The Author 2008. Published by Oxford University Press. All - Human

Stoichiometric imbalance in the receptor complexcontributes to dysfunctional BMPR-II mediatedsignalling in pulmonary arterial hypertension

M. Talat Nasim1,�, Amar Ghouri2, Bhakti Patel2, Victoria James2, Nung Rudarakanchana3,

Nicholas W. Morrell3 and Richard C. Trembath1,�

1Department of Medical and Molecular Genetics, King’s College London, Guy’s Hospital, London SE1 9RT, UK,2Department of Genetics, University of Leicester, Leicester LE1 7RH, UK and 3Department of Medicine, University of

Cambridge, Cambridge, UK

Received December 21, 2007; Revised and Accepted February 22, 2008

Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins(BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hyper-tension (PAH). However, the precise molecular consequences of PAH causing mutations on the function ofthe receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based tech-niques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD)and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD,providing further evidence that haplo-insufficiency is a major molecular consequence of disease-relatedBMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impairedtype I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leadingto perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intra-cellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally andto address the potential for resolution of stoichiometric balance, we investigated an agent that promotestranslational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway.We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor–receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have ident-ified an important target for early therapeutic intervention in familial PAH.

INTRODUCTION

Bone morphogenetic proteins (BMPs) are required for cellulardifferentiation and mammalian development (1). BMP signal-ling is transduced by one of two pathways, each with distinctcellular outcomes. In the first, ligand stimulation triggersassembly of the receptor complex through rapid associationand diffusion. In the second pathway, the receptor and itsassociated components exist in a so-called ‘pre-formedcomplex’. Activation of the receptor complex generates aphosphorylation relay of cytoplasmic signalling proteins,including the Smad family, which translocate to the nucleus

to directly regulate gene transcription (2). BMPs andtransforming growth factors (TGF-b) have also been reportedto activate Smad independent signalling, including ERK, JNKand p38MAPK (3–6).

The BMPR2 gene, comprising 13 exons, encodes a type IIreceptor for bone morphogenetic proteins (BMPR-II), amember of the TGF-b receptor family. Alternative splicingof exon 12 may give rise to a long isoform (LF), generatinga protein product of 1038 amino acids, or a shorter transcriptof poorly defined functional significance. The mature receptorconsists of four functional regions, namely the ligand-binding,transmembrane, kinase and C-terminal domains.

�To whom correspondence should be addressed. Tel: þ44 2071889505; Fax: þ44 2071882585; Email: [email protected] (T.N.);Tel: þ44 2071887994; Fax: þ44 2071888050; Email: [email protected] (R.C.T.)

# The Author 2008. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2008, Vol. 17, No. 11 1683–1694doi:10.1093/hmg/ddn059Advance Access published on March 4, 2008

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Heterozygous mutations of BMPR2 have been identified insubjects with the progressive vascular disorder pulmonary arter-ial hypertension (PAH) (7). PAH is characterized by the abnor-mal proliferation of endothelial and smooth muscle cells. Theresultant vascular remodelling of the small pulmonary arteriesleads to elevated pulmonary artery pressure and, in the absenceof effective therapy, eventual right-heart failure (8,9).

To date, over 200 independent heterozygous mutations havebeen detected in PAH subjects of which �70% introduce apremature termination codon (PTC). Typically, PTC harbour-ing transcripts are degraded by a process termed nonsense-mediated decay (NMD) (10). However, these mutations canalso lead to splicing anomalies including exon skipping oralteration of exonic and intronic splicing enhancer (ESE andISE) and suppressors (ESSs and ISSs) (11–14).

Missense mutations are dispersed across all four domains ofthe receptor and have heterogeneous functional consequences.The predominant type of missense mutations in the ligand-binding domain is cysteine substitutions that impair signallingdue to mutant receptor mislocalization in the cytosol (15).Interestingly, a small number of non-cysteine mutations havebeen reported in PAH subjects with associated congenitalheart disease (CHD) and in patients previously exposed toamphetamine like agents, a known risk factor for disease(16–18). Mutant receptors bearing non-cysteine substitutionslocalize to the cell surface but also exhibit defects in signallingactivity (4,15,19). By contrast, mutations in the cytoplasmicC-terminal domain only moderately inhibit Smad-signalling(4,15,19).

