BMP-2 antagonists emerge from alterations in the low-affinity binding epitope for receptor BMPR-II.
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Bone morphogenetic protein-2 (BMP-2) induces bone formation and regeneration in adult vertebrates and regulates important developmental processes in all animals. BMP-2 is a homodimeric cysteine knot protein that, as a member of the transforming growth factor-beta (TGF-beta) superfamily, signals by oligomerizing type I and type II receptor serine-kinases in the cell membrane. The binding epitopes of BMP-2 for BMPR-IA (type I) and BMPR-II or ActR-II (type II) were characterized using BMP-2 mutant proteins for analysis of interactions with receptor ectodomains. A large epitope 1 for high-affinity BMPR-IA binding was detected spanning the interface of the BMP-2 dimer. A smaller epitope 2 for the low-affinity binding of BMPR-II was found to be assembled by determinants of a single monomer. Symmetry-related pairs of the two juxtaposed epitopes occur near the BMP-2 poles. Mutations in both epitopes yielded variants with reduced biological activity in C2C12 cells; however, only epitope 2 variants behaved as antagonists partially or completely inhibiting BMP-2 activity. These findings provide a framework for the molecular description of receptor recognition and activation in the BMP/TGF-beta superfamily. (+info)
Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene.
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Familial primary pulmonary hypertension is a rare autosomal dominant disorder that has reduced penetrance and that has been mapped to a 3-cM region on chromosome 2q33 (locus PPH1). The phenotype is characterized by monoclonal plexiform lesions of proliferating endothelial cells in pulmonary arterioles. These lesions lead to elevated pulmonary-artery pressures, right-ventricular failure, and death. Although primary pulmonary hypertension is rare, cases secondary to known etiologies are more common and include those associated with the appetite-suppressant drugs, including phentermine-fenfluramine. We genotyped 35 multiplex families with the disorder, using 27 microsatellite markers; we constructed disease haplotypes; and we looked for evidence of haplotype sharing across families, using the program TRANSMIT. Suggestive evidence of sharing was observed with markers GGAA19e07 and D2S307, and three nearby candidate genes were examined by denaturing high-performance liquid chromatography on individuals from 19 families. One of these genes (BMPR2), which encodes bone morphogenetic protein receptor type II, was found to contain five mutations that predict premature termination of the protein product and two missense mutations. These mutations were not observed in 196 control chromosomes. These findings indicate that the bone morphogenetic protein-signaling pathway is defective in patients with primary pulmonary hypertension and may implicate the pathway in the nonfamilial forms of the disease. (+info)
Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family.
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BACKGROUND: Primary pulmonary hypertension (PPH), resulting from occlusion of small pulmonary arteries, is a devastating condition. Mutations of the bone morphogenetic protein receptor type II gene (BMPR2), a component of the transforming growth factor beta (TGF-beta) family which plays a key role in cell growth, have recently been identified as causing familial PPH. We have searched for BMPR2 gene mutations in sporadic PPH patients to determine whether the same genetic defect underlies the more common form of the disorder. METHODS: We investigated 50 unrelated patients, with a clinical diagnosis of PPH and no identifiable family history of pulmonary hypertension, by direct sequencing of the entire coding region and intron/exon boundaries of the BMPR2 gene. DNA from available parent pairs (n=5) was used to assess the occurrence of spontaneous (de novo) mutations contributing to sporadic PPH. RESULTS: We found a total of 11 different heterozygous germline mutations of the BMPR2 gene in 13 of the 50 PPH patients studied, including missense (n=3), nonsense (n=3), and frameshift (n=5) mutations each predicted to alter the cell signalling response to specific ligands. Parental analysis showed three occurrences of paternal transmission and two of de novo mutation of the BMPR2 gene in sporadic PPH. CONCLUSION: The sporadic form of PPH is associated with germline mutations of the gene encoding the receptor protein BMPR-II in at least 26% of cases. A molecular classification of PPH, based upon the presence or absence of BMPR2 mutations, has important implications for patient management and screening of relatives. (+info)
Characterization of receptors for osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7) in rat kidneys.
