Characterization of Gαs and Gαolf activation by catechol and non-catechol dopamine D1 receptor agonists

Abstract

The dopamine D1 receptor (D1R) couples to Gα_s and Gα_olf and is crucial in regulating neurological and neuropsychiatric functions. In the brain, Gα_olf is predominantly found in the striatum whereas Gα_s is expressed elsewhere. Our in vitro assays revealed that the tetracyclic catechol agonists dihydrexidine, methyl-dihydrexidine, doxanthrine, and the non-catechol compounds PF-8294, PF-6142 exerted full agonism for Gα_s coupling but only partial agonism for Gα_olf coupling. In contrast, the non-catechol agonist tavapadon acted as a full agonist at Gα_olf and a partial agonist at Gα_s. The effects of these ligands on the thalamocortical and striatonigral electrophysiological events, as well as on the locomotor activity and cognitive function of mice agreed with their selectivity profiles in vitro. These findings suggest the possibility of achieving region-specific pharmacology and open new directions for developing D1R drugs to treat relevant neurological and neuropsychiatric disorders.

Keywords: Molecular biology; Neuroscience.

Graphical abstract

Figure 1

D1R agonist-induced D1R-Gα_s and -Gα_olf engagement Drug-induced BRET ratio change between D1R-Rluc8 and Gα_s-Venus (A–C) or Gα_olf-Venus (D–F) in response to DA (black), SKF 81297 (dark orange), SKF 38393 (light orange) (top row), DHX (blue), m-DHX (light blue), DOX (violet) (middle row), TAV (red), PF-8294 (pink), and PF-6142 (magenta). Results were normalized to the E_max of DA response for D1R-Gα_s (A–C) and D1R-Gα_olf (D–F) and presented as mean ± SEM (n ≥ 5).

Figure 2

D1R agonist-induced D1R-Gα_s and -Gα_olf activation Drug-induced BRET ratio change between γ7-Rluc8 and Gα_s-Venus (A–C) or Gα_olf-Venus (D–F) in response to DA (black), SKF 81297 (dark orange), SKF 38393 (light orange) (top row), DHX (blue), m-DHX (light blue), DOX (violet) (middle row), TAV (red), PF-8294 (pink), and PF-6142 (magenta). Results were normalized to the E_max of DA response for D1R-Gα_s (A–C) and D1R-Gα_olf (D–F) and presented as mean ± SEM (n ≥ 5).

Figure 3

D1R agonist-induced cAMP production via Gα_s and Gα_olf Drug-induced fluorescence change of the cAMP biosensor Pink Flamindo in D1R-Gα_s (A–C) or D1R-Gα_olf (D–F) in response to DA (black), SKF 81297 (dark orange), SKF 38393 (light orange) (top row), DHX (blue), m-DHX (light blue), DOX (violet) (middle row), TAV (red), PF-8294 (pink), and PF-6142 (magenta). Results were normalized to the E_max of DA response for D1R-Gα_s (A–C) and D1R-Gα_olf (D–F) and presented as mean ± SEM (n ≥ 5).

Figure 4

D1R agonist-induced cAMP production via Gα_olf/s chimera (A) Cryo-EM structure of TAV-bound D1R and mini-Gα_s complex (PDB: 7X2D). (B) The ICL2-αN interface of the D1R-Gα_s complex. Molecular graphics were generated in PyMOL 3.1. Drug-induced fluorescence change of Pink Flamindo detecting cAMP level for DA (black), SKF81297 (orange), SKF38393 (light orange) (C), DHX (blue), m-DHX (light blue), DOX (violet) (D), TAV (red), PF-8294 (pink), and PF-6142 (magenta) (E). Results were normalized to the E_max of DA response for Gα_s, Gα_olf/s chimera (αN), Gα_olf and presented as mean ± SEM (n ≥ 5). Comparison of the E_max across Gα_s, αN, and Gα_olf was done using one-way ANOVA followed by post-hoc Tukey test. ∗, ∗∗, ∗∗∗, ∗∗∗∗p < 0.05, 0.01, 0.001, 0.0001, respectively.

Figure 5

Gα_s and Gα_olf activities in brain slice electrophysiology ChR2-YFP injection in the mediodorsal thalamus projecting to the prelimbic/infralimbic mPFC of D1R-tdTomato mouse (A) and in the dorsal striatum projecting to the SNr of WT mouse (D). Scale bar: 500 μm. Average traces of the optically elicited ChR2-driven EPSCs in the mPFC (B) and IPSCs in the SNr (E) following 2 ms of photostimulation by blue light. Analysis of the D1R-mediated potentiation of EPSCs in the mPFC (C) IPSCs in the SNr (F) for 1.0 μM SKF81297, DHX, and TAV (orange, blue, and red) was done using one-way ANOVA followed by post-hoc Tukey test. ∗, ∗∗, ∗∗∗p < 0.05, 0.01, 0.001 respectively.

Figure 6

D1R agonists effect on locomotor activity (A–C) Psychomotor stimulatory effect of SKF-81297 [S] (A), TAV [T] (B), and DHX [D] (C) at 1, 3, 6 mg/kg (S1, S3, S6 etc.) in combination with 1 mg/kg quinpirole (Q1) on DA-depleted reserpinized mice. ∗, ∗∗, ∗∗∗, ∗∗∗∗p < 0.05, 0.01, 0.001, 0.0001, respectively, compared to the effect of saline using one-way ANOVA followed by post-hoc Dunnett test. (D) Comparison of the ambulatory distance induced by these drugs at 6 mg/kg was done using one-way ANOVA followed by post-hoc Tukey test. ∗, ∗∗, ∗∗∗, ∗∗∗∗p < 0.05, 0.01, 0.001, 0.0001, respectively.

Figure 7

D1R agonists effect on cognitive function using the novel object recognition assay Comparison of the discriminatory index (DI) of WT mice following the administration of SKF-81297 [S6], TAV [T6], and DHX [D6] at 6 mg/kg ∗p < 0.05 compared to the SKF 81297-injected group using one-way ANOVA followed by post-hoc Dunnett test.