Engineered D2R Variants Reveal the Balanced and Biased Contributions of G-Protein and β-Arrestin to Dopamine-Dependent Functions

Abstract

The dopamine D2 receptor (D2R), like many G-protein-coupled receptors, signals through G-protein- and β-arrestin-dependent pathways. Preferential activation of one of these pathways is termed functional selectivity or biased signaling and is a promising therapeutic strategy. Though biased signaling through D2Rs has been demonstrated, acquiring the mechanistic details of biased D2R/G-protein and D2R/β-arrestin signaling in vivo has been challenging because of the lack of techniques that specifically target these interactions in discrete cell populations. To address this question, we employed a cell type-specific viral expression approach to restore D2R variants that preferentially engage either G-protein or β-arrestin signaling in 'indirect pathway' medium spiny neurons (iMSNs), because of their central role in dopamine circuitry. We found that the effect of haloperidol antagonism on D2R metabolic signaling events is largely mediated by acute blockade of D2R/G-protein signaling. We show that a D2R-driven behavior, nestlet shredding, is similarly driven by D2R/G-protein signaling. On the other hand, D2R-driven locomotion and rearing require coordinated D2R/G-protein and D2R/β-arrestin signaling. The acute locomotor response to amphetamine and cocaine similarly depend on both G-protein and β-arrestin D2R signaling. Surprisingly, another psychotropic drug, phencyclidine, displayed a selective D2R/β-arrestin potentiation of locomotion. These findings highlight how D2R mostly relies upon balanced G-protein and β-arrestin signaling in iMSNs. However, the response to haloperidol and phencyclidine indicates that normal D2R signaling homeostasis can be dramatically altered, indicating that targeting a specific D2R signal transduction pathway could allow for more precise modulation of dopamine circuit function.

Figure 1

Viral restoration approach for expressing biased D2R variants in iMSNs. (a) Schematic of a viral transgene packaged in AAV, containing a double-floxed inverted open reading frame. Drd2^flox/flox:: A2Acre (iMSN-D2^KO) mice lack endogenous D2R expression in iMSNs while simultaneously allowing Cre-dependent expression of viral transgenes in iMSNs. AAVs induce expression of HA-tagged D2R variants (purple) and label iMSNs with mCerulean. (b) No colocalization between mCerulean+ and ChAT+ neurons (scale bar=100 μm). (c) mCerulean labels iMSN (striatopallidal) projections in external globus pallidus (GPe), but not dMSN (striatonigral) projections in substrantia nigra pars reticulate (SNr) (scale bar=500 μm). (d) Representative images of mCerulean (top) and HA immunostaining (bottom) in the rostral striatum of iMSN-D2^AAV-WT, iMSN-D2^AAV-βarr, iMSN-D2^AAV-Gprot, and iMSN-D2^AAV-D80A mice (scale bar=500 μm). (e) The extent that each virus was expressed was quantified by measuring the volume of dorsal and ventral striatum with expression of mCerulean. iMSN-D2^AAV-WT (n=6), iMSN-D2^{AAV- βarr} (n=7), and iMSN-D2^AAV-D80A mice (n=5) showed similar percent striatum with mCerulean, whereas iMSN-D2^AAV-Gprot (n=7) showed slightly less striatal volume with expression (ANOVA with Bonferroni post hoc test, *p<0.05). (f) HA immunostaining intensity was quantified as a measure of D2R expression. Whereas iMSN-D2^AAV-WT (n= 6), iMSN-D2^{AAV- βarr} (n=7), and iMSN-D2^AAV-D80A mice (n=5) do not differ, HA immunostaining intensity was significantly reduced in iMSN-D2^AAV-Gprot (n=7) (ANOVA with Bonferroni post hoc test, ***p<0.001). Values represent mean±SEM.

