Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists

Abstract

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar(1), Ile(4), Ile(8)]-AngII (SII), and [Sar(1), D-Ala(8)]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between "G protein-dependent" and "β-arrestin-dependent" signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization "G protein-dependent" vs. "β-arrestin-dependent" signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

Keywords: GPCR; biased agonism; kinases; medicinal chemistry; signal transduction.

Figure 1

Schematic representation of (A) a reference agonist and (B) the hypothesis of downstream transduction of a “biased message” after receptor activation by a biased agonist. AngII, angiotensin II; SII, [Sar¹, Ile⁴, Ile⁸]-AngII; TRV, TRV027 ([Sar¹, D-Ala⁸]-AngII).

Figure 2

Analysis of 13 human kinase substrate phosphorylation levels whose phosphorylation status changed after stimulation with AngII, SII or TRV027. (A) Quantification of kinase substrate phosphorylation levels in response to AngII, and two biased agonists for 10 min. For clarity, spot intensities were converted to a false color scale to facilitate the visualization of the differences. (B) Venn diagram representing the set of kinase substrates differentially modulated in the three experimental conditions used. (C) Schematic representation of the obtained data, showing that no clear biased message was observed at the level of kinase substrate phosphorylation after stimulation with biased agonists.

Figure 3

Cluster analysis of target genes differentially modulated by the three ligands assayed. (A) The five gene clusters identified using the Self Organizing Tree Algorithm (Herrero et al., 2001). Fold-changes were calculated comparing to untreated samples and are represented on a log scale. (B) Schematic representation of the obtained data, showing that no clear biased message was observed at the level of gene expression modulation after stimulation with biased agonists.

Figure 4

Analyses of β-arrestin-1 and -2 conformational changes after stimulation with the three ligands assayed. Kinetics of conformational changes for β-arrestin-1 (A) and β-arrestin-2 (B) were evaluated using a fixed concentration of each ligand, the same concentrations used in the kinase phosphorylation array and the PCR array. Concentration-response curves were performed after 10 min stimulation and conformational changes were analyzed for β-arrestin-1 (C) and 2 (D). One-Way ANOVA, p = 0.05: (#) AngII vs. SII; ($) AngII vs. TRV; (Φ) SII vs. TRV; (n.s.) non-significant. One-Way ANOVA was performed using the area under the curve (A,B) or the curve bottom plateaus (C,D).