Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension
Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension
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DOI：
10.1093/hmg/11.13.1517
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发表时间：
2002-06-15
影响因子：
3.5
通讯作者：
Morrell, NW
中科院分区：
文献类型：
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作者：
Rudarakanchana, N;Flanagan, JA;Morrell, NW
骨形态发生蛋白 II 型受体 (BMPR-II) 的多种突变已被证明是原发性肺动脉高压的基础。为了确定 BMPR-II 功能改变的机制，我们使用绿色荧光蛋白 (GFP) 标记的野生型和突变型 BMPR2 构建体以及共聚焦显微镜来定位受体，对肺血管平滑肌细胞的细胞系和原代培养物进行瞬时转染研究。配体结合或激酶结构域中半胱氨酸残基的取代可阻止 BMPR-II 运输至细胞表面，并减少 I-125-BMP4 的结合。此外，与野生型 BMPR-II 相比，半胱氨酸取代的 BMPR-II 的转染显着降低了 BMP/Smad 响应性荧光素酶报告基因 (3GC2wt-Lux) 的基础和 BMP4 刺激的转录活性，表明这些突变体对 Smad 信号传导具有显性负效应。相比之下，在激酶结构域中含有非半胱氨酸取代的 BMPR-II 定位于细胞膜，尽管这些也抑制了 3GC2wt-Lux 的活性。有趣的是，细胞质尾部内的 BMPR-II 突变转移到细胞表面，但保留了激活 3GC2wt-Lux 的能力。将突变体而非野生型构建体转染到小鼠上皮细胞系（NMuMG 细胞）中会导致 p38(MAPK) 激活，并且与野生型受体相比，血清诱导的增殖增加，部分 p38(MAPK) 依赖性。我们得出结论，BMPR-II 的突变通过不同的分子机制异质性地抑制 BMP/Smad 介导的信号传导。然而，所有研究的突变体均表现出涉及 p38(MAPK) 依赖性增殖途径上调的功能获得。
A wide range of mutations in the type II receptor for bone morphogenetic protein (BMPR-II) have been shown to underlie primary pulmonary hypertension. To determine the mechanism of altered BMPR-II function, we employed transient transfection studies in cell lines and primary cultures of pulmonary vascular smooth muscle cells using green fluorescent protein (GFP)-tagged wild-type and mutant BMPR2 constructs and confocal microscopy to localize receptors. Substitution of cysteine residues in the ligand binding or kinase domain prevented trafficking of BMPR-II to the cell surface, and reduced binding of I-125-BMP4. In addition, transfection of cysteine-substituted BMPR-II markedly reduced basal and BMP4-stimulated transcriptional activity of a BMP/Smad responsive luciferase reporter gene (3GC2wt-Lux), compared with wild-type BMPR-II, suggesting a dominant-negative effect of these mutants on Smad signalling. In contrast, BMPR-II containing non-cysteine substitutions in the kinase domain were localized to the cell membrane, although these also suppressed the activity of 3GC2wt-Lux. Interestingly, BMPR-II mutations within the cytoplasmic tail trafficked to the cell surface, but retained the ability to activate 3GC2wt-Lux. Transfection of mutant, but not wild-type, constructs into a mouse epithelial cell line (NMuMG cells) led to activation of p38(MAPK) and increased serum-induced proliferation compared with the wild-type receptor, which was partly p38(MAPK)-dependent. We conclude that mutations in BMPR-II heterogeneously inhibit BMP/Smad-mediated signalling by diverse molecular mechanisms. However, all mutants studied demonstrate a gain of function involving upregulation of p38(MAPK)-dependent proproliferative pathways.