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BACKGROUND: Osteogenic protein-1/bone morphogenetic protein-7 (OP-1/BMP-7), a member of the transforming growth factor-beta superfamily, has been shown to prevent kidney damage from ischemia/reperfusion injury in rats. The molecular events involved in OP-1 action on kidney are not yet understood. METHODS: In this study, we evaluated the biodistribution of (125)I-labeled OP-1 in rat kidneys. Adult rats received a single intravenous injection of 250 microg (125)I-labeled OP-1 per kg body wt, a dose that was effective in protecting kidneys from ischemic injury. Tissue localization, in situ hybridization, and immunostaining with a specific receptor antibody were performed to identify OP-1 cellular targets. Also, isolated plasma membranes from kidney cortex and medulla regions were analyzed to identify and characterize receptor structural components that recognize OP-1. RESULTS: At 10 and 180 minutes following injection, the relative uptake of (125)I-labeled OP-1 was consistently higher in kidney cortex than in medulla region. Upon autoradiography, kidney tissue sections revealed that OP-1 bound to the convoluted tubule epithelium, glomeruli, and collecting ducts. Moreover, in situ hybridization and immunostaining methods have shown localization of mRNA transcripts and the protein for BMP receptor type II in the cortex and medulla in similar areas as (125)I-labeled OP-1. Bulk membranes (enriched with plasma membranes) isolated from the cortex and medulla regions of kidney each bound specifically to (125)I-OP-1, and the binding of (125)I-labeled OP-1 was inhibited by unlabeled OP-1 in a dose-dependent manner. However, platelet-derived growth factor, transforming growth factor-beta, insulin-like growth factor, fibroblast growth factors, and other members of BMP family such as BMP-2 and cartilage-derived morphogenetic protein-1/growth and differentiation factor-5 (CDMP-1/GDF-5) failed to inhibit the binding of (125)I-labeled OP-1 to receptors, suggesting a high degree of specificity with which OP-1 bound to kidney receptors. Scatchard analysis of quantitative binding data indicated that the OP-1 receptors of kidney contained a single class of high-affinity binding sites for OP-1 with an association constant (Ka) of 2.26 x 109 mol/L-1 and a binding capacity of 1.01 pmol of OP-1 per mg membrane protein. When analyzed by a ligand blot technique, plasma membranes isolated from kidney cortex and medulla each showed the presence of a prominent specific band with a relative molecular mass (Mr) of 100 kD. Further analysis by Western blotting indicated that an antibody raised against BMP type II receptor effectively recognized the 100 kD OP-1 binding component of kidney plasma membranes. CONCLUSIONS: We demonstrated, to our knowledge for the first time, the presence of membrane-bound, specific, high-affinity OP-1 receptors in rat kidney tissues, which are likely to mediate OP-1 actions in the kidney. The major OP-1-binding component of the kidney appears to be a long form of BMP type II receptor with a Mr of 100 kD. In vivo and in vitro evidence suggests that the cellular targets for OP-1 are convoluted tubule epithelium, glomeruli, and collecting ducts. OP-1 does not share receptor binding properties with other growth factors, including BMP-2 and CDMP-1, suggesting that its mode of action in kidney appears to be specific. (+info)
BMPR2 haploinsufficiency as the inherited molecular mechanism for primary pulmonary hypertension.
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Primary pulmonary hypertension (PPH) is a potentially lethal disorder, because the elevation of the pulmonary arterial pressure may result in right-heart failure. Histologically, the disorder is characterized by proliferation of pulmonary-artery smooth muscle and endothelial cells, by intimal hyperplasia, and by in situ thrombus formation. Heterozygous mutations within the bone morphogenetic protein type II receptor (BMPR-II) gene (BMPR2), of the transforming growth factor beta (TGF-beta) cell-signaling superfamily, have been identified in familial and sporadic cases of PPH. We report the molecular spectrum of BMPR2 mutations in 47 additional families with PPH and in three patients with sporadic PPH. Among the cohort of patients, we have identified 22 novel mutations, including 4 partial deletions, distributed throughout the BMPR2 gene. The majority (58%) of mutations are predicted to lead to a premature termination codon. We have also investigated the functional impact and genotype-phenotype relationships, to elucidate the mechanisms contributing to pathogenesis of this important vascular disease. In vitro expression analysis demonstrated loss of BMPR-II function for a number of the identified mutations. These data support the suggestion that haploinsufficiency represents the common molecular mechanism in PPH. Marked variability of the age at onset of disease was observed both within and between families. Taken together, these studies illustrate the considerable heterogeneity of BMPR2 mutations that cause PPH, and they strongly suggest that additional factors, genetic and/or environmental, may be required for the development of the clinical phenotype. (+info)
Mutation in the gene for bone morphogenetic protein receptor II as a cause of primary pulmonary hypertension in a large kindred.
