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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 β2-AR internalization depending on the hydroxylase activity. (A) β2-AR-293 cells were incubated in 21% or 1% O2 or treated with DMOG (1 mM) for 12 hrs, and then stimulated with ISO (10 μM) for 10, 30 or 40 min. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with cells incubated in 21% O2 (P < 0.05). (B) β2-AR-293 cells were transfected with PHD1/PHD2 siRNA, PHD2/PHD3 siRNA, PHD1/PHD3 siRNA or scramble siRNA (Ctrl) for 36 hrs, and then a portion of these cells were treated with DMOG (1 mM) for an additional 12 hrs. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with the cells transfected with scramble siRNA (Ctrl). ‘#’ denotes a significant difference between cells treated with or without DMOG in the same siRNA transfection group. (C) FLAG-β-arrestin 2 was purified from β2-AR-293 cells cultured in 21% or 1% O2, digested with trypsin and analysed by LC-MS. Monoisotopic precursor ions (shown in red), at m/z = 958.3, 966.3, 974.3 and 982.3, correspond to [M+2H]+2 for the unhydroxylated, monoproline hydroxylated, diproline hydroxylated and triproline hydroxylated VQFAPETPGPQPSAETTR peptides, respectively. (D) A tandem mass spectrum of the precursor ion at m/z = 971.5. A large red ‘P’ indicates hydroxylated proline residues. The peak heights show the relative abundances of the corresponding fragment ions with the annotations identifying the matching N terminus-containing ions (b ions) in blue and C terminus-containing ions (y ions) in magenta. For clarity, only the major peaks are labelled.
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fig04: PHD2 regulates β2-AR internalization depending on the hydroxylase activity. (A) β2-AR-293 cells were incubated in 21% or 1% O2 or treated with DMOG (1 mM) for 12 hrs, and then stimulated with ISO (10 μM) for 10, 30 or 40 min. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with cells incubated in 21% O2 (P < 0.05). (B) β2-AR-293 cells were transfected with PHD1/PHD2 siRNA, PHD2/PHD3 siRNA, PHD1/PHD3 siRNA or scramble siRNA (Ctrl) for 36 hrs, and then a portion of these cells were treated with DMOG (1 mM) for an additional 12 hrs. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with the cells transfected with scramble siRNA (Ctrl). ‘#’ denotes a significant difference between cells treated with or without DMOG in the same siRNA transfection group. (C) FLAG-β-arrestin 2 was purified from β2-AR-293 cells cultured in 21% or 1% O2, digested with trypsin and analysed by LC-MS. Monoisotopic precursor ions (shown in red), at m/z = 958.3, 966.3, 974.3 and 982.3, correspond to [M+2H]+2 for the unhydroxylated, monoproline hydroxylated, diproline hydroxylated and triproline hydroxylated VQFAPETPGPQPSAETTR peptides, respectively. (D) A tandem mass spectrum of the precursor ion at m/z = 971.5. A large red ‘P’ indicates hydroxylated proline residues. The peak heights show the relative abundances of the corresponding fragment ions with the annotations identifying the matching N terminus-containing ions (b ions) in blue and C terminus-containing ions (y ions) in magenta. For clarity, only the major peaks are labelled.

Mentions: PHD2 is an important oxygen sensor that modifies the corresponding target proteins through its hydroxylase activity [5]. Hypoxia condition or dimethyloxalyglycine (DMOG) treatment resulted in the inhibition of PHD activity, and in turn obviously accelerated β2-AR internalization compared with the cells cultured under nomoxic conditions, suggesting that PHD activity plays a negative role in the regulation of β2-AR internalization (Fig. 4A). However, hypoxia or DMOG is a broad-spectrum PHD inhibitor, and these treatments failed to distinguish which PHD isoform activity participated in the regulation of β2-AR internalization. Next, we adopted double gene knockdown technique combined with DMOG treatment to solve this problem. As shown in Fig 4B, compared with the untreated cells (Ctrl), PHD1/PHD3 double knockdown (PHD1−/PHD3−) did not affect the rate of β2-AR internalization upon β2-AR activation, whereas subsequent DMOG treatment could inhibit PHD2 hydroxylase activity, and accelerate β2-AR internalization in response to agonist stimulation. In contrast, compared with the untreated cells, PHD1/PHD2 (PHD1−/PHD2−) or PHD2/PHD3 (PHD2−/PHD3−) double knockdown obviously accelerated β2-AR internalization, but the effect was mainly mediated by PHD2 depletion. Although subsequent DMOG treatment resulted in the inhibition of PHD1 or PHD3 hydroxylase activity, the rate of β2-AR internalization did not further change. Collectively, these results suggest that the hydroxylase activity of PHD2, but not PHD1 or PHD3, is involved 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 β2-AR internalization depending on the hydroxylase activity. (A) β2-AR-293 cells were incubated in 21% or 1% O2 or treated with DMOG (1 mM) for 12 hrs, and then stimulated with ISO (10 μM) for 10, 30 or 40 min. