Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation

Abstract

The dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor's downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G protein-dependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in "indirect pathway" medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wild-type D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wild-type D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics.

Figure 1.. An extremely arrestin-biased D2R mutant receptor, D2R(L123W:R132L).

A) Snake-plot representation of D2R highlighting mutated residues R132 (red) to L and L123 (blue) to W in transmembrane segment 3 (TM3). B) Ribbon representation of D2R co-crystallized with the D2-like receptor antagonist risperidone. Risperidone (orange) is shown bound to the DA-binding orthosteric binding site (OBS) of the receptor, which is formed by the TM bundle and lies above the mutated residues R132 (red) and L123 (blue). C) Schematic representation of a bioluminescence resonance energy transfer (BRET)-based assay that measures agonist-induced recruitment of Gα_i1 (α) fused to mVenus (V) in its α-helical domain to D2R fused to Renilla luciferase 8 (RL) at its C-terminus (inset). Gα_i1-recruitment to D2R-WT, D2R(R132L), D2R(L123W), or D2R(L123W:R132L) in response to increasing concentrations of DA. Dose-response curves are representative of independent experiments (n= 6, 6, 3, and 3, respectively) performed in triplicate. D) Schematic representation of a BRET-based assay that measures agonist-induced recruitment of arrestin-3 (AR) fused at its N-terminus to mVenus (V) to D2R-Rluc8 (inset). Arrestin-3- recruitment to D2R-WT, D2R(R132L), D2R(L123W), or D2R(L123W:R132L) in response to increasing concentrations of DA (refer to Figure 1C for figure legend). Dose-response curves are representative of independent experiments (n= 6, 6, 3, and 3, respectively) performed in triplicate. E) Surface expression of D2R(R132L), D2R(L123W) and D2R(L123W:R132L) in HEK293T cells as a percentage of D2R-WT. Bars represent the average of six to seven independent experiments. F) Gγ-mVenus recruitment to D2R-WT and D2R(L123W:R132L) with co-expression of pertussis toxin insensitive variants of human Gα_i1–3 or Gα_oA or wildtype human Gα_z in response to 100 μΜ DA. Cells were treated with 100 ng/mL pertussis toxin prior to measurement of response. Responses represent a change in BRET normalized to that of 100 μΜ DA at the D2R-WT with coexpression of Gαi1. Bars represent the average of four to five independent experiments. G) G_i/o/z-mediated inhibition of forskolin (30 μM)-induced cyclic AMP accumulation (cAMPi) for D2R-WT, D2R(R132L), D2R(L123W), and D2R(L123W:R132L) in response to increasing concentrations of DA (refer to Figure 1C for figure legend). Dose-response curves are representative of three independent experiments performed in triplicate. H) The percentage of D2R-WT, D2R(R132L), D2R(L123W), and D2R(L123W:R132L) that internalizes in response to DA (10 μM) after one hour. Bars represent the average of four to six independent experiments. The short isoform of D2R (D2Rs) was used for all experiments with the exception of the Gγ- mVenus recruitment assay. Error bars represent S.E.M.

Figure 2.. Arrestin bias downstream of D2R in iMSNs of the NAc.

A) Schematic representation of AAV1/2-hSyn1-DIO-SF-D2R-IRES-mVenus. The expression cassette is oriented in the forward direction in the presence of Cre-recombinase, which recognizes flox sites (black and white arrows) (top). The AAV was injected into the NAc of Drd2^−/−A2A-Cre mice (bottom). B) Schematic of striatal neurons from mice lacking native D2Rs. D2R-WT or D2R-ARB (red ovals) was selectively expressed in NAc-iMSNs by injecting Cre-dependent AAVs into the ventral striatum of Drd2^−/−A2A-Cre mice. C) AAV-induced expression of EGFP, D2R-WT, or D2R-ARB in NAc four weeks after injection. Shown are D2R immunoreactivity (left) and EGFP (top right) or mVenus (middle, bottom right) fluorescence. Mean D2R immunofluorescence intensity analysis showed similar D2R levels in both D2R groups (D2R-WT: 1.70 ± 0.07 A.U., n=4 mice; D2R-ARB: 1.83 ± 0.07 A.U., n=5 mice; p = 0.21). Scale bar equals 100 μm. (D) Quinpirole- or (E) CP55,940-induced ³⁵S-GTPγS binding in dorsal striatal membranes prepared from Drd2^+/-A2A-Cre (D2R-HET) or Drd2^−/−mice. Drd2^−/−A2A-Cre mice were injected with Cre-dependent AAVs encoding EGFP, D2R-WT, or D2R-ARB. Dose-response curves were fit globally from three independent experiments performed in duplicate. Dose-response curves were normalized to a vehicle control. F) Baseline ³⁵S-GTPγS incorporation did not differ between the samples in assays performed in parallel.