Figure 2

D2R antagonist-induced signaling is mostly mediated by D2R/G-protein interaction. The five commonly used cellular response markers were assessed 1 h following i.p. injection of 0.5 mg/kg haloperidol (n=6 iMSN-D2^AAV-WT, n=5 iMSN-D2^AAV-Gprot, n=5 iMSN-D2^AAV-βarr, and n=4 iMSN-D2^AAV-D80A), 2 mg/kg haloperidol (n=5 iMSN-D2^AAV-WT, n=5 iMSN-D2^AAV-Gprot, n=5 iMSN-D2^AAV-βarr, and n=4 iMSN-D2^AAV-D80A), or vehicle (n=4 iMSN-D2^AAV-WT, n=5 iMSN-D2^AAV-Gprot, n=3 iMSN-D2^AAV-βarr, and n=3 iMSN-D2^AAV-D80A). mCerulean+ iMSNs in the dorsal striatum that were immunopositive for the cellular response marker were counted and are expressed as a percentage of total mCerulean+ iMSNs. Activation was defined as a significant increase over iMSN-D2^AAV-D80A mice. Representative images of vehicle and 2 mg/kg haloperidol conditions are shown (scale bar=50 μm). (a) The 0.5 mg/kg haloperidol caused c-fos expression in iMSN-D2^AAV-WT mice (two-way ANOVA of viral genotype × drug dose interaction with Bonferroni post hoc test, **p<0.01), but not in iMSN-D2^AAV-βarr or iMSN-D2^AAV-Gprot mice. Similarly, 2 mg/kg haloperidol caused c-fos expression in iMSN-D2^AAV-WT mice (***p<0.001), but not in iMSN-D2^AAV-βarr or iMSN-D2^AAV-Gprot mice. (b) The 0.5 mg/kg haloperidol increased S6 phosphorylation in iMSN-D2^AAV-WT mice (two-way ANOVA of viral genotype × drug dose interaction with Bonferroni post hoc test, **p<0.01), but not in iMSN-D2^AAV-βarr or iMSN-D2^AAV-Gprot mice. The 2 mg/kg haloperidol increased S6 phosphorylation in iMSN-D2^AAV-WT mice (***p<0.001) and iMSN-D2^AAV-Gprot mice (**p<0.01) but not in iMSN-D2^AAV-βarr mice. (c) The 0.5 mg/kg haloperidol did not increase H3 phosphorylation in any of the viral genotypes. Alternatively, 2 mg/kg haloperidol increased H3 phosphorylation in iMSN-D2^AAV-Gprot mice (two-way ANOVA of viral genotype × drug dose interaction with Bonferroni post hoc test, **p<0.01), but not in iMSN-D2^AAV-WT or iMSN-D2^AAV-βarr mice. (d) The 0.5 mg/kg haloperidol did not increase egr-1 expression in any of the viral genotypes. The 2 mg/kg haloperidol, however, increased egr-1 expression in iMSN-D2^AAV-WT mice (two-way ANOVA on effect of viral genotype with Bonferroni post hoc test, **p<0.01), iMSN-D2^AAV-Gprot mice (*p<0.05), and iMSN-D2^AAV-βarr mice (*p<0.05). (e) The 0.5 mg/kg did not increase c-Jun expression in any of the viral genotypes. The 2 mg/kg haloperidol induced c-Jun expression in iMSN-D2^AAV-WT mice (two-way ANOVA on viral genotype × drug dose interaction with Bonferroni post hoc test, ***p<0.001), and iMSN-D2^AAV-Gprot (***p<0.001). Values represent mean±SEM.

Figure 3

PCA in the cell activation markers over iMSN-D2^AAV-D80A following 2 mg/kg haloperidol in iMSN-D2^AAV-WT (n=5), iMSN-D2^AAV-βarr (n=5), and iMSN-D2^AAV-Gprot mice (n=5). PC1 distinguishes the iMSN-D2^AAV-βarr from iMSN-D2^AAV-WT and iMSN-D2^AAV-Gprot. PC2 distinguishes iMSN-D2^AAV-WT from iMSN-D2^AAV-Gprot. Points represent the signaling responses of individual animals. White=iMSN-D2^AAV-WT, red=iMSN-D2^AAV-βarr, and green=iMSN-D2^AAV-Gprot mice.

