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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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Characterization of the endogenously expressed Bmpr2 mutant product in pulmonary endothelial cells from Bmpr2ΔEx2/+ mice.Studies were performed using conditionally immortalized PECs (ciPECs) isolated from wild type control and Bmpr2ΔEx2/+ mice and replicated at least three times. A, Western blot using the Clone 18 anti-BMPR2 antibody in wild type (WT) and Bmpr2ΔEx2/+ (ΔEx2/+) ciPEC lysates. Both cell lines expressed a 150 kDa wild type Bmpr2 product (WT). Bmpr2ΔEx2/+ ciPECs also expressed a 130 kDa product (ΔEx2). B, Western blot using ASQ anti-BMPR2 antibody. Wild type control and Bmpr2ΔEx2/+ ciPECs expressed 150 kDa WT Bmpr2, but the 130 kDa product was not detected. C, Cell surface expression of Bmpr2 in ciPECs. ciPECs were labeled with membrane impermeable biotin and cell surface expression of Bmpr2 detected in streptavidin pull-down of cell lysates. Anti-BMPR2 Clone 18 antibody detected the 150 kDa wild type Bmpr2 in control and Bmpr2ΔEx2/+ ciPECs after streptavidin pull-down but not the 130kDa Bmpr2ΔEx2 mutant product. Lower panel, Western blot for Bmpr2 in the supernatant remaining after depletion of cell surface proteins by streptavidin pull-down. The ratios of 150 kDa WT Bmpr2 and the 130 kDa Bmpr2ΔEx2 mutant band intensities in Bmpr2ΔEx2/+ ciPECs supernatants after depletion of cell surface proteins are indicated below the lower panel.
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pone-0080319-g002: Characterization of the endogenously expressed Bmpr2 mutant product in pulmonary endothelial cells from Bmpr2ΔEx2/+ mice.Studies were performed using conditionally immortalized PECs (ciPECs) isolated from wild type control and Bmpr2ΔEx2/+ mice and replicated at least three times. A, Western blot using the Clone 18 anti-BMPR2 antibody in wild type (WT) and Bmpr2ΔEx2/+ (ΔEx2/+) ciPEC lysates. Both cell lines expressed a 150 kDa wild type Bmpr2 product (WT). Bmpr2ΔEx2/+ ciPECs also expressed a 130 kDa product (ΔEx2). B, Western blot using ASQ anti-BMPR2 antibody. Wild type control and Bmpr2ΔEx2/+ ciPECs expressed 150 kDa WT Bmpr2, but the 130 kDa product was not detected. C, Cell surface expression of Bmpr2 in ciPECs. ciPECs were labeled with membrane impermeable biotin and cell surface expression of Bmpr2 detected in streptavidin pull-down of cell lysates. Anti-BMPR2 Clone 18 antibody detected the 150 kDa wild type Bmpr2 in control and Bmpr2ΔEx2/+ ciPECs after streptavidin pull-down but not the 130kDa Bmpr2ΔEx2 mutant product. Lower panel, Western blot for Bmpr2 in the supernatant remaining after depletion of cell surface proteins by streptavidin pull-down. The ratios of 150 kDa WT Bmpr2 and the 130 kDa Bmpr2ΔEx2 mutant band intensities in Bmpr2ΔEx2/+ ciPECs supernatants after depletion of cell surface proteins are indicated below the lower panel.

Mentions: To evaluate expression and trafficking of the same, endogenously expressed, BMPR2ΔEx2 mutant product in a more physiologically relevant cell type, we isolated conditionally immortalized pulmonary endothelial cells (ciPECs) from Bmpr2ΔEx2/+ mice. Bmpr2ΔEx2/+ mice carry the same, heterozygous in-frame deletion of Exon 2 found in F108 HPAH patients [26]. The anti-BMPR2 antibody Clone 18 detected a 150 kDa band in control (wild type) and Bmpr2ΔEx2/+ ciPECs, but an additional 130 kDa band was only detected in the Bmpr2ΔEx2/+ ciPECs (Figure 2A). To determine if the 130 kDa band was the predicted product resulting from an in-frame deletion of Bmpr2 Exon2, we performed western blot analysis using the anti-BMPR2 antibody ASQ, raised against the peptide sequence in Exon2 (Figure 1A). The wild type 150kDa Bmpr2 band was detected in both the control and Bmpr2ΔEx2/+ ciPECs, but the 130kDa product was not detectable in the Bmpr2ΔEx2/+ciPECs using this antibody (Figure 2B). These data indicate that Bmpr2ΔEx2/+ ciPECs expressed high levels of the Bmpr2ΔEx2 mutant product, which is detected as a 130 kDa band on western blot and is distinct from the 150 kDa wild type Bmpr2 product.


