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Effect of a toggle switch mutation in TM6 of the human adenosine A₃ receptor on Gi protein-dependent signalling and Gi-independent receptor internalization.

Stoddart LA, Kellam B, Briddon SJ, Hill SJ - Br. J. Pharmacol. (2014)

Bottom Line: We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A₃ receptor.Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor.Investigation of the pharmacology of the W6.48F mutant of the adenosine A₃ receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, School of Life Sciences, The University of Nottingham, Nottingham, UK.

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Visualization and quantitative analysis of agonist-stimulated A3 receptor or A3 W243F receptor BiFC with β-arrestin2. (A) Confocal images of A3-vYc/β-arrestin2-vYnL- (top panels) and A3 W243F-vYc/β-arrestin2-vYnL- (bottom panels) expressing cells were obtained in the absence (left-hand panels) of agonist. Cells were stimulated with 10 μM NECA (middle panels) or 10 μM HEMADO for 60 min (left-hand panels). Images were obtained using a Zeiss 710 confocal microscope and are representative of those obtained in three separate experiments. Quantitative analysis of agonist-stimulated BiFC was carried out on images obtained from the ImageXpress Ultra confocal plate reader. A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with increasing concentrations of (B) NECA or (C) HEMADO for 60 min before imaging and automated image analysis as described in the Methods section. To examine the time course of BiFC formation, A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with 10 μM NECA (D) or HEMADO (E) for increasing amounts of time (2.5–120 min). Each data point represents the mean ± SEM of at least five separate experiments performed in triplicate.
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fig10: Visualization and quantitative analysis of agonist-stimulated A3 receptor or A3 W243F receptor BiFC with β-arrestin2. (A) Confocal images of A3-vYc/β-arrestin2-vYnL- (top panels) and A3 W243F-vYc/β-arrestin2-vYnL- (bottom panels) expressing cells were obtained in the absence (left-hand panels) of agonist. Cells were stimulated with 10 μM NECA (middle panels) or 10 μM HEMADO for 60 min (left-hand panels). Images were obtained using a Zeiss 710 confocal microscope and are representative of those obtained in three separate experiments. Quantitative analysis of agonist-stimulated BiFC was carried out on images obtained from the ImageXpress Ultra confocal plate reader. A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with increasing concentrations of (B) NECA or (C) HEMADO for 60 min before imaging and automated image analysis as described in the Methods section. To examine the time course of BiFC formation, A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with 10 μM NECA (D) or HEMADO (E) for increasing amounts of time (2.5–120 min). Each data point represents the mean ± SEM of at least five separate experiments performed in triplicate.

Mentions: Subtle differences in the efficacies of NECA and HEMADO at the signalling pathways tested thus far, and the inability of HEMADO to stimulate sequestration of A3 W243F-YFP from the cell surface, raises the possibility that these agonists may differentially recruit β-arrestin2 to the receptor. To quantify β-arrestin2 recruitment, we used a BiFC approach to trap receptor-β-arrestin complexes through interaction between two fragments of venus YFP (vYFP) and subsequent visualization of the resulting mature vYFP chromophore (Rose et al., 2010). We generated stable CHO cell lines expressing β-arrestin2-vYnL (residues 1-173 of vYFP) in combination with A3-vYc fusion protein (residues 155-238 of vYFP) or A3-vYc containing the W243F point mutation (A3 W243F-vYc). Confocal imaging of the resulting cell lines indicated low levels of BiFC under control conditions in both A3-vYc/β-arrestin2-vYnL and A3 W234F-vYc/β-arrestin2-vYnL cell lines, suggesting some degree of basal receptor-β-arrestin association (Figure 10). Both NECA and HEMADO caused a substantial increase in BiFC fluorescence in both cell lines, which was localized in perinuclear compartments indicating internalized receptor-β-arrestin complexes (Figure 10). To investigate the time- and concentration-dependence of A3-vYc/β-arrestin2-vYnL and A3 W243F-vYc/β-arrestin2-vYnL BiFC to both agonists, quantification of BiFC was carried out using a MD ImageXpress Ultra confocal plate reader. NECA-stimulated receptor β-arrestin BiFC with similar potency in both A3-vYc/β-arrestin2-vYnL- and A3 W243F-vYc/β-arrestin2-vYnL-expressing cells (Table 3). HEMADO also caused a concentration-dependent increase in BiFC although with significantly reduced maximal levels of BiFC compared with NECA (Figure 10 and Table 3). BiFC occurs rapidly when the two fragments of the fluorescent protein are within close proximity (Rose et al., 2010); therefore, it can be used to examine the recruitment of β-arrestin by agonist-stimulated receptor. Both NECA and HEMADO stimulated the formation of receptor/β-arrestin complexes rapidly in both cell lines. In addition, longer exposure to HEMADO did not increase the maximal BiFC observed in both A3-vYc/β-arrestin2-vYnL and A3 W243F-vYc/β-arrestin2-vYnL-expressing cells (% maximal internalization vs. 10 μM NECA at 2 h, 84.3 ± 8.2 and 58.0 ± 5.8 for A3-vYc/β-arrestin2-vYnL and A3 W243F-vYc/β-arrestin2-vYnL BiFC, respectively, P > 0.05, Student's unpaired t-test vs. maximal internalization at 60 min).


Effect of a toggle switch mutation in TM6 of the human adenosine A₃ receptor on Gi protein-dependent signalling and Gi-independent receptor internalization.

