Cytoskeletal defects in Bmpr2-associated pulmonary arterial hypertension

The heritable form of pulmonary arterial hypertension (PAH) is typically caused by a mutation in bone morphogenic protein receptor type 2 (BMPR2), and mice expressing Bmpr2 mutations develop PAH with features similar to human disease. BMPR2 is known to interact with the cytoskeleton, and human array studies in PAH patients confirm alterations in cytoskeletal pathways. The goal of this study was to evaluate cytoskeletal defects in BMPR2-associated PAH. Expression arrays on our Bmpr2 mutant mouse lungs revealed cytoskeletal defects as a prominent molecular consequence of universal expression of a Bmpr2 mutation (Rosa26-Bmpr2(R899X)). Pulmonary microvascular endothelial cells cultured from these mice have histological and functional cytoskeletal defects. Stable transfection of different BMPR2 mutations into pulmonary microvascular endothelial cells revealed that cytoskeletal defects are common to multiple BMPR2 mutations and are associated with activation of the Rho GTPase, Rac1. Rac1 defects are corrected in cell culture and in vivo through administration of exogenous recombinant human angiotensin-converting enzyme 2 (rhACE2). rhACE2 reverses 77% of gene expression changes in Rosa26-Bmpr2(R899X) transgenic mice, in particular, correcting defects in cytoskeletal function. Administration of rhACE2 to Rosa26-Bmpr2(R899X) mice with established PAH normalizes pulmonary pressures. Together, these findings suggest that cytoskeletal function is central to the development of BMPR2-associated PAH and that intervention against cytoskeletal defects may reverse established disease.     

BMP signaling controls PASMC KV channel expression in vitro and in vivo

Bone morphogenetic proteins (BMPs) have been implicated in the pathogenesis of familial pulmonary arterial hypertension. The type 2 receptor (BMPR2) is required for recognition of all BMPs. Transgenic mice with a smooth muscle cell-targeted mutation in this receptor (SM22-tet-BMPR2(delx4+)) developed increased pulmonary artery pressure, associated with a modest increase in arterial muscularization, after 8 wk of transgene activation (West J, Fagan K, Steudel W, Fouty B, Lane K, Harral J, Hoedt-Miller M, Tada Y, Ozimek J, Tuder R, and Rodman DM. Circ Res 94: 1109-1114, 2004). In the present study, we show that these transgenic mice developed increased right ventricular pressures after only 1 wk of transgene activation, without significant remodeling of the vasculature. We then tested the hypothesis that the increased pulmonary artery pressure due to loss of BMPR2 signaling was mediated by reduced K(V) channel expression. There was decreased expression of K(V)1.1, K(V)1.5, and K(V)4.3 mRNA isolated from whole lung. Western blot confirmed decreased K(V)1.5 protein in these lungs. Human pulmonary artery smooth muscle cells (PASMC) treated with recombinant BMP2 had increased K(V)1.5 protein and macroscopic K(V) current density, which was blocked by anti-K(V)1.5 antibody. In vivo, nifedipine, a selective L-type Ca(2+) channel blocker, reduced RV systolic pressure in these dominant-negative BMPR2 mice to levels seen in control animals. This suggests that activation of L-type Ca(2+) channels caused by reduced K(V)1.5 mediates increased pulmonary artery pressure in these animals. These studies suggest that BMP regulates K(V) channel expression and that loss of this signaling pathway in PASMC through a mutation in BMPR2 is sufficient to cause pulmonary artery vasoconstriction.     

Overexpression of human bone morphogenetic protein receptor 2 does not ameliorate monocrotaline pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is associated with mutations of bone morphogenetic protein receptor 2 (BMPR2), and BMPR2 expression decreases with the development of experimental PAH. Decreased BMPR2 expression and impaired intracellular BMP signaling in pulmonary artery (PA) smooth muscle cells (PASMC) suppresses apoptosis and promotes proliferation, thereby contributing to the pathogenesis of PAH. We hypothesized that overexpression of BMPR2 in resistance PAs would ameliorate established monocrotaline PAH. Human BMPR2 was inserted into a serotype 5 adenovirus with a green fluorescent protein (GFP) reporter. Dose-dependent transgene expression was confirmed in PASMC using fluorescence microscopy, quantitative RT-PCR, and immunoblots. PAH was induced by injecting Sprague-Dawley rats with monocrotaline (60 mg/kg ip) or saline. On day 14, post-monocrotaline (MCT) rats received 5 x 10(9) plaque-forming units of either Ad-human BMPR2 (Ad-hBMPR2) or Ad-GFP. Transgene expression was confirmed by fluorescence microscopy, quantitative RT-PCR of whole lung samples, and laser-capture microdissected resistance PAs. Invasive hemodynamic and echocardiographic end points of pulmonary hypertension were assessed on day 24. Endogenous BMPR2 mRNA levels were greatest in resistance PAs, and expression declined with MCT PAH. Despite robust hBMPR2 expression in all lung lobes and within resistance PAs of treated rats, hBMPR2 did not lower mean PA pressure, pulmonary vascular resistance index, right ventricular hypertrophy, or remodeling of resistance PAs. Nebulized intratracheal adenoviral gene therapy with hBMPR2 reliably distributed hBMPR2 to resistance PAs but did not ameliorate PAH. Depressed BMPR2 expression may be a marker of PAH but is not central to the pathogenesis of this model of PAH.     