In this report, we investigated the functional consequencesof a wide range of BMPR2 mutations, identified in subjectswith familial, spontaneous or ‘idiopathic’ and associatedforms of PAH. In addition, we assessed an agent for its capa-bility to promote translational read-through in constructs har-bouring BMPR2 nonsense mutations. Together, these studiessuggest that dysregulated stoichiometry of the multi-proteinreceptor complex contributes to aberrant BMPR-II signallingin PAH and that restoration of the stoichiometric balance bystimulating translational readthrough of mutant alleles mightprovide an effective therapeutic intervention prior to or fol-lowing the onset of disease.

RESULTS

Effect of mutations introducing PTC on BMPR2 mRNAexpression

As 70% of mutations are predicted to introduce PTC in theBMPR2 reading frame, we first investigated the impact oftypical mutations upon BMPR2 mRNA expression. Usingcomputer simulation, such as GENSCAN, we first investigatedthe consequence of nonsense mutations on BMPR2 codingstructure. GENSCAN predicted that insertion (c.2292insA),deletion (c.2386delG) and nonsense mutations (c.2620G.Tand c.2695C.T) within exon 12 of BMPR2, each might acti-vate cryptic splice sites (see supplementary Material,Table S2). If correct, this would lead to the skipping of thenonsense codon and maintenance of a reading frame (Fig. 1A).

To further investigate the consequence of mutations pre-dicted by GENSCAN, we utilized a novel dual-fluorescence-

based assay system (Nasim et al., 2008, submitted forpublication). In this system, successful splicing would leadto the production of DsRed-Express-GFP fusion protein(Fig. 1B). Expression of only the upstream reporter wouldoccur should a pre-mature termination codon (PTC) be recog-nized as a translation stop signal. By way of contrast, shouldPTC trigger aberrant splicing, a shorter form of the fusionprotein would be produced. Fluorescence microscopy revealedthat the wild-type sequence underwent splicing leading to theexpression of both fluorescence proteins (Fig. 1C). However,each of the BMPR2 mutations tested namely, c.2292insA,c.2386delG, c.2620G.T and c.2695C.T failed to producethe dual-fluorescence reporter indicating that mutation hadled to the incorporation of a translation stop signal withinexon 12. Direct RNA analysis confirmed that the dual-fluorescence was consequent upon efficient splicing of wild-type sequence (Supplementary Material).

Mutations introducing PTCs are subject tononsense-mediated RNA decay

We next sought to investigate whether PTC bearing mutationsof BMPR2 triggered the NMD machinery. To study this, weemployed a novel enzyme-based NMD assay (Nasim et al.,2008, submitted for publication). The system was developedin such a way that the incorporation of the PTC would leadto the truncation of a full-length protein (Fig. 2A). Thus, ifthe mRNA is subjected to NMD, inhibition of translationusing cycloheximide or puromycin would lead to the increasedlevel of PTC bearing mRNA with a consequence of increasedexpression of the truncated protein. Transfection of the repor-ter constructs into HEK293 cells led to a significant increase inthe reporter protein following treatment with cycloheximide(5-fold) and puromycin (20-fold) (Fig. 2B). To confirm thatthese mutations were subject to NMD, plasmids encodingsiRNA that inhibit expression of hUpf1 (20) were transfectedtogether with the reporter plasmid. Compared with cells trans-fected with the reporter alone, the gal-luc ratio increasedbetween 3 and 6-fold when hUpf1 RNA was knocked downby RNAi (Fig. 2C). Finally, we demonstrated NMD in pul-monary artery smooth muscle cell lines (21) harbouring a non-sense mutation (exons 4 and 5 deleted) derived frombmpr2þ/2 mice (22) (Fig. 2D).

Mutation in the exon or intron modulates BMPR2pre-mRNA splicing

Point mutations may dysregulate splicing through the creationof ESSs or by disruption of ESEs (23,24). To investigate spli-cing efficiency, we examined PAH causing mutations ofBMPR2 exon 9 by constructing a splicing reporter adaptedfrom a recently reported method (25). The efficiency of spli-cing of the wild-type BMPR2 sequence was established as100%. These studies revealed that mutation at c.1270T.Chad no significant impact yet and in contrast, c.1259G.A sub-stantially reduced splicing efficiency (Fig. 3B).

