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Prolyl hydroxylase 2: a novel regulator of β2 -adrenoceptor internalization.

Yan B, Huo Z, Liu Y, Lin X, Li J, Peng L, Zhao H, Zhou ZN, Liang X, Liu Y, Zhu W, Liang D, Li L, Sun Y, Cui J, Chen YH - J. Cell. Mol. Med. (2011)

Bottom Line: However, it remains to be clarified whether or how PHDs are involved in the regulation of β(2) -adrenoceptor (β(2) -AR) signalling.Here we show that PHD2 can modulate the rate of β(2) -AR internalization through interactions with β-arrestin 2.PHD2 hydroxylates β-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of β-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with β(2) -AR into the cytosol. β(2) -AR internalization is critical to control the temporal and spatial aspects of β(2) -AR signalling.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Arrhythmias, Ministry of Education, China (East Hospital, Tongji University School of Medicine), Shanghai, China.

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PHD2 overexpression retards the recruitment of β-arrestin 2 to the plasma membrane and its co-internalization with β2-AR into the cytosol. (A) β2-AR-293 cells were transfected with β-arrestin 2 with or without PHD2. β-arrestin 2 dynamics upon receptor stimulation were imaged by confocal microscopy and categorized after imaging into three groups: I, cytosolic β-arrestin 2 (resting state); II, β-arrestin 2 translocated to the plasma membrane and clustered with β2-AR at the plasma membrane; and III, β-arrestin 2 co-internalized with β2-AR into the cytosol. Representative images of cells in each state are shown. (B) For quantitative analysis, the percentage of cells conforming to the phenotypes outlined in Fig. 5A was determined 1, 10 or 40 min. after agonist stimulation. The graph represents the mean ± S.E.M. (n = 4) with statistically significant differences marked with * (P < 0.05). (C) β2-AR-293 cells were transfected with PHD2, PHD2 siRNA, or scramble siRNA (Scr siRNA) or left untreated (control) for 48 hrs, and then these cells were lysed. Western blots were conducted to determine the level of β-arrestin 2 expression. GAPDH expression was used as the loading control. β-arrestin 2 expression was determined as the ratio of densitometric value compared to GAPDH expression. Representative immunoblots are shown along with quantitative data showing the mean ± S.E.M. from four separate blots.
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fig05: PHD2 overexpression retards the recruitment of β-arrestin 2 to the plasma membrane and its co-internalization with β2-AR into the cytosol. (A) β2-AR-293 cells were transfected with β-arrestin 2 with or without PHD2. β-arrestin 2 dynamics upon receptor stimulation were imaged by confocal microscopy and categorized after imaging into three groups: I, cytosolic β-arrestin 2 (resting state); II, β-arrestin 2 translocated to the plasma membrane and clustered with β2-AR at the plasma membrane; and III, β-arrestin 2 co-internalized with β2-AR into the cytosol. Representative images of cells in each state are shown. (B) For quantitative analysis, the percentage of cells conforming to the phenotypes outlined in Fig. 5A was determined 1, 10 or 40 min. after agonist stimulation. The graph represents the mean ± S.E.M. (n = 4) with statistically significant differences marked with * (P < 0.05). (C) β2-AR-293 cells were transfected with PHD2, PHD2 siRNA, or scramble siRNA (Scr siRNA) or left untreated (control) for 48 hrs, and then these cells were lysed. Western blots were conducted to determine the level of β-arrestin 2 expression. GAPDH expression was used as the loading control. β-arrestin 2 expression was determined as the ratio of densitometric value compared to GAPDH expression. Representative immunoblots are shown along with quantitative data showing the mean ± S.E.M. from four separate blots.

Mentions: The process of β-arrestin 2-mediated β2-AR internalization involves the initial translocation of β-arrestin 2 to the plasma membrane, the subsequent formation of the membrane cluster, and β-arrestin 2/β2-AR co-internalization into the cytosol [19]. To determine how PHD2 affected β-arrestin 2 movement, we monitored the agonist-dependent trafficking of β-arrestin 2-GFP in β2-AR-293 cells in response to receptor activation (Fig. 5A). Membrane clusters were detected as early as 1 min. after the addition of ISO. Compared to the control group, PHD2 overexpression exhibited delayed formation of membrane clusters (34.44%± 1.92% versus 25.56 ± 3.85%, Fig. 5B). The differences in internal clusters were first observed at 10 min., with a greater internalization percentage in control cells than in PHD2-overexpressing cells (28.89 ± 3.85% versus 20 ± 3.33%). The greatest internalization percentages (46.67 ± 3.33% versus 33.33 ± 3.33%) were observed at 40 min. for both the control and PHD2 overexpressing cells, but the value is significantly higher in control cells. These results support and strengthen the findings observed in Fig. 1A, namely that PHD2 retards β2-AR internalization. During the experiment, Western blots revealed that PHD2 expression level had no effect on the endogenous β-arrestin 2 expression (Fig. 5C). Taken together, these results suggest that PHD2 delays β2-AR internalization by retarding the recruitment of β-arrestin 2 to the plasma membrane and its subsequent co-internalization with β2-AR.


