Bias analyses of preclinical and clinical D2 dopamine ligands: studies with immediate and complex signaling pathways

Abstract

G protein-coupled receptors (GPCRs) often activate multiple signaling pathways, and ligands may evoke functional responses through individual pathways. These unique responses provide opportunities for biased or functionally selective ligands to preferentially modulate one signaling pathway over another. Studies with several GPCRs have suggested that selective activation of signaling pathways downstream of a GPCR may lead to safer and more effective drug therapies. The dopamine D2 receptor (D2R) is one of the main drug targets in the therapies for Parkinson's disease and schizophrenia. Recent studies suggest that selective modulation of individual signaling pathways downstream of the D2R may lead to safer antipsychotic drugs. In the present study, immediate effectors of the D2R (i.e., Gαi/o, Gβγ, β-arrestin recruitment) and more complex signaling pathways (i.e., extracellular signal-regulated kinase phosphorylation, heterologous sensitization, and dynamic mass redistribution) were examined in response to a series of D2R ligands. This was accomplished using Chinese hamster ovary cells stably expressing the human D2L dopamine receptor in the PathHunter β-Arrestin GPCR Assay Platform. The use of a uniform cellular background was designed to eliminate potential confounds associated with cell-to-cell variability, including expression levels of receptor as well as other components of signal transduction, including G protein subunits. Several well characterized and clinically relevant D2R ligands were evaluated across each signaling pathway in this cellular model. The most commonly used methods to measure ligand bias were compared. Functional selectivity analyses were also used as tools to explore the relative contribution of immediate D2R effectors for the activation of more complex signaling pathways.

Fig. 1.

Activation of three signaling pathways downstream of the D2R in CHO-D_2L cells. (A) Recruitment of β-arrestin to the D2R was measured using the PathHunter assay from DiscoveRx. (B) Activation of Gα_i/o by the D2R was measured by assessing inhibition of forskolin-mediated cAMP production. (C) Activation of Gβγ by the D2R was assessed by measuring potentiation of PMA-stimulated cAMP accumulation. Data represent the average and S.E.M. of at least three independent experiments.

Fig. 2.

Bias analyses of β-arrestin recruitment in comparison with Gα_i/o activation by the D2R. (A) Equimolar comparison. (B) Equiactive comparison. (C) Transduction coefficient. (D) Sigma comparison. Dopamine was used as the reference compound for all the analyses. For the quantitative analyses positive values indicate bias for β-arrestin; negative values indicate bias for Gα_i/o. Data represent the average and S.E.M. of at least three independent experiments. *P < 0.05.

Fig. 3.

Bias analyses of Gβγ in comparison with Gα_i/o activation by the D2R. (A) Equimolar comparison. (B) Equiactive comparison. (C) Transduction coefficient. (D) Sigma comparison. Dopamine was used as the reference compound for all the analyses. For the quantitative analyses positive values indicate bias for Gβγ; negative values indicate bias for Gα_i/o. Data represent the average and S.E.M. of at least three independent experiments. *P < 0.05.

Fig. 4.

Activation of complex signaling pathways downstream of the D2R. (A) Heterologous sensitization was assessed by pretreating the cells with the D2R ligand for 2 hours and then stimulating them with forskolin. (B) ERK phosphorylation was measured after treating the cells with the D2R ligand for 10 minutes using Cisbio’s Cellul’erk kit. (C) Dynamic mass redistribution was measured during stimulation with D₂ ligands, and the maximal peak height was determined. Data represent the average and S.E.M. of at least three independent experiments.

Fig. 5.

Bias analyses using the equiactive comparison and transduction coefficients of heterologous sensitization in comparison with effectors of the D2R. (A) Equiactive comparison of heterologous sensitization and Gα_i/o activation. (B) Equiactive comparison of heterologous sensitization and Gβγ activation. (C) Equiactive comparison of heterologous sensitization and β-arrestin recruitment. (D) Analyses using the transduction coefficients of heterologous sensitization in comparison with Gα_i/o activation. (E) Analyses using the transduction coefficients of heterologous sensitization in comparison with Gβγ activation. (F) Analyses using the transduction coefficients of heterologous sensitization in comparison with β-arrestin recruitment. Dopamine was used as the reference compound for all the analyses. Positive values indicate bias for heterologous sensitization; negative values indicate bias for the D2R effector under analysis. Data represent the average and S.E.M. of at least three independent experiments. *P < 0.05.

Fig. 6.

Bias analyses using the equiactive comparison and transduction coefficients of ERK phosphorylation in comparison with effectors of the D2R. (A) Equiactive comparison of ERK phosphorylation and Gα_i/o activation. (B) Equiactive comparison of ERK phosphorylation and Gβγ activation. (C) Equiactive comparison of ERK phosphorylation and β-arrestin recruitment. (D) Analyses using the transduction coefficients of ERK phosphorylation in comparison with Gα_i/o activation. (E) Analyses using the transduction coefficients of ERK phosphorylation in comparison with Gβγ activation. (F) Analyses using the transduction coefficients of ERK phosphorylation in comparison with β-arrestin recruitment. Dopamine was used as the reference compound for all the analyses. Positive values indicate bias for ERK phosphorylation; negative values indicate bias for the immediate D2R effector under analysis. Data represent the average and S.E.M. of at least three independent experiments. *P < 0.05.

Fig. 7.

Bias analyses using the equiactive comparison and transduction coefficients of DMR in comparison with effectors of the D2R. (A) Equiactive comparison of DMR and Gα_i/o activation. (B) Equiactive comparison of DMR and Gβγ activation. (C) Equiactive comparison of DMR and β-arrestin recruitment. (D) Analyses using the transduction coefficients of DMR in comparison with Gα_i/o activation. (E) Analyses using the transduction coefficients of DMR in comparison with Gβγ activation. (F) Analyses using the transduction coefficients of DMR in comparison with β-arrestin recruitment. Dopamine was used as the reference compound for all the analyses. Positive values indicate bias for DMR; negative values indicate bias for the immediate D2R effector under analysis. Data represent the average and S.E.M. of at least three independent experiments. *P < 0.05.