Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension

Hum Mol Genet. 2002 Jun 15;11(13):1517-25. doi: 10.1093/hmg/11.13.1517.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Protein Receptors, Type II
  • Bone Morphogenetic Proteins / metabolism
  • Cell Line
  • Cells, Cultured
  • Genes, Reporter
  • HeLa Cells
  • Humans
  • Hypertension, Pulmonary / genetics*
  • Hypertension, Pulmonary / metabolism
  • Ligands
  • Mice
  • Mitogen-Activated Protein Kinases
  • Mutation
  • Protein Serine-Threonine Kinases / genetics*
  • Protein Serine-Threonine Kinases / metabolism
  • Protein Structure, Tertiary
  • Protein Transport / physiology
  • Recombinant Proteins / metabolism
  • Transforming Growth Factor beta*
  • p38 Mitogen-Activated Protein Kinases

Substances

  • BMP2 protein, human
  • Bmp2 protein, mouse
  • Bone Morphogenetic Protein 2
  • Bone Morphogenetic Proteins
  • Ligands
  • Recombinant Proteins
  • Transforming Growth Factor beta
  • recombinant human bone morphogenetic protein-2
  • Protein Serine-Threonine Kinases
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • BMPR2 protein, human
  • Bmpr2 protein, mouse
  • Bone Morphogenetic Protein Receptors, Type II