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with cells incubated in 21% O2 (P < 0.05). (B) β2-AR-293 cells were transfected with PHD1/PHD2 siRNA, PHD2/PHD3 siRNA, PHD1/PHD3 siRNA or scramble siRNA (Ctrl) for 36 hrs, and then a portion of these cells were treated with DMOG (1 mM) for an additional 12 hrs. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with the cells transfected with scramble siRNA (Ctrl). ‘#’ denotes a significant difference between cells treated with or without DMOG in the same siRNA transfection group. (C) FLAG-β-arrestin 2 was purified from β2-AR-293 cells cultured in 21% or 1% O2, digested with trypsin and analysed by LC-MS. Monoisotopic precursor ions (shown in red), at m/z = 958.3, 966.3, 974.3 and 982.3, correspond to [M+2H]+2 for the unhydroxylated, monoproline hydroxylated, diproline hydroxylated and triproline hydroxylated VQFAPETPGPQPSAETTR peptides, respectively. (D) A tandem mass spectrum of the precursor ion at m/z = 971.5. A large red ‘P’ indicates hydroxylated proline residues. The peak heights show the relative abundances of the corresponding fragment ions with the annotations identifying the matching N terminus-containing ions (b ions) in blue and C terminus-containing ions (y ions) in magenta. For clarity, only the major peaks are labelled.
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Related In: Results  -  Collection

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fig04: PHD2 regulates β2-AR internalization depending on the hydroxylase activity. (A) β2-AR-293 cells were incubated in 21% or 1% O2 or treated with DMOG (1 mM) for 12 hrs, and then stimulated with ISO (10 μM) for 10, 30 or 40 min. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with cells incubated in 21% O2 (P < 0.05). (B) β2-AR-293 cells were transfected with PHD1/PHD2 siRNA, PHD2/PHD3 siRNA, PHD1/PHD3 siRNA or scramble siRNA (Ctrl) for 36 hrs, and then a portion of these cells were treated with DMOG (1 mM) for an additional 12 hrs. Receptor internalization was determined as described in ‘Materials and methods’. The graph shows values as the means ± S.E.M. for four independent experiments. Asterisk (*) denotes a significant difference compared with the cells transfected with scramble siRNA (Ctrl). ‘#’ denotes a significant difference between cells treated with or without DMOG in the same siRNA transfection group. (C) FLAG-β-arrestin 2 was purified from β2-AR-293 cells cultured in 21% or 1% O2, digested with trypsin and analysed by LC-MS. Monoisotopic precursor ions (shown in red), at m/z = 958.3, 966.3, 974.3 and 982.3, correspond to [M+2H]+2 for the unhydroxylated, monoproline hydroxylated, diproline hydroxylated and triproline hydroxylated VQFAPETPGPQPSAETTR peptides, respectively. (D) A tandem mass spectrum of the precursor ion at m/z = 971.5. A large red ‘P’ indicates hydroxylated proline residues. The peak heights show the relative abundances of the corresponding fragment ions with the annotations identifying the matching N terminus-containing ions (b ions) in blue and C terminus-containing ions (y ions) in magenta. For clarity, only the major peaks are labelled.
Mentions: PHD2 is an important oxygen sensor that modifies the corresponding target proteins through its hydroxylase activity [5]. Hypoxia condition or dimethyloxalyglycine (DMOG) treatment resulted in the inhibition of PHD activity, and in turn obviously accelerated β2-AR internalization compared with the cells cultured under nomoxic conditions, suggesting that PHD activity plays a negative role in the regulation of β2-AR internalization (Fig. 4A). However, hypoxia or DMOG is a broad-spectrum PHD inhibitor, and these treatments failed to distinguish which PHD isoform activity participated in the regulation of β2-AR internalization. Next, we adopted double gene knockdown technique combined with DMOG treatment to solve this problem. As shown in Fig 4B, compared with the untreated cells (Ctrl), PHD1/PHD3 double knockdown (PHD1−/PHD3−) did not affect the rate of β2-AR internalization upon β2-AR activation, whereas subsequent DMOG treatment could inhibit PHD2 hydroxylase activity, and accelerate β2-AR internalization in response to agonist stimulation. In contrast, compared with the untreated cells, PHD1/PHD2 (PHD1−/PHD2−) or PHD2/PHD3 (PHD2−/PHD3−) double knockdown obviously accelerated β2-AR internalization, but the effect was mainly mediated by PHD2 depletion. Although subsequent DMOG treatment resulted in the inhibition of PHD1 or PHD3 hydroxylase activity, the rate of β2-AR internalization did not further change. Collectively, these results suggest that the hydroxylase activity of PHD2, but not PHD1 or PHD3, is involved 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