We next investigated whether BMPR2 mutations maytrigger alternative splicing using an exon-trapping reporterderived from a previously described system (26). Mutationswere selected as follows; an exonic mutation (c1241G.A)

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predicted to introduce a PTC (p.W414X) and an intronic(c.1276þ4A.G) mutation located in a conserved (70–75%)consensus 50 splice site. RNA analysis revealed that the wild-type sequence led to significant incorporation of exon 9. Incontrast, mutation in the intron at position c.1276þ4A.Gtriggered exon skipping whereas the c.1241G.A mutationhad no discernible effect on exon incorporation (Fig. 3D).

BMPR-II mutations exhibit differences in transcriptionalactivity of a BMP-responsive reporter

Having established the consequence of mutation on receptorexpression, we next sought to investigate the functional

activities of wild-type and mutant BMPR-II. Smad-inducedtranscriptional activity mediated by wild-type BMPR-II inthe presence and absence of BMP4 ligand were examined using3Gc2wt-Lux in HEK 293 cells. The activity of 3Gc2wt-Luxreporter, a BMP responsive promoter–reporter construct (27)was enhanced between 3 and 6-fold following the overexpres-sion of BMPR-II and BMP4 stimulation, respectively(Fig. 4A), and was further increased to 20-fold when cellstransfected with BMPR-II were stimulated by BMP4. Wenext tested a range of mutations (Fig. 4B) including atypicalnon-cysteine substitutions within the ligand-binding domain(p.Q42R, p.G47N, p.Q82H), the linker region between trans-membrane and the kinase domain (p.G182D, p.M186V), the

Figure 1. Effect of mutation on BMPR2 transcript integrity. (A) A schematic comparison between the wild type (left panel) and mutant pre-mRNA processing(right panel). GENSCAN predicts that mutation activates cryptic splice sites in the exon 12 which leads to the skipping of the PTC. 50 and 30 splice sites areindicated by 50ss and 30ss, respectively. (B) Diagram of the single-cell-based dual-fluorescence assay system. The detail of system was described elsewhere(Nasim et al., 2008, submitted for publication). In brief, the Exon 12 of BMPR2 was introduced in a manner such that efficient splicing produced a DsRed-GFPfusion protein. Effect of mutation in the exon 12 may impact on transcript integrity in either of the two ways. In the event of PTC-associated skipping as pre-dicted by Genscan (Supplementary Material, Table S2), a dual-fluorescence protein was produced. In the event that recognized PTC as translational stop codon,would lead to the production of the DsRed-Express protein. The location of mutation is indicated. (C) HEK 293 cells transfected with the wild-type construct(pTN139; Fig. 1Ci) generated both DsRed-Express and GFP fluorescence proteins whereas frameshift (c.2292insA; pTN140; Fig. 1Cii), deletion (c.2386delG;pTN141; Fig. 1Ciii) and nonsense mutations (c.2620G.T; pTN142; Fig. iCiv and c.2695C.T; pTN143; Fig. 1Cv) only produced DsRed-Express protein.Figure 1Cii and Ciii is described elsewhere (Nasim et al., 2008, submitted for publication) and included here for comparison.

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kinase domain (p.D485G, p.E503D) and the C-terminaldomain (p.R899X, p.R899P) for their ability to stimulate thereporter. Of interest, the transcription activities stimulated bythe mutants at position p.Q42R, p.G47N, p.G182D,p.M186V and p.E503D were comparable with that stimulatedby the wild-type receptor (Fig. 4C). In contrast, p.Q82H,p.D485G and p.R899X mutants were unable to activate repor-ter expression, while p.R899P construct generated a slightincrease in activity. These data indicate that the basal activityof these three constructs as measured by stimulation of thereporter was abrogated. We next investigated whether stimu-lation with BMP4 ligand was capable of inducing a response.BMP4 upregulated the activity of the mutant p.R899X recep-tor albeit to a level lower than that of wild type (Fig. 4D),whereas p.Q82H, p.D485G and p.R899P mutants remainedseverely functionally impaired. In contrast, the remainingmutations displayed similar signalling characteristics to thewild-type receptor.

