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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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HPAH patient-derived lymphocytes express mutant BMPR2 products.A, Schematic of the BMPR2 protein. Areas recognized by the anti-BMPR2 antibodies clone 18 and ASQ are indicated by black arrows. Exons 1-13 are represented by alternating gray and white boxes, and numbers indicate corresponding amino acids. LBD, represents the ligand binding domain, TM the transmembrane domain, KD the kinase domain and CT the cytoplasmic tail. B, Detection of BMPR2 products in HPAH patient-derived lymphocytes, image representative of three experiments. Western blot using anti-BMPR2 antibody, Clone 18. A 130-145 kDa wild type BMPR2 product (WT) was detected in normal control and Family 108 (F108) HPAH patient-derived lymphoblasts. F108 cells expressed an additional 120 kDa BMPR2 mutant product (ΔEx2). C, Representative western blot from three experiments using the ASQ anti-BMPR2 antibody. The 130-145 kDa product was detected in both control and F108 lymphocytes, but the 120 kDa band was not detected. D, Cell surface expression of BMPR2 in HPAH patient-derived lymphocytes labeled with a membrane impermeable biotin. Experiment replicated three times. Left panel, input cell lysates before the streptavidin pull-down. Right panel, cell surface proteins detected in streptavidin pull-down by Western blot using Clone 18 anti-BMPR2 antibody. A 130-145 kDa wild type BMPR2 product was detected in control and F108 cultured lymphocytes in streptavidin pull-down but not 120kDa BMPR2ΔEx2 mutant product.
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pone-0080319-g001: HPAH patient-derived lymphocytes express mutant BMPR2 products.A, Schematic of the BMPR2 protein. Areas recognized by the anti-BMPR2 antibodies clone 18 and ASQ are indicated by black arrows. Exons 1-13 are represented by alternating gray and white boxes, and numbers indicate corresponding amino acids. LBD, represents the ligand binding domain, TM the transmembrane domain, KD the kinase domain and CT the cytoplasmic tail. B, Detection of BMPR2 products in HPAH patient-derived lymphocytes, image representative of three experiments. Western blot using anti-BMPR2 antibody, Clone 18. A 130-145 kDa wild type BMPR2 product (WT) was detected in normal control and Family 108 (F108) HPAH patient-derived lymphoblasts. F108 cells expressed an additional 120 kDa BMPR2 mutant product (ΔEx2). C, Representative western blot from three experiments using the ASQ anti-BMPR2 antibody. The 130-145 kDa product was detected in both control and F108 lymphocytes, but the 120 kDa band was not detected. D, Cell surface expression of BMPR2 in HPAH patient-derived lymphocytes labeled with a membrane impermeable biotin. Experiment replicated three times. Left panel, input cell lysates before the streptavidin pull-down. Right panel, cell surface proteins detected in streptavidin pull-down by Western blot using Clone 18 anti-BMPR2 antibody. A 130-145 kDa wild type BMPR2 product was detected in control and F108 cultured lymphocytes in streptavidin pull-down but not 120kDa BMPR2ΔEx2 mutant product.

Mentions: To determine if NMD negative BMPR2 mutations are expressed and incorrectly trafficked to the cell surface in patients with HPAH, we evaluated BMPR2 protein expression in HPAH patient-derived lymphocytes [16,30]. Cultured immortalized lymphocytes were used because these cells are easily isolated and stored. Additionally, we have a repository of frozen, HPAH patient-derived cultured lymphocytes at Vanderbilt from participants in the Vanderbilt Prospective Pulmonary Hypertension Research Cohort study [16,19,30,32-35]. For these studies we evaluated BMPR2 expression in lymphocytes derived from an HPAH patient from Vanderbilt PAH Family 108 (F108) who carry a splice site mutation predicted to result in an in-frame deletion of BMPR2 EXON2, which encodes residues 26-82 of the 1038 full length BMPR2 protein[30]. If expressed, the mutant product, BMPR2ΔEx2, would be distinguishable from the wild type allelic product by a mobility shift on western blot resulting from deletion of 56 amino acids encoded by EXON2. We obtained lymphocytes from one normal control and one F108 HPAH patient. The anti-BMPR2 antibody, Clone 18, which is a mouse monoclonal antibody raised against a recombinant fragment (residues 803-996) from the cytoplasmic tail of human BMPR2 (Figure 1A), detected a strong 130-145 kDa wild type BMPR2 band in control and F108 HPAH patient lymphocytes (Figure 1B). An additional weaker 120 kDa band was detected in F108 HPAH, but not in normal control cells. To determine if the 120 kDa band was the predicted product resulting from in-frame deletion of BMPR2 EXON2 in F108 HPAH lymphocytes, we generated an affinity purified rabbit polyclonal anti-BMPR2 antibody, ASQ, raised against the peptide sequence of the first 14 amino acids encoded by BMPR2 EXON2 (Figure 1A). The 130-145 kDa wild type BMPR2 band was detected in control and F108 lymphocytes, but the 120 kDa BMPR2 band was not detected in F108 cells using this antibody (Figure 1C). These data indicate that lymphocytes from a F108 HPAH patient express a mutant BMPR2 product resulting from an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2).