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BACKGROUND: Most patients with primary pulmonary hypertension are thought to have sporadic, not inherited, disease. Because clinical disease develops in only 10 to 20 percent of persons carrying the gene for familial primary pulmonary hypertension, we hypothesized that many patients with apparently sporadic primary pulmonary hypertension may actually have familial primary pulmonary hypertension. METHODS: In a study conducted over 20 years, we developed a registry of 67 families affected by familial primary pulmonary hypertension. Through patient referrals, extensive family histories, and correlation of family pedigrees, we discovered shared ancestry among five subfamilies. We established the diagnosis of primary pulmonary hypertension by direct evaluation of patients and review of autopsy material and medical records. We assessed some family members for mutations in the gene encoding bone morphogenetic protein receptor II (BMPR2), which has recently been found to cause familial primary pulmonary hypertension. RESULTS: We linked five separately identified subfamilies that included 394 known members spanning seven generations, which were traced back to a founding couple in the mid-1800s. Familial primary pulmonary hypertension has been diagnosed in 18 family members, 12 of whom were first thought to have sporadic disease. The conditions of 7 of the 18 were initially misdiagnosed as other cardiopulmonary diseases. Six members affected with familial primary pulmonary hypertension and 6 of 10 at risk for carriage have been undergone genotype analysis, and they have the same mutation in BMPR2, a transversion of thymine to guanine at position 354 in exon 3. CONCLUSIONS: Many cases of apparently sporadic primary pulmonary hypertension may be familial. Failure to detect familial primary pulmonary hypertension results from incomplete expression within families, skipped generations, and incomplete family pedigrees. The recent discovery of mutations in BMPR2 should make it possible to identify those with susceptibility to disease. (+info)
Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia.
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BACKGROUND: Most patients with familial primary pulmonary hypertension have defects in the gene for bone morphogenetic protein receptor II (BMPR2), a member of the transforming growth factor beta (TGF-beta) superfamily of receptors. Because patients with hereditary hemorrhagic telangiectasia may have lung disease that is indistinguishable from primary pulmonary hypertension, we investigated the genetic basis of lung disease in these patients. METHODS: We evaluated members of five kindreds plus one individual patient with hereditary hemorrhagic telangiectasia and identified 10 cases of pulmonary hypertension. In the two largest families, we used microsatellite markers to test for linkage to genes encoding TGF-beta-receptor proteins, including endoglin and activin-receptor-like kinase 1 (ALK1), and BMPR2. In subjects with hereditary hemorrhagic telangiectasia and pulmonary hypertension, we also scanned ALK1 and BMPR2 for mutations. RESULTS: We identified suggestive linkage of pulmonary hypertension with hereditary hemorrhagic telangiectasia on chromosome 12q13, a region that includes ALK1. We identified amino acid changes in activin-receptor-like kinase 1 that were inherited in subjects who had a disorder with clinical and histologic features indistinguishable from those of primary pulmonary hypertension. Immunohistochemical analysis in four subjects and one control showed pulmonary vascular endothelial expression of activin-receptor-like kinase 1 in normal and diseased pulmonary arteries. CONCLUSIONS: Pulmonary hypertension in association with hereditary hemorrhagic telangiectasia can involve mutations in ALK1. These mutations are associated with diverse effects, including the vascular dilatation characteristic of hereditary hemorrhagic telangiectasia and the occlusion of small pulmonary arteries that is typical of primary pulmonary hypertension. (+info)
Severe pulmonary hypertension after the discovery of the familial primary pulmonary hypertension gene.
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The recent discoveries of the familial primary pulmonary hypertension gene and somatic mutations in key cell growth and cell death regulatory genes in primary pulmonary hypertension have added a new dimension to severe pulmonary hypertension research. These findings have already impacted on how the disease is viewed, and ultimately, how severe pulmonary hypertension is diagnosed and treated. However, this new information raises several fundamental questions related to the role of bone morphogenetic protein receptor signalling in the control of lung vascular cell function. Furthermore, additional genes and gene products may also be involved in the pathogenesis of the disease. The way severe pulmonary hypertension is viewed and studied is on the verge of shifting from a vasoconstrictive to a cell growth paradigm. (+info)