title: Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension.
abstract: 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.
authors.affiliation: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge CB2 2QQ, UK.
authors.forename: Nung
authors.initials: N
authors.lastname: Rudarakanchana
authors.forename: Julia A
authors.initials: JA
authors.lastname: Flanagan
authors.forename: Hailan
authors.initials: H
authors.lastname: Chen
authors.forename: Paul D
authors.initials: PD
authors.lastname: Upton
authors.forename: Rajiv
authors.initials: R
authors.lastname: Machado
authors.forename: D
authors.initials: D
authors.lastname: Patel
authors.forename: Richard C
authors.initials: RC
authors.lastname: Trembath
authors.forename: Nicholas W
authors.initials: NW
authors.lastname: Morrell
mesh_terms: D000818:Animals; D055396:Bone Morphogenetic Protein 2; D052006:Bone Morphogenetic Protein Receptors, Type II; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D006976:Hypertension, Pulmonary / Q000235:genetics* / Q000378:metabolism; D008024:Ligands; D051379:Mice; D020928:Mitogen-Activated Protein Kinases; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D016212:Transforming Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D008024:Ligands; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C498218:BMPR2 protein, human; C498219:Bmpr2 protein, mouse; D052006:Bone Morphogenetic Protein Receptors, Type II