Figure 4

Examination of iMSN-D2R-biased signaling pathways in basic D2R-driven behaviors. (a) iMSN-D2^KO and Cre- littermate controls were tested in a novel open field for 1 h (n=8/genotype). Locomotion was significantly reduced in iMSN-D2 ^KO (two-way repeated-measures ANOVA of genotype × time interaction with Bonferroni post hoc test, ***p<0.001). (b) Next, iMSN-D2^AAV-WT (n=10), iMSN-D2^AAV-Gprot (n=12), iMSN-D2^AAV-βarr (n=12), and iMSN-D2^AAV-D80A (n=9) were tested in the novel open field. iMSN-D2^AAV-WT showed restoration of open-field locomotion compared with iMSN-D2^AAV-D80A (two-way repeated-measures ANOVA on effect of time with Bonferroni post hoc test, *p<0.05). iMSN-D2^AAV-Gprot and iMSN-D2^AAV-βarr mice showed intermediate restoration effects, with no significant difference found between these viral genotypes and iMSN-D2^AAV-WT or iMSN-D2^AAV-D80A mice. (c) Rearing was assessed concurrently to locomotion. Rearing was significantly reduced in iMSN-D2^KO compared with Cre- littermates (Student’s t-test, **p<0.01). (d) Rearing showed nonsignificant trend toward restoration in iMSN-D2^AAV-WT compared with iMSN-D2^AAV-D80A mice (ANOVA with Bonferroni post hoc test p=0.06), but not in iMSN-D2^AAV-Gprot and iMSN-D2^AAV-βarr mice. (e) Nestlet shredding behavior was assessed in iMSN-D2^KO and Cre- littermate controls. iMSN-D2^KO showed a reduction in this behavior (Student’s t-test, **p<0.01). (f) Nestlet shredding was significantly restored in iMSN-D2^AAV-Gprot mice (ANOVA with Bonferroni post hoc test, *p<0.05). Values represent mean±SEM.

Figure 5

Examination of iMSN-D2R-biased signaling pathways in psychomotor drug effects. (a) iMSN-D2^KO (n=8) and Cre- (n=8) littermate controls were tested for their locomotor response to 3 mg/kg amphetamine. Amphetamine-induced locomotion was significantly reduced in iMSN-D2^KO mice following 3 mg/kg compared with Cre- controls (two-way repeated-measures ANOVA on genotype × time interaction with Bonferroni post hoc tests within time points, ***p<0.001). (b) Next, iMSN-D2^AAV-WT (n=10), iMSN-D2^AAV-Gprot (n=12), iMSN-D2^AAV-βarr (n=12), and iMSN-D2^AAV-D80A (n=9) were tested for amphetamine-induced locomotion. iMSN-D2^AAV-WT mice showed significant restoration of amphetamine-induced locomotion over iMSN-D2^AAV-D80A (two-way repeated-measures ANOVA on viral genotype × time interaction with Bonferroni post hoc tests, *p<0.05 and **p<0.01). iMSN-D2^AAV-Gprot and iMSN-D2^AAV-βarr mice showed intermediate restoration effects, with no significant difference found between these viral genotypes and iMSN-D2^AAV-WT or iMSN-D2^AAV-D80A mice. (c) Locomotion was significantly reduced in iMSN-D2^KO following 10 mg/kg cocaine (two-way RMANOVA on genotype × time interaction with Bonferroni post hoc tests, ***p<0.001). (d) iMSN-D2^AAV-WT (n=6), iMSN-D2^AAV-Gprot (n=8), iMSN-D2^AAV-βarr (n=8), and iMSN-D2^AAV-D80A (n=6) were similarly tested following 10 mg/kg cocaine. The iMSN-D2^AAV-WT, iMSN-D2^AAV-Gprot, and iMSN-D2^AAV-βarr mice all showed significant restoration of cocaine induced locomotion (two-way repeated-measures ANOVA on genotype × time interaction with Bonferroni post hoc tests, **p<0.01 and ***p<0.001). (e) Finally, PCP-induced locomotion was tested in iMSN-D2^KO and Cre- mice that was significantly reduced in iMSN-D2^KO mice (two-way repeated-measures ANOVA on genotype × time interaction with Bonferroni post hoc tests, *p<0.05 and **p<0.001). (f) iMSN-D2^AAV-βarr mice (n=8) showed significant restoration of PCP-induced locomotion over iMSN-D2^AAV-D80A mice (n=6) (two-way repeated-measures ANOVA on genotype × time interaction with Bonferroni post hoc tests, *p<0.05 and **p<0.01 Bonferroni post hoc tests). iMSN-D2^AAV-WT (n=6) and iMSN-D2^AAV-Gprot mice (n=8), however, only showed intermediate restoration, not significantly differing from iMSN-D2^AAV-D80A mice. Values represent mean±SEM.