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Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

Frump AL, Lowery JW, Hamid R, Austin ED, de Caestecker M - PLoS ONE (2013)

Bottom Line: However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described.The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum.These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

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Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface.A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.
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pone-0080319-g004: Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface.A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.

Mentions: Since the retention of mutant protein in the ER is often a consequence of incorrect protein folding [37], we wanted to evaluate the effects of two chemical chaperones known to aid in protein folding, 4-PBA and TUDCA [24,25,38-43] , on trafficking of the Bmpr2ΔEx2 mutant product to the cell surface. Bmpr2ΔEx2/+ ciPECs treated with increasing concentrations of 4-PBA show a dose-dependent increase expression of the 130kDa Bmpr2ΔEx2 mutant product in the streptavidin pull-down (Figure 4A/B). Treatment with 4-PBA also increased the cell surface expression of the 150 kDa wild type Bmpr2 protein in Bmpr2ΔEx2/+ ciPECs, but to a relatively lesser extent than Bmpr2ΔEx2. An aliquot of cell lysates taken before the streptavidin pull-down showed no change in expression of wild type Bmpr2 or Bmpr2ΔEx2 with the addition of 4-PBA (Figure 4A, lower panel). An additional chemical chaperone, TUDCA added to cells prior to biotinylation also increased cell surface expression of the 130kDa Bmpr2ΔEx2 mutant protein in Bmpr2ΔEx2/+ ciPECs (Figure 4C/D). There was also a small increase in cell surface expression of wild type Bmpr2 after treatment with TUDCA (a 1.4 fold increase with 500µM TUDCA versus untreated cells). These data indicate that chemical chaperones increase cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs, and suggest that ER retention of Bmpr2ΔEx2 results from mis-folding of the mutant protein.


Abnormal trafficking of endogenously expressed BMPR2 mutant allelic products in patients with heritable pulmonary arterial hypertension.

Frump AL, Lowery JW, Hamid R, Austin ED, de Caestecker M - PLoS ONE (2013)

Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface.A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818254&req=5

pone-0080319-g004: Chemical chaperones partially restore Bmpr2ΔEx2 mutant product expression at the cell surface.A, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with 4-PBA. Bmpr2ΔEx2/+ ciPECs were treated for 48 hours with 100µM, 250µM, 500µM or 1mM of 4-PBA. Monolayers were then labeled with membrane impermeable biotin and biotinylated cell surface proteins pulled-down with streptavidin agarose beads. Western Blot was performed with Clone 18 anti-BMPR2 antibody. The 150 kDa wild type Bmpr2 product was detected in the streptavidin pull-down in control and Bmpr2ΔEx2/+ ciPECs, but the 130 kDa Bmpr2ΔEx2 mutant product was not detected. After treating with the chemical chaperone 4-PBA, the 130kDa Bmpr2ΔEx2 mutant product was detected and there was increased expression of the wild type Bmpr2 product in the streptavidin pull-down. Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with 4-PBA. Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with 4-PBA treatment. B, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after 4-PBA treatment relative to untreated controls from three independent experiments, standard error is indicated. C, Cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs treated with TUDCA. Wild type and Bmpr2ΔEx2/+ ciPECs were treated for 5 hours with 50µM 100µM, 250µM or 500µM of TUDCA. Streptavidin pull-down shows that the 130kDa Bmpr2ΔEx2 mutant product was partially restored at the cell surface and there was a slight increase in wild type Bmpr2 with TUDCA treatment (1.4 fold increase with 500µM TUDCA versus untreated cells). Numbers shown below the upper panel indicate the ratio of the 130 kDa Bmpr2ΔEx2 band before and after treatment with TUDCA Lower panel, expression of wild type Bmpr2 and Bmpr2ΔEx2 in ciPEC cell lysates with TUDCA treatment. D, Quantification of wild type Bmpr2 and Bmpr2ΔEx2 band densities after TUDCA treatment relative to untreated controls from three independent experiments.
Mentions: Since the retention of mutant protein in the ER is often a consequence of incorrect protein folding [37], we wanted to evaluate the effects of two chemical chaperones known to aid in protein folding, 4-PBA and TUDCA [24,25,38-43] , on trafficking of the Bmpr2ΔEx2 mutant product to the cell surface. Bmpr2ΔEx2/+ ciPECs treated with increasing concentrations of 4-PBA show a dose-dependent increase expression of the 130kDa Bmpr2ΔEx2 mutant product in the streptavidin pull-down (Figure 4A/B). Treatment with 4-PBA also increased the cell surface expression of the 150 kDa wild type Bmpr2 protein in Bmpr2ΔEx2/+ ciPECs, but to a relatively lesser extent than Bmpr2ΔEx2. An aliquot of cell lysates taken before the streptavidin pull-down showed no change in expression of wild type Bmpr2 or Bmpr2ΔEx2 with the addition of 4-PBA (Figure 4A, lower panel). An additional chemical chaperone, TUDCA added to cells prior to biotinylation also increased cell surface expression of the 130kDa Bmpr2ΔEx2 mutant protein in Bmpr2ΔEx2/+ ciPECs (Figure 4C/D). There was also a small increase in cell surface expression of wild type Bmpr2 after treatment with TUDCA (a 1.4 fold increase with 500µM TUDCA versus untreated cells). These data indicate that chemical chaperones increase cell surface expression of Bmpr2ΔEx2 in Bmpr2ΔEx2/+ ciPECs, and suggest that ER retention of Bmpr2ΔEx2 results from mis-folding of the mutant protein.

Bottom Line: However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described.The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum.These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
More than 200 heterozygous mutations in the type 2 BMP receptor gene, BMPR2, have been identified in patients with Heritable Pulmonary Arterial Hypertension (HPAH). More severe clinical outcomes occur in patients with BMPR2 mutations by-passing nonsense-mediated mRNA decay (NMD negative mutations). These comprise 40% of HPAH mutations and are predicted to express BMPR2 mutant products. However expression of endogenous NMD negative BMPR2 mutant products and their effect on protein trafficking and signaling function have never been described. Here, we characterize the expression and trafficking of an HPAH-associated NMD negative BMPR2 mutation that results in an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2) in HPAH patient-derived lymphocytes and in pulmonary endothelial cells (PECs) from mice carrying the same in-frame deletion of Exon 2 (Bmpr2 (ΔEx2/+) mice). The endogenous BMPR2ΔEx2 mutant product does not reach the cell surface and is retained in the endoplasmic reticulum. Moreover, chemical chaperones 4-PBA and TUDCA partially restore cell surface expression of Bmpr2ΔEx2 in PECs, suggesting that the mutant product is mis-folded. We also show that PECs from Bmpr2 (ΔEx2/+) mice have defects in the BMP-induced Smad1/5/8 and Id1 signaling axis, and that addition of chemical chaperones restores expression of the Smad1/5/8 target Id1. These data indicate that the endogenous NMD negative BMPRΔEx2 mutant product is expressed but has a folding defect resulting in ER retention. Partial correction of this folding defect and restoration of defective BMP signaling using chemical chaperones suggests that protein-folding agents could be used therapeutically in patients with these NMD negative BMPR2 mutations.

Show MeSH
Related in: MedlinePlus