As type I receptors are involved in BMP mediated signal-ling events, we next investigated their impact on reporter acti-vation. The effects of type I receptors, namely ALK3 (also

known as BMPR1A) and ALK6 (BMPR1B) on the ability ofBMPR-II to stimulate transcription were examined by overex-pressing plasmids encoding ALK3 and ALK6 along withBMPR-II (Fig. 4E). The activities of the BMPR-II stimulatedreporter were further increased between 5 and 6-fold by over-expression of ALK3 and ALK6, respectively. Both ALK3and ALK6 promoted the activity of p.Q82H mutant to alevel comparable to the wild type. In the presence of thetype I receptors, the p.R899P and p.R899X mutants exhibitedincreased signalling activity. However, the level of augmenta-tion was lower than that of wild type. Neither ALK3 norALK6 stimulated the p.D485G mutant. We next tested the

Figure 2. (A) The outline of the test system for determining nonsense-mediated RNA decay was described elsewhere (Nasim et al., 2008, submittedfor publication). The system is based on the reporter gene encodingb-galactosidase, which was fused in-frame with the PTC (c.2386delG,c.2292insA) bearing recombinant double intron splicing unit (for details seeMaterials and Methods and Supplementary Material, Fig. S2). The locationsof the mutations are indicated. Plasmids bearing c.2292insA (pTN147) andc.2386delG (pTN148) mutations were transfected into HEK293 cells andgrown in the presence or absence of NMD inhibitors including puromycinand cycloheximide. The treatments increased the reporter activity between 5and 20-fold (Fig. 2B). Compared with cells transfected with the reporteralone, the activity increased between 3 and 6-fold when hUpf1 RNA wasknock down by RNAi (Fig. 2C). Transfection efficiency was normalized bytransfecting an independent plasmid expressing luciferase gene. Dataderived from 4 to 10 individual experiments are presented as the ratio ofbgal and luciferase activities. The data of chemical treatments and Upf1/IsiRNA on pTN148 were described elsewhere (Nasim et al., 2008, submittedfor publication) as proof-of-principle and are included here for comparison.Standard deviations are indicated by error bars. Quantitative RT–PCR on apulmonary artery smooth muscle cell (PASMC) line harbouring a nonsensemutation (exons 4 and 5 deletion) derived from bmpr2þ/2 mice treated withpuromycin rescued PTC bearing mRNA (Fig. 2D).

Figure 1. Continued.


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impact of BMP4 on ALK3þBMPR-II and ALK6þBMPR-IIstimulated reporter. With the exception of p.D485G mutant,BMP4þALK3 increased observed activity of all mutants, toa level comparable to wild type. BMP4þALK6 mediatedincrease for mutants p.R899X and p.R899P was significantlylower than that of wild type (Fig. 4F). The p.D485G mutantwas not simulated at all.

Mutation in the kinase domain of BMPR-II affects type Ireceptor binding and phosphorylation

As concomitant overexpression of type I and type II receptorsenhanced transcriptional activation of a reporter construct, wenext examined whether these two receptors physically inter-acted and sought the consequence of mutation upon the inter-action. To investigate whether BMPR-II binds ALK3, weperformed co-immunoprecipitation experiments in Hela cellsusing plamids encoding myc-tagged wild-type BMPR-II andmutant receptors (p.C118W, p.C483R, p.D485G, p.N519K)along with HA-tagged ALK3. As shown in Figure 5A, thewild-type BMPR-II binds to ALK3. Mutation in the ligand-binding domain has no discernible effect on the interaction

but the ability of the p.D485G, p.C483R and p.N519Kmutants to bind ALK3 is markedly reduced. We then testedthe ability of wild-type and mutant BMPR-II constructs tophosphorylate the type I receptor (Fig. 5B). By comparisonto wild type, both the p.R899X and p.N519K mutants demon-strated a reduced ability to phosphorylate ALK6, whereas bothp.D485G and p.C483R were effectively unable to mediatephosphorylation.

The efficiency of type I and type II receptor interactions

To identify the functional domains required for receptor inter-actions and determine the efficiency of interactions, we devel-oped a novel method based on a dual-light reporter system(28). In brief, the red fluorescence protein signals regardlessof receptor–receptor interactions (Fig. 6A) and acts as a refer-ence standard. In the event of a protein–protein interactionboth red and green fluorescence proteins are produced.Expression of the GFP protein was not activated when reporterwas transfected alone (6Bi) or co-transfected with LF (6Bii)and p.D485G (6Biii). The expression of GFP was activatedwhen ALK3 was transfected with BMPR-II (6Bv), which

