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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 regulates the rate of β2-AR internalization. (A) Overexpression of PHD2, but not PHD1 or PHD3, inhibits β2-AR internalization. β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or the vector (pcDNA3.0) plasmid. Forty-eight hours after transfection, these cells were stimulated with ISO (10 μM) for the indicated time to determine β2-AR internalization as described in ‘Materials and methods’. Internalized receptors are considered to be those removed from the plasma membrane after agonist treatment. (B) β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or scramble (Scram) siRNA for 48 hrs. The expression of each PHD isoform was determined by Western blots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (C) PHD2 silencing promotes β2-AR internalization. Endogenous PHD expression in β2-AR-293 cells was silenced by specific siRNA. Forty-eight hours after transfection, β2-AR was stimulated with ISO (10 μM) for the indicated time to conduct a β2-AR internalization assay. (D) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 or vector virus (control) for 48 hrs, and then these cells were stimulated with ISO for the indicated time to determine β2-AR internalization. (E) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 shRNA-lentiviral constructs or vector virus (control), and then these cells were stimulated with ISO for the indicated time for β2-AR internalization assay. The data are shown as the means ± S.E.M. of representative experiments performed at least three times in triplicate. Asterisk (*) denotes a value that is significantly different from controls (P < 0.05).
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fig01: PHD2 regulates the rate of β2-AR internalization. (A) Overexpression of PHD2, but not PHD1 or PHD3, inhibits β2-AR internalization. β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or the vector (pcDNA3.0) plasmid. Forty-eight hours after transfection, these cells were stimulated with ISO (10 μM) for the indicated time to determine β2-AR internalization as described in ‘Materials and methods’. Internalized receptors are considered to be those removed from the plasma membrane after agonist treatment. (B) β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or scramble (Scram) siRNA for 48 hrs. The expression of each PHD isoform was determined by Western blots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (C) PHD2 silencing promotes β2-AR internalization. Endogenous PHD expression in β2-AR-293 cells was silenced by specific siRNA. Forty-eight hours after transfection, β2-AR was stimulated with ISO (10 μM) for the indicated time to conduct a β2-AR internalization assay. (D) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 or vector virus (control) for 48 hrs, and then these cells were stimulated with ISO for the indicated time to determine β2-AR internalization. (E) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 shRNA-lentiviral constructs or vector virus (control), and then these cells were stimulated with ISO for the indicated time for β2-AR internalization assay. The data are shown as the means ± S.E.M. of representative experiments performed at least three times in triplicate. Asterisk (*) denotes a value that is significantly different from controls (P < 0.05).

Mentions: To determine the role for PHDs in the regulation of β2-AR internalization, PHD1, PHD2, PHD3 or the vector plasmid was transfected into β2-AR-293 cells, respectively. After 48 hrs of expression, these cells were treated with ISO (10 μM) to activate β2-AR. In PHD1- or PHD3-overexpressing cells, ISO stimulation resulted in the loss of surface receptors with a t1/2 of approximately 8 min., similar to the cells expressing β2-AR only (Control). In contrast, PHD2 overexpression resulted in a dramatic decrease in β2-AR internalization rate (t1/2 of approximately 12 min.; Fig. 1A) in response to receptor activation, suggesting a substantial role of PHD2 in the regulation of β2-AR internalization.


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 regulates the rate of β2-AR internalization. (A) Overexpression of PHD2, but not PHD1 or PHD3, inhibits β2-AR internalization. β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or the vector (pcDNA3.0) plasmid. Forty-eight hours after transfection, these cells were stimulated with ISO (10 μM) for the indicated time to determine β2-AR internalization as described in ‘Materials and methods’. Internalized receptors are considered to be those removed from the plasma membrane after agonist treatment. (B) β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or scramble (Scram) siRNA for 48 hrs. The expression of each PHD isoform was determined by Western blots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (C) PHD2 silencing promotes β2-AR internalization. Endogenous PHD expression in β2-AR-293 cells was silenced by specific siRNA. Forty-eight hours after transfection, β2-AR was stimulated with ISO (10 μM) for the indicated time to conduct a β2-AR internalization assay. (D) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 or vector virus (control) for 48 hrs, and then these cells were stimulated with ISO for the indicated time to determine β2-AR internalization. (E) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 shRNA-lentiviral constructs or vector virus (control), and then these cells were stimulated with ISO for the indicated time for β2-AR internalization assay. The data are shown as the means ± S.E.M. of representative experiments performed at least three times in triplicate. Asterisk (*) denotes a value that is significantly different from controls (P < 0.05).
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Related In: Results  -  Collection

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fig01: PHD2 regulates the rate of β2-AR internalization. (A) Overexpression of PHD2, but not PHD1 or PHD3, inhibits β2-AR internalization. β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or the vector (pcDNA3.0) plasmid. Forty-eight hours after transfection, these cells were stimulated with ISO (10 μM) for the indicated time to determine β2-AR internalization as described in ‘Materials and methods’. Internalized receptors are considered to be those removed from the plasma membrane after agonist treatment. (B) β2-AR-293 cells were transfected with PHD1, PHD2, PHD3 or scramble (Scram) siRNA for 48 hrs. The expression of each PHD isoform was determined by Western blots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. (C) PHD2 silencing promotes β2-AR internalization. Endogenous PHD expression in β2-AR-293 cells was silenced by specific siRNA. Forty-eight hours after transfection, β2-AR was stimulated with ISO (10 μM) for the indicated time to conduct a β2-AR internalization assay. (D) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 or vector virus (control) for 48 hrs, and then these cells were stimulated with ISO for the indicated time to determine β2-AR internalization. (E) Neonatal rat cardiomyocytes were infected with PHD1, PHD2, PHD3 shRNA-lentiviral constructs or vector virus (control), and then these cells were stimulated with ISO for the indicated time for β2-AR internalization assay. The data are shown as the means ± S.E.M. of representative experiments performed at least three times in triplicate. Asterisk (*) denotes a value that is significantly different from controls (P < 0.05).
Mentions: To determine the role for PHDs in the regulation of β2-AR internalization, PHD1, PHD2, PHD3 or the vector plasmid was transfected into β2-AR-293 cells, respectively. After 48 hrs of expression, these cells were treated with ISO (10 μM) to activate β2-AR. In PHD1- or PHD3-overexpressing cells, ISO stimulation resulted in the loss of surface receptors with a t1/2 of approximately 8 min., similar to the cells expressing β2-AR only (Control). In contrast, PHD2 overexpression resulted in a dramatic decrease in β2-AR internalization rate (t1/2 of approximately 12 min.; Fig. 1A) in response to receptor activation, suggesting a substantial role of PHD2 in the regulation of β2-AR internalization.

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
Related in: MedlinePlus