Abstract 5659: Heterozygous Mutation in the Carboxyl-Terminal Domain of the Type 2 Bone Morphogenetic Protein Receptor Leads to Ligand-Specific Gain of Function
Heterozygous mutations in the gene encoding the bone morphogenetic protein (BMP) type II receptor (BMPR2) are associated with familial and idiopathic pulmonary arterial hypertension. These mutations are found to affect all domains of the protein, including the amino-terminal ligand-binding domain, the intracellular kinase domain, and the long cytoplasmic carboxyl-terminal (CT) domain. Binding of BMP ligands to BMPR2 and BMP type I receptors leads to phosphorylation of SMAD 1, 5, and 8 and expression of genes including Id1. In pulmonary artery smooth muscle cells (PaSMC) isolated from mice with a heterozygous nonsense mutation in the BMPR2 amino-terminal domain (BMPR2+/−), BMP4 and BMP7 signaling is reduced, whereas deletion of both BMPR2 alleles paradoxically augments BMP7 signaling. However, the impact of heterozygous mutations in the BMPR2 CT domain on BMP signaling is still unknown. To characterize the function of the BMPR2 CT domain, we generated a mouse (BMPR2SG/+) in which BMPR2 exon 12 (encoding the CT domain) was deleted, and a sequence specifying green fluorescent protein (GFP) was inserted in frame after exon 11, resulting in a BMPR2 short form-GFP fusion protein (SG). Using qRT-PCR and primers for exons 4 and 5, similar levels of BMPR2 transcripts were detected in PaSMC isolated from wild-type (WT) and BMPR2SG/+ mice. In contrast, using primers for exon 12, there was a 50% reduction of BMPR2 transcripts in BMPR2SG/+ PaSMC. Similarly, immunoblots performed with a CT-specific antibody revealed that WT BMPR2 protein levels were 50% less in BMPR2SG/+ than in WT PaSMC. SG protein expression was confirmed using an anti-GFP antibody. Induction of SMAD1/5/8 phosphorylation and Id1 gene expression in response to BMP4 (10 ng/ml) did not differ between WT and BMPR2SG/+ PaSMC. However, the induction of SMAD1/5/8 phosphorylation and Id1 gene expression by BMP7 were both more than 2-fold greater in BMPR2SG/+ than WT PaSMC. On the other hand, BMPR2+/− PaSMC had reduced SMAD1/5/8 activation and Id1 transcription in response to both BMP4 and BMP7. In conclusion, our findings suggest that heterozygous mutations in the BMPR2 CT domain may lead to gain of signaling for a subset of BMP ligands, including BMP7.