Figure 3. Effects of mutation on splicing efficiency and alternative splicing. (A) Diagram of the dual-reporter construct to determine the splicing efficiency of theBMPR2 exon 9. Unspliced but transported RNA was translated to produce b-galactosidase protein, whereas the spliced RNA was translated to produceluciferase-b-galactosidase fusion protein. XXX indicates translation termination signals located in the intron. (B) Effect of mutation on splicing efficiency.The locations of the mutation are indicated. Wild-type and mutant plasmids were transfected into HEK293 cells and luciferase and b-galactosidase activitieswere determined and expressed as ratios, normalized to value of 100 with wild type. Standard deviations of normalized ratios are indicated by error bars.(C) Diagram of the exon-trapping system to determine alternative splicing (exon incorporation versus skipping). In the events of exon skipping and incorporationan mRNA is produced, where exon 9 is excluded and included, respectively. (D) Analysis of wild-type and mutant mRNAs by RT–PCR and gel electrophoresisthat had skipped (lower band and filled arrow) and included (higher band and empty arrow) exon 9 after tranfection of the respective plasmids into HEK293 cellline.


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demonstrated that the intracellular domains of both receptorsare necessary and sufficient for interactions. The expressionof GFP was not detected following the overexpression ofALK3 together with the p.D485G mutant form of aBMPR-II receptor.

To determine the efficiency of interaction of BMPR-II andALK3, we employed a gal-luc-based dual-reporter assaysystem. The dual-reporter (28) (pTN114) along with therespective plasmids was transfected into HEK293 cells andtheir luciferase and b-galactosidase activities were deter-mined. Cells overexpressing recombinant wild type,p.D485G and ALK3 alone failed to activate luciferase

expression (Fig. 6C). However, that the expression of lucifer-ase was activated by overexpression of plasmids containingALK3 and BMPR-II further confirmed that both proteins inter-acted with each other via the intracellular domains. We nextinvestigated the intensity of interaction of BMPR-II withALK3 or ALK6. The gal-luc ratio indicated that ALK3 inter-acts more efficiently with BMPR-II compared to ALK6(Fig. 6D). Since the reporter assays suggest that BMPR-IIinteracts with ALK3 and ALK6, we next sought to investigatethe consequences of a series of missense mutations located inthe kinase domain of BMPRII on receptor complex inter-actions. For the mutations at position p.A313P, p.C347Y,

Figure 4. (A) Cells transfected with a BMP/Smad luciferase (Lux) plasmid along with or without a plasmid harbouring the BMPR2 gene demonstrated increasedluciferase activity in response to BMP-4 stimulation. Luciferase activity was normalized with b-galactosidase activity to a value of 100 with Lux. (B) An outlineof the BMPR-II mutations. (C) Overexpression of BMPR-II increased luciferase activity but mutations at positions p.Q82H, p.D485G, p.R899X and p.R899Pfailed to stimulate Smad mediated transcription. (D) As (C), but cells were stimulated with BMP-4 (50 ng/ml). BMP-4 stimulated normalized value of the Luxwas set as 100. (E) Effects of ALK3 and ALK6 on the ability of BMPR-II to stimulate transcription. Overexpression of ALK3 and ALK6 enhanced BMPR-IIstimulated reporter activity. The ability of BMPR-II to stimulate transcription is completely abolished when p.D485G was mutated. Mutations at positionsp.R899X and p.R899P were able to stimulate reporter expression but at reduced level. BMPR-II stimulated normalized value of the Lux was set as 100. (F)As (E) but cells were stimulated with BMP-4. Plasmids encoding wild-type and mutant BMPR2 activated reporter activity but mutation at position p.D485Ghad severely abolished the activity. BMP-4 and BMPR-II stimulated normalized value was set as 100.


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p.D485G and p.R491Q, interactions with ALK3 and ALK6were impaired (Fig. 6E). In contrast, p.L401S and p.E503Dhad no discernible effect upon these receptor interactions.

Translational readthrough of nonsense codons byaminoglycosides

Having demonstrated that PAH causing nonsense mutation ofBMPR2 triggers the NMD pathway, we next progressed toinvestigate an approach by which a significant amount func-tional protein can be generated from a nonsense transcript.As the NMD pathway depends on a set of factors that modu-