Mice expressing BMPR2R899X transgene in smooth muscle develop pulmonary vascular lesions

Familial pulmonary arterial hypertension (PAH) is associated with mutations in bone morphogenetic protein type II receptor (BMPR2). Many of these mutations occur in the BMPR2 tail domain, leaving the SMAD functions intact. To determine the in vivo consequences of BMPR2 tail domain mutation, we created a smooth muscle-specific doxycycline-inducible BMPR2 mutation with an arginine to termination mutation at amino acid 899. When these SM22-rtTA x TetO(7)-BMPR2(R899X) mice had transgene induced for 9 wk, starting at 4 wk of age, they universally developed pulmonary vascular pruning as assessed by fluorescent microangiography. Approximately one-third of the time, the induced animals developed elevated right ventricular systolic pressures (RVSP), associated with extensive pruning, muscularization of small pulmonary vessels, and development of large structural pulmonary vascular changes. These lesions included large numbers of macrophages and T cells in their adventitial compartment as well as CD133-positive cells in the lumen. Small vessels filled with CD45-positive and sometimes CD3-positive cells were a common feature in all SM22-rtTA x TetO(7)-BMPR2(R899X) mice. Gene array experiments show changes in stress response, muscle organization and function, proliferation, and apoptosis and developmental pathways before RVSP increases. Our results show that the primary phenotypic result of BMPR2 tail domain mutation in smooth muscle is pulmonary vascular pruning leading to elevated RVSP, associated with early dysregulation in multiple pathways with clear relevance to PAH. This model should be useful to the research community in examining early molecular and physical events in the development of PAH and as a platform to validate potential treatments.     

Serotonin 2B Receptor Antagonism Prevents Heritable Pulmonary Arterial Hypertension

Serotonergic anorexigens are the primary pharmacologic risk factor associated with pulmonary arterial hypertension (PAH), and the resulting PAH is clinically indistinguishable from the heritable form of disease, associated with BMPR2 mutations. Both BMPR2 mutation and agonists to the serotonin receptor HTR2B have been shown to cause activation of SRC tyrosine kinase; conversely, antagonists to HTR2B inhibit SRC trafficking and downstream function. To test the hypothesis that a HTR2B antagonist can prevent BMRP2 mutation induced PAH by restricting aberrant SRC trafficking and downstream activity, we exposed BMPR2 mutant mice, which spontaneously develop PAH, to a HTR2B antagonist, SB204741, to block the SRC activation caused by BMPR2 mutation. SB204741 prevented the development of PAH in BMPR2 mutant mice, reduced recruitment of inflammatory cells to their lungs, and reduced muscularization of their blood vessels. By atomic force microscopy, we determined that BMPR2 mutant mice normally had a doubling of vessel stiffness, which was substantially normalized by HTR2B inhibition. SB204741 reduced SRC phosphorylation and downstream activity in BMPR2 mutant mice. Gene expression arrays indicate that the primary changes were in cytoskeletal and muscle contractility genes. These results were confirmed by gel contraction assays showing that HTR2B inhibition nearly normalizes the 400% increase in gel contraction normally seen in BMPR2 mutant smooth muscle cells. Heritable PAH results from increased SRC activation, cellular contraction, and vascular resistance, but antagonism of HTR2B prevents SRC phosphorylation, downstream activity, and PAH in BMPR2 mutant mice.     

Physiologic and molecular consequences of endothelial Bmpr2 mutation

Background

Pulmonary arterial hypertension (PAH) is thought to be driven by dysfunction of pulmonary vascular microendothelial cells (PMVEC). Most hereditary PAH is associated with BMPR2 mutations. However, the physiologic and molecular consequences of expression of BMPR2 mutations in PMVEC are unknown.