Figure 3.. Selective expression of D2R-ARB in iMSNs of the NAc reverses blunted locomotor responses in D2R-knockout mice.

Time course of distance traveled (cm) by Drd2^−/−A2A-Cre mice expressing EGFP, D2R-WT, or D2R-ARB 90 minutes following the injection of saline on day 1 (A, top), saline on day 2 (B, top), and cocaine (15 mg/kg) on day 3 (C, top). Total distance traveled by EGFP, D2R-WT, or D2RARB mice in an open field over 90 minutes after the injection of saline on day 1 (A, bottom), saline on day 2 (B, bottom), and cocaine on day 3 (C, bottom). One-way ANOVA, Bonferroni. *p<0.05, **p<0.01, EGFP (n=11), D2R-WT (n=11), and D2R-ARB (n=12), respectively. Error bars represent S.E.M.

Figure 4.. Overexpression of an arrestin-biased D2R in the NAc of mice enhances locomotion.

A) Schematic depicting neurons in the striatum that express native D2R (blue ovals). These include iMSNs, cholinergic interneurons (ChIs) as well as dopaminergic (DA) and glutamatergic (GIu) neurons, which express D2Rs at their terminals. D2R-WT or D2R-ARB (red ovals) were upregulated selectively in postsynaptic D2R-expressing iMSNs of D2-Cre mice. B) AAV-induced expression of EGFP, D2R-WT, or D2-ARB in NAc four weeks following injection. Shown are D2R immunoreactivity (left), EGFP (top right), or mVenus (middle, bottom right) fluorescence. Mean D2R immunofluorescence intensity analysis showed similar D2R upregulation in both D2R groups (D2R-WT: 5.70 ± 0.30 A.U., n=3 mice; D2R-ARB: 5.95 ± 0.77 A.U., n=3 mice; p = 0.77). Scale bar equals 100 μm. C, D) Distance traveled (cm) over 90 minutes in an open field by D2-Cre mice expressing EGFP, D2R-WT, or D2R-ARB. One-way ANOVA, Bonferroni. **p<0.01, EGFP (n=10), D2R-WT (n=9), and D2R-ARB (n=8), respectively. Error bars represent S.E.M.

Figure 5.. Overexpression of an arrestin-biased D2R in the NAc of mice does not enhance motivation.

A) Average lever presses in a progressive ratio (PR)-task by D2-Cre mice expressing EGFP, D2R-WT, or D2R-ARB and (B) breakpoint were significantly increased in D2R-WT relative to D2R-ARB or EGFP. One-way ANOVA, Bonferroni. *p<0.05 and ***p<0.001, EGFP (n=10), D2RWT (n=9), and D2R-ARB (n=9), respectively. C) Effect of overexpressing D2R-WT or D2R-ARB on PR session duration. Kaplan-Meier survival function showing the percentage of mice that continue to respond on the PR schedule as a function of session time. Average data of four PR sessions are shown. Log-rank test showed overall group effect (χ²=13.33, p<0.005), and pairwise log-rank tests comparing between groups indicated that D2R-ARB (n=8) and EGFP (n=10) mice lasted significantly less time in the PR session than D2R-WT (n = 8) mice (EGFP vs D2R-WT, χ²=12.54, p<0.0005; D2R-WT vs. D2RARB, χ²=7.407, p=0.0065; EGFP vs. D2R-ARB, χ²=2.222, p=0.1361). D) Press rate, plotted here as a function of the ratio requirement, was not altered. One-way ANOVA, p=0.26, EGFP (n=10), D2R-WT (n=9), and D2R-ARB (n=9), respectively. Error bars represent S.E.M.