late both RNA stability and translation termination efficiency,inactivation of any of these stabilizes nonsense transcripts andpromotes translation readthrough (10). Aminoglycosides suchas gentamicin have been shown to promote translational read-through of PTC in mammalian cells and animal models ofnonsense-associated diseases permitting expression of full-length functional protein (29–31). We, therefore, sought todetermine whether such agents might protect BMPR2 non-sense transcripts from NMD. To investigate the effect of ami-noglycosides on transcript stability, pulmonary artery smoothmuscle cells derived from a PAH subject harbouring a non-sense mutation (p.W9X) were grown in the presence andabsence of gentamicin. QPCR data indicated that the levelof BMPR2 transcript was significantly reduced in p.W9Xcell line, providing evidence of NMD. We observed no signifi-cant increase in the level of BMPR2 transcript following gen-tamicin treatment (Fig. 7A). In the absence of an antibody forreliable detection of endogenous full-length BMPR-II proteinin a quantitative assay, we adapted a cell-based approach tomeasure the impact of gentamicin exposure. We employed adouble reporter to determine the efficiency of stop codon sup-pression (Fig. 7B). We found that the treatment enhancedtranslational readthrough of the reporter construct (Fig. 7C).Finally, a reporter construct containing PTC introduced by aBMPR2 insertion mutation (Fig. 7D) showed an enhancedlevel of beta-galactosidase activity following gentamicinexposure (Fig. 7E).

DISCUSSION

The demonstration that heterozygous mutations of the BMPR2gene underlie inherited PAH has provided a significant oppor-

Figure 5. (A) Co-immunoprecipitation of myc-tagged BMPR-II wild-type andmutant protein with HA-tagged ALK3 from transfected HeLa cells. Bottomtwo panels, expression of ALK3 and BMPR-II protein was confirmed byimmunoprecipitation followed by immunoblotting with anti-HA andanti-myc antibodies, respectively. Top panel, cell lysates were immunopreci-pitated for one epitope tag followed by immunoblotting for the secondepitope tag. These data represent results from six individual experiments.Wt, wild type; L1, p.C118W; K1, p.C483R; K2, p.D485G; C1, p.N519K,T1, p.S532X. (B) In vitro kinase assay. BMPR-II wild type, mutant or shortform protein kinase activity as assessed by phosphorylation of ALK6. Top,an autoradiograph of 32P-labelled phosphorylated ALK6. Middle, the sameALK6 protein band stained with coomassie blue. Bottom, a bar graph repre-senting the 32P counts per minute values above control measurements of thelabelled ALK6 gel protein bands as assessed by phosphorImage analysis.These results are representative of three individual experiments.

Figure 6. (A) An outline of the dual-light reporter system to determine theefficiency of type I and type II receptors interactions. In the absence of inter-acting receptors the reporter A is expressed, whereas reporters A and B bothare expressed in the event of an interaction. (B) Upon transfection of the dual-fluorescence plasmid (pTN126), the expression of GFP protein was activatedas shown, where cells were transfected with (i) reporter alone, orco-transfected with (ii) BMPR-II and (iii) p.D485G mutant. The expressionof GFP was visualized when ALK3 was transfected with wild-typeBMPR-II (v) but not with the D485G mutant (vi). (C) The reporter plasmid(pTN114) along with plasmids encoding BMPR-II and ALK3 were overex-pressed into HEK 293 cells and both reporter activities were normalized toa value of 100 with BMPR-II and ALK3. Overexpression of plasmids encod-ing mutant BMPR-II (p.D485G) and ALK3 could not activate luciferaseactivity. (D) BMPR-II interacted more efficiently with ALK3 than that ofALK6. (E) Mutation at position p.A313P, p.C347Y, p.D485G and p.R491Qseverely affected ALK3 and ALK6 interactions.


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tunity to advance our understanding of the molecular basis ofthis typically fatal disease. However, it remains a significantchallenge to resolve the consequence of BMPR2 defects inpredisposing to vascular disease. In an earlier study, wehypothesized that PAH causing nonsense mutations ofBMPR2 are likely to follow the NMD pathway and thus gen-erate a state of haploinsufficiency for the BMPR-II protein(32). We have used a number of independent approaches togenerate a body of data to support this proposal. First, utilizingnovel fluorescence-based assays, we found that mutations thatintroduce a PTC fail to stimulate aberrant splicing (Sup-plementary Material, Figs S1 and S2), a possible consequenceof nonsense mutation and one predicted by computer simu-lation (see Supplementary Material). In this study, we investi-gated a range a disease causing defects that reflects themutation diversity identified in PAH cohorts (33). Secondly,

we wished to perform quantitative assay of BMPR2 tran-scripts, but were hampered by the limited availability ofsamples of relevant cell type from subjects with PAH. We,therefore, developed an enzyme-based reporter assay that pro-vided evidence of transcripts loss which was shown by use ofinhibitors to be due to activation of NMD pathway (Fig. 2A–B). We confirmed these data by analysis of ex-vivo mouse (22)and human cells (34) (Figs 2D–E and 7A). Taken together, weconclude that the nonsense mutations reduce the abundance ofmRNA through the NMD pathway and thereby generatehaplo-insufficiency for BMPR-II protein, a situation that con-tributes to stoichiometric imbalance of the receptor complex(Fig. 8).