Methods

In vivo experiments were performed on adult mice with conditional endothelial-specific expression of the truncation mutation Bmpr2^delx4+, with age-matched transactivator-only mice as controls. Phenotype was assessed by RVSP, counts of muscularized vessels and proliferating cells, and staining for thromboses, inflammatory cells, and apoptotic cells. The effects of BMPR2 knockdown in PMVEC by siRNA on rates of apoptosis were assessed. Affymetrix expression arrays were performed on PMVEC isolated and cultured from triple transgenic mice carrying the immortomouse gene, a transactivator, and either control, Bmpr2^delx4+ or Bmpr2^R899X mutation.

Results

Transgenic mice showed increased RVSP and corresponding muscularization of small vessels, with histologic alterations including thrombosis, increased inflammatory cells, increased proliferating cells, and a moderate increase in apoptotic cells. Expression arrays showed alterations in specific pathways consistent with the histologic changes. Bmpr2^delx4+ and Bmpr2^R899X mutations resulted in very similar alterations in proliferation, apoptosis, metabolism, and adhesion; Bmpr2^delx4+ cells showed upregulation of platelet adhesion genes and cytokines not seen in Bmpr2^R899X PMVEC. Bmpr2 mutation in PMVEC does not cause a loss of differentiation markers as was seen with Bmpr2 mutation in smooth muscle cells.

Conclusions

Bmpr2 mutation in PMVEC in vivo may drive PAH through multiple, potentially independent, downstream mechanisms, including proliferation, apoptosis, inflammation, and thrombosis.     

Deletion of the Sequence Encoding the Tail Domain of the Bone Morphogenetic Protein type 2 Receptor Reveals a Bone Morphogenetic Protein 7-Specific Gain of Function

The bone morphogenetic protein (BMP) type II receptor (BMPR2) has a long cytoplasmic tail domain whose function is incompletely elucidated. Mutations in the tail domain of BMPR2 are found in familial cases of pulmonary arterial hypertension. To investigate the role of the tail domain of BMPR2 in BMP signaling, we generated a mouse carrying a Bmpr2 allele encoding a non-sense mediated decay-resistant mutant receptor lacking the tail domain of Bmpr2. We found that homozygous mutant mice died during gastrulation, whereas heterozygous mice grew normally without developing pulmonary arterial hypertension. Using pulmonary artery smooth muscle cells (PaSMC) from heterozygous mice, we determined that the mutant receptor was expressed and retained its ability to transduce BMP signaling. Heterozygous PaSMCs exhibited a BMP7‑specific gain of function, which was transduced via the mutant receptor. Using siRNA knockdown and cells from conditional knockout mice to selectively deplete BMP receptors, we observed that the tail domain of Bmpr2 inhibits Alk2‑mediated BMP7 signaling. These findings suggest that the tail domain of Bmpr2 is essential for normal embryogenesis and inhibits Alk2‑mediated BMP7 signaling in PaSMCs.     

Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells

Bone morphogenetic protein (BMP) ligands signal by binding the BMP type II receptor (BMPR2) or the activin type II receptors (ActRIIa and ActRIIb) in conjunction with type I receptors to activate SMADs 1, 5, and 8, as well as members of the mitogen-activated protein kinase family. Loss-of-function mutations in Bmpr2 have been implicated in tumorigenesis and in the etiology of primary pulmonary hypertension. Because several different type II receptors are known to recognize BMP ligands, the specific contribution of BMPR2 to BMP signaling is not defined. Here we report that the ablation of Bmpr2 in pulmonary artery smooth muscle cells, using an ex vivo conditional knock-out (Cre-lox) approach, as well as small interfering RNA specific for Bmpr2, does not abolish BMP signaling. Disruption of Bmpr2 leads to diminished signaling by BMP2 and BMP4 and augmented signaling by BMP6 and BMP7. Using small interfering RNAs to inhibit the expression of other BMP receptors, we found that wild-type cells transduce BMP signals via BMPR2, whereas BMPR2-deficient cells transduce BMP signals via ActRIIa in conjunction with a set of type I receptors distinct from those utilized by BMPR2. These findings suggest that disruption of Bmpr2 leads to the net gain of signaling by some, but not all, BMP ligands via the activation of ActRIIa.     

