts of YFP-tagged β-arrestin 2, treated (with dopamine 1 μM ▾ or quinpirole 1 μM (■) or not (♦). Specificity of the BRET signal between D2LR and the β-arrestin 2 was controlled by measuring BRET between _R_luc-tagged D2LR and YFP alone, treated or not with dopamine and quinpirole (see

Fig. S2

). (B) Kinetic of β-arrestin 2 recruitment to D2LR after addition, at time 0, of quinpirole 1 μM (■) or PBS (○). As a negative control, similar experiments were carried out on cells transiently expressing only _R_luc-tagged D2LR. (C) Dose-response curve of quinpirole for β-arrestin 2 recruitment to D2LR. Cells coexpressing _R_luc-tagged D2LR and YFP-tagged β-arrestin 2 were stimulated with dopamine (○) or quinpirole (■). Results are expressed in Net BRET as described in the Methods section. Data represent the mean ± SEM of 3 to 7 independent experiments each performed in duplicate.

Fig. 3.

Fig. 3.

Intrinsic activity of haloperidol, clozapine, and aripiprazole on Gi/o activation and D2LR -mediated β-arrestin 2 recruitment. (A) Dose–response of haloperidol, clozapine, and aripiprazole on adenylylcyclase inhibition through D2R. As described in Methods section, HEK 293 cells stably expressing EPAC biosensor and D2R were stimulated with haloperidol (Upper Left), clozapine (Middle Left) or aripiprazole (Bottom Left) in presence of forskolin (25 μM). cAMP production was normalized to the percentage of forskolin-stimulated cAMP accumulation (set at 100%). (B) BRET measured in cells coexpressing _R_luc-tagged D2LR and YFP-tagged β-arrestin 2 and stimulated with haloperidol (Upper Right), clozapine and aripiprazole (Middle and Bottom Right, respectively). Data are expressed as the percentage of quinpirole maximum effect (1 μM) and represent the mean ± SEM of 3–5 independent experiments each performed in duplicate.

Fig. 4.

Fig. 4.

Antagonist activity of haloperidol, clozapine, and aripiprazole on Gi/o activation and β-arrestin 2 recruitment induced by quinpirole. (A) Dose-response curve for haloperidol, clozapine and aripiprazole (Upper, Middle, and Bottom Left) for inhibiting adenylylcyclase inhibition induced by quinpirole. HEK 293 cells stably coexpressing EPAC biosensor and D2LR were treated with the different antipsychotics and quinpirole (1 μM) in the presence of forskolin (25 μM). (B) Dose-response curve for haloperidol (Upper Right), clozapine and aripiprazole (M_iddle and Bottom Right_, respectively) for inhibiting β-arrestin 2 recruitment induced by 1 μM of quinpirole. BRET was measured in cells coexpressing _R_luc-tagged D2LR and YFP-tagged β-arrestin 2. Data represent the mean ± SEM of 3–5 independent experiments each performed in duplicate.

Fig. 5.

Fig. 5.

β-arrestin 2-YFP translocation to D2LR. Fluorescence microscopy was used to visualize β-arrestin 2-YFP recruitment to the human D2R long form in cells incubated for 30 min with: no drug (Ctr), haloperidol (10 μM) (Halo), clozapine (10 μM) (Cloza), aripiprazole (10 μM) (Ari), quinpirole (10 μM) (Quin). For antagonistic activity (Bottom), cells were pretreated with the different antipsychotics for 30 min and then stimulated for 30 min with quinpirole (10 μM). Representative pictures of each condition are shown (n = 3).

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