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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 interacts specifically with β-arrestin 2. (A) β2-AR-293 cells were transfected with His-tagged PHD2 for 48 hrs and then treated with ISO (10 μM) for the indicated time. The total cell lysates were immunoprecipitated with anti-His antibody (for PHD2). Western blots were performed with antibodies against β2-AR or His (for PHD2). (B) β2-AR-293 cells were cotransfected with His-tagged PHD2 and FLAG-tagged β-arrestin 1 or 2. Forty-eight hours after transfection, these cells were treated as shown in the figure, and then the cell lysates were immunoprecipitated with anti-His antibody (for PHD2). The immunoprecipitates and cell lysates were blotted with the indicated antibodies. (C) β2-AR-293 cells were transiently cotransfected with FLAG-tagged β-arrestin 2 along with the full length PHD2, PHD2ΔMYND or PHD2ΔCD plasmids. Cell lysates were immunoprecipitated with the anti-FLAG antibody for β-arrestin 2. Western blots were performed with the antibody against FLAG (for β-arrestin 2) or GFP (for PHD2 and truncated mutants). (D, E) The cell lysate of β2-AR-293 cells or neonatal rat cardiomyocytes was subjected to immunoprecipitation with anti-IgG or anti-PHD2 antibody, respectively. The immunoprecipitates were then blotted with the indicated antibodies. (F) Purified PHD2 protein was incubated with an equal amount of GST or GST-β-arrestin 2 coupled to GSH-Sepharose. Proteins retained on Sepharose were then detected with the indicated antibodies. A representative image is shown.
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fig03: PHD2 interacts specifically with β-arrestin 2. (A) β2-AR-293 cells were transfected with His-tagged PHD2 for 48 hrs and then treated with ISO (10 μM) for the indicated time. The total cell lysates were immunoprecipitated with anti-His antibody (for PHD2). Western blots were performed with antibodies against β2-AR or His (for PHD2). (B) β2-AR-293 cells were cotransfected with His-tagged PHD2 and FLAG-tagged β-arrestin 1 or 2. Forty-eight hours after transfection, these cells were treated as shown in the figure, and then the cell lysates were immunoprecipitated with anti-His antibody (for PHD2). The immunoprecipitates and cell lysates were blotted with the indicated antibodies. (C) β2-AR-293 cells were transiently cotransfected with FLAG-tagged β-arrestin 2 along with the full length PHD2, PHD2ΔMYND or PHD2ΔCD plasmids. Cell lysates were immunoprecipitated with the anti-FLAG antibody for β-arrestin 2. Western blots were performed with the antibody against FLAG (for β-arrestin 2) or GFP (for PHD2 and truncated mutants). (D, E) The cell lysate of β2-AR-293 cells or neonatal rat cardiomyocytes was subjected to immunoprecipitation with anti-IgG or anti-PHD2 antibody, respectively. The immunoprecipitates were then blotted with the indicated antibodies. (F) Purified PHD2 protein was incubated with an equal amount of GST or GST-β-arrestin 2 coupled to GSH-Sepharose. Proteins retained on Sepharose were then detected with the indicated antibodies. A representative image is shown.

Mentions: Subsequently, we began to explore the mechanism of PHD2 regulation of β2-AR internalization. Previous studies revealed that decreased oxygen availability contributes to the stability of β2-AR in response to agonist stimulation. β2-AR stability is tightly associated with β2-AR responsiveness and also affects the rate of β2-AR internalization [17]. Thus, we first estimated the levels of β2-AR expression during our experiments. Western blots analysis revealed that PHD2 expression did not result in any change in β2-AR expression (Fig. S2). Moreover, no treatment caused HIF-1α induction (Fig. S3), a hypoxia indicator, implicating that all of the experiments were performed in the normoxic state and that changes in β2-AR internalization are not mediated by the actions of HIF-1α but are conferred by PHD2. In addition, consistent with previous study [11], β2-AR-PHD2 association was not detected in β2-AR-293 cells through coimmunoprecipitation experiments (Fig. 3A). Collectively, these results suggest that PHD2 may affect the downstream effectors of β2-AR signalling.


