Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension.
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 (125)I-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.
Mesh Terms:
Animals, Bone Morphogenetic Protein 2, Bone Morphogenetic Protein Receptors, Type II, Bone Morphogenetic Proteins, Cell Line, Cells, Cultured, Genes, Reporter, HeLa Cells, Humans, Hypertension, Pulmonary, Ligands, Mice, Mitogen-Activated Protein Kinases, Mutation, Protein Structure, Tertiary, Protein Transport, Protein-Serine-Threonine Kinases, Recombinant Proteins, Transforming Growth Factor beta, p38 Mitogen-Activated Protein Kinases
Animals, Bone Morphogenetic Protein 2, Bone Morphogenetic Protein Receptors, Type II, Bone Morphogenetic Proteins, Cell Line, Cells, Cultured, Genes, Reporter, HeLa Cells, Humans, Hypertension, Pulmonary, Ligands, Mice, Mitogen-Activated Protein Kinases, Mutation, Protein Structure, Tertiary, Protein Transport, Protein-Serine-Threonine Kinases, Recombinant Proteins, Transforming Growth Factor beta, p38 Mitogen-Activated Protein Kinases
Hum. Mol. Genet.
Date: Jun. 15, 2002
PubMed ID: 12045205
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