Bone morphogenetic protein receptor II is a novel mediator of endothelial nitric-oxide synthase activation

Activation of bone morphogenetic protein (BMP) receptor II (BMPRII) promotes pulmonary artery endothelial cell (PAEC) survival, proliferation, and migration. Mutations to BMPRII are associated with the development of pulmonary arterial hypertension (PAH). Endothelial dysfunction, including decreased endothelial nitric-oxide synthase (eNOS) activity and loss of bioactive nitric oxide (NO), plays a prominent role in the development of PAH. We hypothesized that stimulation of BMPRII promotes normal PAEC function by activating eNOS. We report that BMPRII ligands, BMP2 and BMP4, (i) stimulate eNOS phosphorylation at a critical regulatory site, (ii) increase eNOS activity, and (iii) result in canonical changes in eNOS protein-protein interactions. The stimulation of eNOS activity by BMPRII ligands was largely dependent on protein kinase A (PKA) activation, as demonstrated using the PKA inhibitors H89 and myristoylated PKI(6-22) amide. PAEC migration stimulated by BMP2 and BMP4 was inhibited by the NOS inhibitor l-nitroarginine methyl ester, providing functional evidence of eNOS activation. Furthermore, BMP2 and BMP4 failed to stimulate eNOS phosphorylation when BMPRII was knocked down by siRNA. Most important to the pathophysiology of the disease, BMP2 and BMP4 failed to stimulate eNOS phosphorylation in PAECs isolated from patients with mutations in the BMPR2 gene. These data demonstrate a new action of BMPs/BMPRII in the pulmonary endothelium and provide novel mechanistic insight into the pathogenesis of PAH.     

Risk Factors for Hemoptysis in Idiopathic and Hereditary Pulmonary Arterial Hypertension

Introduction

When hemoptysis complicates pulmonary arterial hypertension (PAH), it is assumed to result from bronchial artery hypertrophy. In heritable PAH, the most common mutation is in the BMPR2 gene, which regulates growth, differentiation and apoptosis of mesenchymal cells. The aim of this study is to determine the relationship in PAH between the occurrence of hemoptysis, and disease progression, bronchial artery hypertrophy, pulmonary artery dilation and BMPR2 mutations.

Methods

129 IPAH patients underwent baseline pulmonary imaging (CT angio or MRI) and repeated right-sided heart catheterization. Gene mutations were assessed in a subset of patients.

Results

Hemoptysis was associated with a greater presence of hypertrophic bronchial arteries and more rapid hemodynamic deterioration. The presence of a BMPR2 mutation did not predispose to the development of hemoptysis, but was associated with a greater number of hypertrophic bronchial arteries and a worse baseline hemodynamic profile.

Conclusion

Hemoptysis in PAH is associated with bronchial artery hypertrophy and faster disease progression. Although the presence of a BMPR2 mutation did not correlate with a greater incidence of hemoptysis in our patient cohort, its association with worse hemodynamics and a trend of greater bronchial arterial hypertrophy may increase the risk of hemoptysis.     

异氟醚通过Nrf2/HO-1/ROS途径抑制缺氧引起肺动脉平滑肌细胞焦亡

研究发现,异氟醚吸入麻醉可明显减轻由缺血-再灌注引起的肺动脉高压(PAH),提示其对肺循环功能有一定保护效应。肺动脉平滑肌细胞(PASMC)是肺动脉血管重塑和PAH发生的主要参与者,其结构改变和功能异常均可显著影响肺动脉高压病情进展。本研究探讨异氟醚对缺氧诱导的PASMC焦亡的影响及其调控机制,旨在为肺动脉高压治疗提供潜在分子靶点。PASMC于37℃、5%CO₂、3%O₂条件下静置培养24 h建立缺氧模型。RT-PCR和Western印迹等结果显示,缺氧致使PASMC内紅系衍生的核转录因子2(Nrf2)核转位减少,血红素加氧酶-1(HO-1)表达水平下调,而焦亡相关蛋白质,包括NOD样受体蛋白3(NLRP3)、胱天蛋白酶1(caspase-1)、凋亡相关斑点样蛋白(ASC)及消皮素D(GSDMD)等表达上调,活性氧(ROS)生成、胱天蛋白酶1活性和乳酸脱氢酶(LDH)释放水平升高,Hoechst/PI染色显示,焦亡孔洞增加。ELISA结果表明,IL-1β、IL-6、IL-18和TNF-α分泌增加(P<0.05)。异氟醚处理可显著激活Nr...     