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 interacts specifically with β-arrestin 2. (A) β2-AR-293 cells were transfected with His-tagged PHD2 for 48 hrs and then treated with ISO (10 μM) for the indicated time. The total cell lysates were immunoprecipitated with anti-His antibody (for PHD2). Western blots were performed with antibodies against β2-AR or His (for PHD2). (B) β2-AR-293 cells were cotransfected with His-tagged PHD2 and FLAG-tagged β-arrestin 1 or 2. Forty-eight hours after transfection, these cells were treated as shown in the figure, and then the cell lysates were immunoprecipitated with anti-His antibody (for PHD2). The immunoprecipitates and cell lysates were blotted with the indicated antibodies. (C) β2-AR-293 cells were transiently cotransfected with FLAG-tagged β-arrestin 2 along with the full length PHD2, PHD2ΔMYND or PHD2ΔCD plasmids. Cell lysates were immunoprecipitated with the anti-FLAG antibody for β-arrestin 2. Western blots were performed with the antibody against FLAG (for β-arrestin 2) or GFP (for PHD2 and truncated mutants). (D, E) The cell lysate of β2-AR-293 cells or neonatal rat cardiomyocytes was subjected to immunoprecipitation with anti-IgG or anti-PHD2 antibody, respectively. The immunoprecipitates were then blotted with the indicated antibodies. (F) Purified PHD2 protein was incubated with an equal amount of GST or GST-β-arrestin 2 coupled to GSH-Sepharose. Proteins retained on Sepharose were then detected with the indicated antibodies. A representative image is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4373440&req=5

fig03: PHD2 interacts specifically with β-arrestin 2. (A) β2-AR-293 cells were transfected with His-tagged PHD2 for 48 hrs and then treated with ISO (10 μM) for the indicated time. The total cell lysates were immunoprecipitated with anti-His antibody (for PHD2). Western blots were performed with antibodies against β2-AR or His (for PHD2). (B) β2-AR-293 cells were cotransfected with His-tagged PHD2 and FLAG-tagged β-arrestin 1 or 2. Forty-eight hours after transfection, these cells were treated as shown in the figure, and then the cell lysates were immunoprecipitated with anti-His antibody (for PHD2). The immunoprecipitates and cell lysates were blotted with the indicated antibodies. (C) β2-AR-293 cells were transiently cotransfected with FLAG-tagged β-arrestin 2 along with the full length PHD2, PHD2ΔMYND or PHD2ΔCD plasmids. Cell lysates were immunoprecipitated with the anti-FLAG antibody for β-arrestin 2. Western blots were performed with the antibody against FLAG (for β-arrestin 2) or GFP (for PHD2 and truncated mutants). (D, E) The cell lysate of β2-AR-293 cells or neonatal rat cardiomyocytes was subjected to immunoprecipitation with anti-IgG or anti-PHD2 antibody, respectively. The immunoprecipitates were then blotted with the indicated antibodies. (F) Purified PHD2 protein was incubated with an equal amount of GST or GST-β-arrestin 2 coupled to GSH-Sepharose. Proteins retained on Sepharose were then detected with the indicated antibodies. A representative image is shown.
Mentions: Subsequently, we began to explore the mechanism of PHD2 regulation of β2-AR internalization. Previous studies revealed that decreased oxygen availability contributes to the stability of β2-AR in response to agonist stimulation. β2-AR stability is tightly associated with β2-AR responsiveness and also affects the rate of β2-AR internalization [17]. Thus, we first estimated the levels of β2-AR expression during our experiments. Western blots analysis revealed that PHD2 expression did not result in any change in β2-AR expression (Fig. S2). Moreover, no treatment caused HIF-1α induction (Fig. S3), a hypoxia indicator, implicating that all of the experiments were performed in the normoxic state and that changes in β2-AR internalization are not mediated by the actions of HIF-1α but are conferred by PHD2. In addition, consistent with previous study [11], β2-AR-PHD2 association was not detected in β2-AR-293 cells through coimmunoprecipitation experiments (Fig. 3A). Collectively, these results suggest that PHD2 may affect the downstream effectors of β2-AR signalling.

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