Stoddart LA, Kellam B, Briddon SJ, Hill SJ - Br. J. Pharmacol. (2014)

Visualization and quantitative analysis of agonist-stimulated A3 receptor or A3 W243F receptor BiFC with β-arrestin2. (A) Confocal images of A3-vYc/β-arrestin2-vYnL- (top panels) and A3 W243F-vYc/β-arrestin2-vYnL- (bottom panels) expressing cells were obtained in the absence (left-hand panels) of agonist. Cells were stimulated with 10 μM NECA (middle panels) or 10 μM HEMADO for 60 min (left-hand panels). Images were obtained using a Zeiss 710 confocal microscope and are representative of those obtained in three separate experiments. Quantitative analysis of agonist-stimulated BiFC was carried out on images obtained from the ImageXpress Ultra confocal plate reader. A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with increasing concentrations of (B) NECA or (C) HEMADO for 60 min before imaging and automated image analysis as described in the Methods section. To examine the time course of BiFC formation, A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with 10 μM NECA (D) or HEMADO (E) for increasing amounts of time (2.5–120 min). Each data point represents the mean ± SEM of at least five separate experiments performed in triplicate.
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fig10: Visualization and quantitative analysis of agonist-stimulated A3 receptor or A3 W243F receptor BiFC with β-arrestin2. (A) Confocal images of A3-vYc/β-arrestin2-vYnL- (top panels) and A3 W243F-vYc/β-arrestin2-vYnL- (bottom panels) expressing cells were obtained in the absence (left-hand panels) of agonist. Cells were stimulated with 10 μM NECA (middle panels) or 10 μM HEMADO for 60 min (left-hand panels). Images were obtained using a Zeiss 710 confocal microscope and are representative of those obtained in three separate experiments. Quantitative analysis of agonist-stimulated BiFC was carried out on images obtained from the ImageXpress Ultra confocal plate reader. A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with increasing concentrations of (B) NECA or (C) HEMADO for 60 min before imaging and automated image analysis as described in the Methods section. To examine the time course of BiFC formation, A3-vYc/β-arrestin2-vYnL (closed symbols) and A3 W243F-vYc/β-arrestin2-vYnL- (open symbols) expressing cells were stimulated with 10 μM NECA (D) or HEMADO (E) for increasing amounts of time (2.5–120 min). Each data point represents the mean ± SEM of at least five separate experiments performed in triplicate.
Mentions: Subtle differences in the efficacies of NECA and HEMADO at the signalling pathways tested thus far, and the inability of HEMADO to stimulate sequestration of A3 W243F-YFP from the cell surface, raises the possibility that these agonists may differentially recruit β-arrestin2 to the receptor. To quantify β-arrestin2 recruitment, we used a BiFC approach to trap receptor-β-arrestin complexes through interaction between two fragments of venus YFP (vYFP) and subsequent visualization of the resulting mature vYFP chromophore (Rose et al., 2010). We generated stable CHO cell lines expressing β-arrestin2-vYnL (residues 1-173 of vYFP) in combination with A3-vYc fusion protein (residues 155-238 of vYFP) or A3-vYc containing the W243F point mutation (A3 W243F-vYc). Confocal imaging of the resulting cell lines indicated low levels of BiFC under control conditions in both A3-vYc/β-arrestin2-vYnL and A3 W234F-vYc/β-arrestin2-vYnL cell lines, suggesting some degree of basal receptor-β-arrestin association (Figure 10). Both NECA and HEMADO caused a substantial increase in BiFC fluorescence in both cell lines, which was localized in perinuclear compartments indicating internalized receptor-β-arrestin complexes (Figure 10). To investigate the time- and concentration-dependence of A3-vYc/β-arrestin2-vYnL and A3 W243F-vYc/β-arrestin2-vYnL BiFC to both agonists, quantification of BiFC was carried out using a MD ImageXpress Ultra confocal plate reader. NECA-stimulated receptor β-arrestin BiFC with similar potency in both A3-vYc/β-arrestin2-vYnL- and A3 W243F-vYc/β-arrestin2-vYnL-expressing cells (Table 3). HEMADO also caused a concentration-dependent increase in BiFC although with significantly reduced maximal levels of BiFC compared with NECA (Figure 10 and Table 3). BiFC occurs rapidly when the two fragments of the fluorescent protein are within close proximity (Rose et al., 2010); therefore, it can be used to examine the recruitment of β-arrestin by agonist-stimulated receptor. Both NECA and HEMADO stimulated the formation of receptor/β-arrestin complexes rapidly in both cell lines. In addition, longer exposure to HEMADO did not increase the maximal BiFC observed in both A3-vYc/β-arrestin2-vYnL and A3 W243F-vYc/β-arrestin2-vYnL-expressing cells (% maximal internalization vs. 10 μM NECA at 2 h, 84.3 ± 8.2 and 58.0 ± 5.8 for A3-vYc/β-arrestin2-vYnL and A3 W243F-vYc/β-arrestin2-vYnL BiFC, respectively, P > 0.05, Student's unpaired t-test vs. maximal internalization at 60 min).

Bottom Line: We set out to characterize the effect of this mutation on the efficacy of two agonists at multiple signalling pathways downstream of the adenosine A₃ receptor.Mutation of W6.48F therefore resulted in differential effects on agonist efficacy, and introduced signalling pathway bias for HEMADO at the adenosine A3 receptor.Investigation of the pharmacology of the W6.48F mutant of the adenosine A₃ receptor confirms that this region is important in forming the active conformation of the receptor for stimulating a number of different signalling pathways and that mutations in this residue can lead to changes in agonist efficacy and signalling bias.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, School of Life Sciences, The University of Nottingham, Nottingham, UK.

Show MeSH
Related in: MedlinePlus