Type II BMP and activin receptors BMPR2 and ACVR2A share a conserved mode of growth factor recognition

BMPR2 is a type II Transforming Growth Factor (TGF)-β family receptor that is fundamentally associated with pulmonary arterial hypertension (PAH) in humans. BMPR2 shares functional similarities with the type II activin receptors ACVR2A and ACVR2B, as it interacts with an overlapping group of TGF-β family growth factors (GFs). However, how BMPR2 recognizes GFs remains poorly understood. Here, we solved crystal structures of BMPR2 in complex with the GF activin B and of ACVR2A in complex with the related GF activin A. We show that both BMPR2 and ACVR2A bind GFs with nearly identical geometry using a conserved hydrophobic hot spot, while differences in contacting residues are predominantly found in loop areas. Upon further exploration of the GF-binding spectrum of the two receptors, we found that although many GFs bind both receptors, the high-affinity BMPR2 GFs comprise BMP15, BMP10, and Nodal, whereas those of ACVR2A are activin A, activin B, and GDF11. Lastly, we evaluated GF-binding domain BMPR2 variants found in human PAH patients. We demonstrate that mutations within the GF-binding interface resulted in loss of GF binding, while mutations in loop areas allowed BMPR2 to retain the ability to bind cognate GFs with high affinity. In conclusion, the in vitro activities of BMPR2 variants and the crystal structures reported here indicate biochemically relevant complexes that explain how some GF-binding domain variants can lead to PAH.     

Truncating and missense BMPR2 mutations differentially affect the severity of heritable pulmonary arterial hypertension

Background

Autosomal dominant inheritance of germline mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are a major risk factor for pulmonary arterial hypertension (PAH). While previous studies demonstrated a difference in severity between BMPR2 mutation carriers and noncarriers, it is likely disease severity is not equal among BMPR2 mutations. We hypothesized that patients with missense BMPR2 mutations have more severe disease than those with truncating mutations.

Methods

Testing for BMPR2 mutations was performed in 169 patients with PAH (125 with a family history of PAH and 44 with sporadic disease). Of the 106 patients with a detectable BMPR2 mutation, lymphocytes were available in 96 to functionally assess the nonsense-mediated decay pathway of RNA surveillance. Phenotypic characteristics were compared between BMPR2 mutation carriers and noncarriers, as well as between those carriers with a missense versus truncating mutation.

Results

While there was a statistically significant difference in age at diagnosis between carriers and noncarriers, subgroup analysis revealed this to be the case only for females. Among carriers, there was no difference in age at diagnosis, death, or survival according to exonic location of the BMPR2 mutation. However, patients with missense mutations had statistically significant younger ages at diagnosis and death, as well as shorter survival from diagnosis to death or lung transplantation than those with truncating mutations. Consistent with this data, the majority of missense mutations were penetrant prior to age 36 years, while the majority of truncating mutations were penetrant after age 36 years.

Conclusion

In this cohort, BMPR2 mutation carriers have more severe PAH disease than noncarriers, but this is only the case for females. Among carriers, patients with missense mutations that escape nonsense-mediated decay have more severe disease than those with truncating mutations. These findings suggest that treatment and prevention strategies directed specifically at BMPR2 pathway defects may need to vary according to the type of mutation.     

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca²⁺ influx in response to endothelin‐1 (ET‐1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH‐derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phospho‐antibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET‐1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results, whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC, while it continues in IPAH. Importantly, the data suggest that caution must be taken when applying ET‐1 receptor antagonist therapy to PAH patients. J. Cell. Physiol. 228: 322–329, 2013. © 2012 Wiley Periodicals, Inc.     

Chronic allergic inflammation causes vascular remodeling and pulmonary hypertension in BMPR2 hypomorph and wild-type mice

Loss-of-function mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene have been identified in patients with heritable pulmonary arterial hypertension (PAH); however, disease penetrance is low, suggesting additional factors play a role. Inflammation is associated with PAH and vascular remodeling, but whether allergic inflammation triggers vascular remodeling in individuals with BMPR2 mutations is unknown. Our goal was to determine if chronic allergic inflammation would induce more severe vascular remodeling and PAH in mice with reduced BMPR-II signaling. Groups of Bmpr2 hypomorph and wild-type (WT) Balb/c/Byj mice were exposed to house dust mite (HDM) allergen, intranasally for 7 or 20 weeks to generate a model of chronic inflammation. HDM exposure induced similar inflammatory cell counts in all groups compared to controls. Muscularization of pulmonary arterioles and arterial wall thickness were increased after 7 weeks HDM, more severe at 20 weeks, but similar in both groups. Right ventricular systolic pressure (RVSP) was measured by direct cardiac catheterization to assess PAH. RVSP was similarly increased in both HDM exposed groups after 20 weeks compared to controls, but not after 7 weeks. Airway hyperreactivity (AHR) to methacholine was also assessed and interestingly, at 20 weeks, was more severe in HDM exposed Bmpr2 hypomorph mice versus WT. We conclude that chronic allergic inflammation caused PAH and while the severity was mild and similar between WT and Bmpr2 hypomorph mice, AHR was enhanced with reduced BMPR-II signaling. These data suggest that vascular remodeling and PAH resulting from chronic allergic inflammation occurs independently of BMPR-II pathway alterations.     

Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.