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)

HPAH patient-derived lymphocytes express mutant BMPR2 products.A, Schematic of the BMPR2 protein. Areas recognized by the anti-BMPR2 antibodies clone 18 and ASQ are indicated by black arrows. Exons 1-13 are represented by alternating gray and white boxes, and numbers indicate corresponding amino acids. LBD, represents the ligand binding domain, TM the transmembrane domain, KD the kinase domain and CT the cytoplasmic tail. B, Detection of BMPR2 products in HPAH patient-derived lymphocytes, image representative of three experiments. Western blot using anti-BMPR2 antibody, Clone 18. A 130-145 kDa wild type BMPR2 product (WT) was detected in normal control and Family 108 (F108) HPAH patient-derived lymphoblasts. F108 cells expressed an additional 120 kDa BMPR2 mutant product (ΔEx2). C, Representative western blot from three experiments using the ASQ anti-BMPR2 antibody. The 130-145 kDa product was detected in both control and F108 lymphocytes, but the 120 kDa band was not detected. D, Cell surface expression of BMPR2 in HPAH patient-derived lymphocytes labeled with a membrane impermeable biotin. Experiment replicated three times. Left panel, input cell lysates before the streptavidin pull-down. Right panel, cell surface proteins detected in streptavidin pull-down by Western blot using Clone 18 anti-BMPR2 antibody. A 130-145 kDa wild type BMPR2 product was detected in control and F108 cultured lymphocytes in streptavidin pull-down but not 120kDa BMPR2ΔEx2 mutant product.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818254&req=5

pone-0080319-g001: HPAH patient-derived lymphocytes express mutant BMPR2 products.A, Schematic of the BMPR2 protein. Areas recognized by the anti-BMPR2 antibodies clone 18 and ASQ are indicated by black arrows. Exons 1-13 are represented by alternating gray and white boxes, and numbers indicate corresponding amino acids. LBD, represents the ligand binding domain, TM the transmembrane domain, KD the kinase domain and CT the cytoplasmic tail. B, Detection of BMPR2 products in HPAH patient-derived lymphocytes, image representative of three experiments. Western blot using anti-BMPR2 antibody, Clone 18. A 130-145 kDa wild type BMPR2 product (WT) was detected in normal control and Family 108 (F108) HPAH patient-derived lymphoblasts. F108 cells expressed an additional 120 kDa BMPR2 mutant product (ΔEx2). C, Representative western blot from three experiments using the ASQ anti-BMPR2 antibody. The 130-145 kDa product was detected in both control and F108 lymphocytes, but the 120 kDa band was not detected. D, Cell surface expression of BMPR2 in HPAH patient-derived lymphocytes labeled with a membrane impermeable biotin. Experiment replicated three times. Left panel, input cell lysates before the streptavidin pull-down. Right panel, cell surface proteins detected in streptavidin pull-down by Western blot using Clone 18 anti-BMPR2 antibody. A 130-145 kDa wild type BMPR2 product was detected in control and F108 cultured lymphocytes in streptavidin pull-down but not 120kDa BMPR2ΔEx2 mutant product.
Mentions: To determine if NMD negative BMPR2 mutations are expressed and incorrectly trafficked to the cell surface in patients with HPAH, we evaluated BMPR2 protein expression in HPAH patient-derived lymphocytes [16,30]. Cultured immortalized lymphocytes were used because these cells are easily isolated and stored. Additionally, we have a repository of frozen, HPAH patient-derived cultured lymphocytes at Vanderbilt from participants in the Vanderbilt Prospective Pulmonary Hypertension Research Cohort study [16,19,30,32-35]. For these studies we evaluated BMPR2 expression in lymphocytes derived from an HPAH patient from Vanderbilt PAH Family 108 (F108) who carry a splice site mutation predicted to result in an in-frame deletion of BMPR2 EXON2, which encodes residues 26-82 of the 1038 full length BMPR2 protein[30]. If expressed, the mutant product, BMPR2ΔEx2, would be distinguishable from the wild type allelic product by a mobility shift on western blot resulting from deletion of 56 amino acids encoded by EXON2. We obtained lymphocytes from one normal control and one F108 HPAH patient. The anti-BMPR2 antibody, Clone 18, which is a mouse monoclonal antibody raised against a recombinant fragment (residues 803-996) from the cytoplasmic tail of human BMPR2 (Figure 1A), detected a strong 130-145 kDa wild type BMPR2 band in control and F108 HPAH patient lymphocytes (Figure 1B). An additional weaker 120 kDa band was detected in F108 HPAH, but not in normal control cells. To determine if the 120 kDa band was the predicted product resulting from in-frame deletion of BMPR2 EXON2 in F108 HPAH lymphocytes, we generated an affinity purified rabbit polyclonal anti-BMPR2 antibody, ASQ, raised against the peptide sequence of the first 14 amino acids encoded by BMPR2 EXON2 (Figure 1A). The 130-145 kDa wild type BMPR2 band was detected in control and F108 lymphocytes, but the 120 kDa BMPR2 band was not detected in F108 cells using this antibody (Figure 1C). These data indicate that lymphocytes from a F108 HPAH patient express a mutant BMPR2 product resulting from an in-frame deletion of BMPR2 EXON2 (BMPR2ΔEx2).

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