Prolyl hydroxylase 2: a novel regulator of β2 -adrenoceptor internalization.

Yan B, Huo Z, Liu Y, Lin X, Li J, Peng L, Zhao H, Zhou ZN, Liang X, Liu Y, Zhu W, Liang D, Li L, Sun Y, Cui J, Chen YH - J. Cell. Mol. Med. (2011)

PHD2 overexpression retards the recruitment of β-arrestin 2 to the plasma membrane and its co-internalization with β2-AR into the cytosol. (A) β2-AR-293 cells were transfected with β-arrestin 2 with or without PHD2. β-arrestin 2 dynamics upon receptor stimulation were imaged by confocal microscopy and categorized after imaging into three groups: I, cytosolic β-arrestin 2 (resting state); II, β-arrestin 2 translocated to the plasma membrane and clustered with β2-AR at the plasma membrane; and III, β-arrestin 2 co-internalized with β2-AR into the cytosol. Representative images of cells in each state are shown. (B) For quantitative analysis, the percentage of cells conforming to the phenotypes outlined in Fig. 5A was determined 1, 10 or 40 min. after agonist stimulation. The graph represents the mean ± S.E.M. (n = 4) with statistically significant differences marked with * (P < 0.05). (C) β2-AR-293 cells were transfected with PHD2, PHD2 siRNA, or scramble siRNA (Scr siRNA) or left untreated (control) for 48 hrs, and then these cells were lysed. Western blots were conducted to determine the level of β-arrestin 2 expression. GAPDH expression was used as the loading control. β-arrestin 2 expression was determined as the ratio of densitometric value compared to GAPDH expression. Representative immunoblots are shown along with quantitative data showing the mean ± S.E.M. from four separate blots.
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fig05: PHD2 overexpression retards the recruitment of β-arrestin 2 to the plasma membrane and its co-internalization with β2-AR into the cytosol. (A) β2-AR-293 cells were transfected with β-arrestin 2 with or without PHD2. β-arrestin 2 dynamics upon receptor stimulation were imaged by confocal microscopy and categorized after imaging into three groups: I, cytosolic β-arrestin 2 (resting state); II, β-arrestin 2 translocated to the plasma membrane and clustered with β2-AR at the plasma membrane; and III, β-arrestin 2 co-internalized with β2-AR into the cytosol. Representative images of cells in each state are shown. (B) For quantitative analysis, the percentage of cells conforming to the phenotypes outlined in Fig. 5A was determined 1, 10 or 40 min. after agonist stimulation. The graph represents the mean ± S.E.M. (n = 4) with statistically significant differences marked with * (P < 0.05). (C) β2-AR-293 cells were transfected with PHD2, PHD2 siRNA, or scramble siRNA (Scr siRNA) or left untreated (control) for 48 hrs, and then these cells were lysed. Western blots were conducted to determine the level of β-arrestin 2 expression. GAPDH expression was used as the loading control. β-arrestin 2 expression was determined as the ratio of densitometric value compared to GAPDH expression. Representative immunoblots are shown along with quantitative data showing the mean ± S.E.M. from four separate blots.
Mentions: The process of β-arrestin 2-mediated β2-AR internalization involves the initial translocation of β-arrestin 2 to the plasma membrane, the subsequent formation of the membrane cluster, and β-arrestin 2/β2-AR co-internalization into the cytosol [19]. To determine how PHD2 affected β-arrestin 2 movement, we monitored the agonist-dependent trafficking of β-arrestin 2-GFP in β2-AR-293 cells in response to receptor activation (Fig. 5A). Membrane clusters were detected as early as 1 min. after the addition of ISO. Compared to the control group, PHD2 overexpression exhibited delayed formation of membrane clusters (34.44%± 1.92% versus 25.56 ± 3.85%, Fig. 5B). The differences in internal clusters were first observed at 10 min., with a greater internalization percentage in control cells than in PHD2-overexpressing cells (28.89 ± 3.85% versus 20 ± 3.33%). The greatest internalization percentages (46.67 ± 3.33% versus 33.33 ± 3.33%) were observed at 40 min. for both the control and PHD2 overexpressing cells, but the value is significantly higher in control cells. These results support and strengthen the findings observed in Fig. 1A, namely that PHD2 retards β2-AR internalization. During the experiment, Western blots revealed that PHD2 expression level had no effect on the endogenous β-arrestin 2 expression (Fig. 5C). Taken together, these results suggest that PHD2 delays β2-AR internalization by retarding the recruitment of β-arrestin 2 to the plasma membrane and its subsequent co-internalization with β2-AR.

Bottom Line: However, it remains to be clarified whether or how PHDs are involved in the regulation of β(2) -adrenoceptor (β(2) -AR) signalling.Here we show that PHD2 can modulate the rate of β(2) -AR internalization through interactions with β-arrestin 2.PHD2 hydroxylates β-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of β-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with β(2) -AR into the cytosol. β(2) -AR internalization is critical to control the temporal and spatial aspects of β(2) -AR signalling.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Arrhythmias, Ministry of Education, China (East Hospital, Tongji University School of Medicine), Shanghai, China.

Show MeSH