Human Molecular Genetics

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Human Molecular Genetics, 2002, Vol. 11, No. 13 1517-1525
© 2002 Oxford University Press

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

Nung Rudarakanchana^1,, Julia A. Flanagan^2,, Hailan Chen¹, Paul D. Upton¹, Rajiv Machado², D. Patel², Richard C. Trembath^2, and Nicholas W. Morrell^1,*

¹Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's and Papworth Hospitals, Cambridge CB2 2QQ, UK and ²Division of Medical Genetics, University of Leicester, Leicester LE1 7RH, UK

Received February 18, 2002; Accepted April 15, 2002

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 ¹²⁵I-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.

^* To whom correspondence should be addressed at: Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Box 157, Hills Road, Cambridge CB2 2QQ, UK. Tel: +44 1223 336744; Fax: +44 1223 762007; E-mail: nwm23{at}cam.ac.uk

Correspondence may also be addressed to Professor Richard C. Trembath, Division of Medical Genetics, Departments of Medicine and Genetics, Adrian Building, University of Leicester, Leicester LE1 7RH, UK. Tel: +44 116 2522263; E-mail: rtrembat{at}hgmp.mrc.ac.uk

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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