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)

Characterization of the endogenously expressed Bmpr2 mutant product in pulmonary endothelial cells from Bmpr2ΔEx2/+ mice.Studies were performed using conditionally immortalized PECs (ciPECs) isolated from wild type control and Bmpr2ΔEx2/+ mice and replicated at least three times. A, Western blot using the Clone 18 anti-BMPR2 antibody in wild type (WT) and Bmpr2ΔEx2/+ (ΔEx2/+) ciPEC lysates. Both cell lines expressed a 150 kDa wild type Bmpr2 product (WT). Bmpr2ΔEx2/+ ciPECs also expressed a 130 kDa product (ΔEx2). B, Western blot using ASQ anti-BMPR2 antibody. Wild type control and Bmpr2ΔEx2/+ ciPECs expressed 150 kDa WT Bmpr2, but the 130 kDa product was not detected. C, Cell surface expression of Bmpr2 in ciPECs. ciPECs were labeled with membrane impermeable biotin and cell surface expression of Bmpr2 detected in streptavidin pull-down of cell lysates. Anti-BMPR2 Clone 18 antibody detected the 150 kDa wild type Bmpr2 in control and Bmpr2ΔEx2/+ ciPECs after streptavidin pull-down but not the 130kDa Bmpr2ΔEx2 mutant product. Lower panel, Western blot for Bmpr2 in the supernatant remaining after depletion of cell surface proteins by streptavidin pull-down. The ratios of 150 kDa WT Bmpr2 and the 130 kDa Bmpr2ΔEx2 mutant band intensities in Bmpr2ΔEx2/+ ciPECs supernatants after depletion of cell surface proteins are indicated below the lower panel.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818254&req=5

pone-0080319-g002: Characterization of the endogenously expressed Bmpr2 mutant product in pulmonary endothelial cells from Bmpr2ΔEx2/+ mice.Studies were performed using conditionally immortalized PECs (ciPECs) isolated from wild type control and Bmpr2ΔEx2/+ mice and replicated at least three times. A, Western blot using the Clone 18 anti-BMPR2 antibody in wild type (WT) and Bmpr2ΔEx2/+ (ΔEx2/+) ciPEC lysates. Both cell lines expressed a 150 kDa wild type Bmpr2 product (WT). Bmpr2ΔEx2/+ ciPECs also expressed a 130 kDa product (ΔEx2). B, Western blot using ASQ anti-BMPR2 antibody. Wild type control and Bmpr2ΔEx2/+ ciPECs expressed 150 kDa WT Bmpr2, but the 130 kDa product was not detected. C, Cell surface expression of Bmpr2 in ciPECs. ciPECs were labeled with membrane impermeable biotin and cell surface expression of Bmpr2 detected in streptavidin pull-down of cell lysates. Anti-BMPR2 Clone 18 antibody detected the 150 kDa wild type Bmpr2 in control and Bmpr2ΔEx2/+ ciPECs after streptavidin pull-down but not the 130kDa Bmpr2ΔEx2 mutant product. Lower panel, Western blot for Bmpr2 in the supernatant remaining after depletion of cell surface proteins by streptavidin pull-down. The ratios of 150 kDa WT Bmpr2 and the 130 kDa Bmpr2ΔEx2 mutant band intensities in Bmpr2ΔEx2/+ ciPECs supernatants after depletion of cell surface proteins are indicated below the lower panel.
Mentions: To evaluate expression and trafficking of the same, endogenously expressed, BMPR2ΔEx2 mutant product in a more physiologically relevant cell type, we isolated conditionally immortalized pulmonary endothelial cells (ciPECs) from Bmpr2ΔEx2/+ mice. Bmpr2ΔEx2/+ mice carry the same, heterozygous in-frame deletion of Exon 2 found in F108 HPAH patients [26]. The anti-BMPR2 antibody Clone 18 detected a 150 kDa band in control (wild type) and Bmpr2ΔEx2/+ ciPECs, but an additional 130 kDa band was only detected in the Bmpr2ΔEx2/+ ciPECs (Figure 2A). To determine if the 130 kDa band was the predicted product resulting from an in-frame deletion of Bmpr2 Exon2, we performed western blot analysis using the anti-BMPR2 antibody ASQ, raised against the peptide sequence in Exon2 (Figure 1A). The wild type 150kDa Bmpr2 band was detected in both the control and Bmpr2ΔEx2/+ ciPECs, but the 130kDa product was not detectable in the Bmpr2ΔEx2/+ciPECs using this antibody (Figure 2B). These data indicate that Bmpr2ΔEx2/+ ciPECs expressed high levels of the Bmpr2ΔEx2 mutant product, which is detected as a 130 kDa band on western blot and is distinct from the 150 kDa wild type Bmpr2 product.

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