We next sought to gain insight as to the molecular conse-quences of less frequently observed class of defects, namelymissense mutations. Direct association of receptor associationas assayed by co-immunoprecipitation, unexpectedly demon-strated that type I and type II receptor interactions do nottake place through the ligand-binding domain (Fig. 5A). Thekinase domain appears both necessary and sufficient for recep-tor–receptor interactions as proteins containing amino-acidsubstitutions within this region are unable to precipitate thetype I receptor. These findings were further supported bydynamic activity-based assays performed using living cells(Fig. 6). In these studies, we confirmed that type I and typeII receptors are capable of interacting with each otherthrough the intracellular domains, an interaction that appearsindependent of ligand stimulation. We suggest that theseobservations provide further insight as to the mechanisms ofBMPR-II mediated signalling. It has previously been specu-lated that ligand binding to either the type I or type II receptor,brings receptor components into close proximity and therebyleads to the formation of a functional complex. We interpretour data to demonstrate that receptors form a functionalcomplex but do so through their intracellular domains, afinding that provides a molecular basis for the existence ofso-called ‘preformed receptor complex’ (35).

Having demonstrated the significance of the kinase domainfor receptor–receptor interplay, we progressed to study theimportance of specific residues by interrogation of BMPR2missense mutations identified within this region. We foundthat a majority of such defects were severely impaired intype I receptor interactions (Figs 5 and 6), which indicatedthat several residues (e.g. p.A313P, p.C347Y, p.D485G andp.R491Q) are more important than others (p.L401S) to retainthe interaction. Interestingly, among these mutationsp.D485G, pC347Y and p.R491Q were defective inBMPR-II-mediated signalling (15).

We next wished to investigate less frequently observedsequence variation detected in subjects where the developmentof PAH occurred in association with other recognized riskfactors for disease, namely the presence of CHD or exposureto appetite suppressants. As representative of this class of vari-ation, we selected the p.E503D variant. Interestingly, wefound that this amino-acid substitution was capable of interact-ing with type I receptors suggesting that such a receptorcomplex may be capable of conveying signalling. As pre-dicted, p.E503D generated Smad-dependent signalling atlevels more or less comparable to that seen in wild type(Fig. 4). We therefore investigated additional reported



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amino-acid substitutions from this class. Importantly, wefound that many of these DNA variants conveyedBMPR-II-mediated signals following ligand stimulation andtype I receptor overexpression (Fig. 4). We thus concludethese variants do not share functional consequences withthose previously characterized in BMPR2 mutations identifiedin familial PAH (16–18) findings that raise the question as towhether they may more appropriately be described as variantsof unknown clinical significance. We acknowledge, however,that we cannot rule out that such variants may exhibit moresubtle effect on either BMPRII expression or function and

specifically in relation to the recognized critical role of thispathway during mammalian development.

We therefore propose that stoichiometric imbalance due tohaploinsufficiency and/or optimal receptor component inter-action is the major molecular consequence of pathogenicmutations contributing to dysfunctional BMPR-II-mediatedsignalling in PAH (Fig. 8). Restoration of stoichiometryeither by increasing the expression of full-length protein orpromoting the receptor interactions might provide protectionfrom disease development. To this end, we tested whetherthe aminoglycosides can promote readthrough of nonsense

Figure 7. Effect of genatimicin on RNA stability and translational readthrough. (A) PAH cell line harbouring nonsense mutation showed reduced level ofBMPR2 transcript as quantified by QPCR. Gentamicin treatment showed no significant increase in the level of BMPR2 transcript. (B) An outline of thedouble reporter for determining the efficiency of stop codon suppression described elsewhere. Briefly, in the event of translation termination a beta-galactosidaseprotein will be produced, while stop codon suppression will generate a beta-galactosidase and luciferase fusion protein. Thus, the ratio of luciferase and beta-galactosidase activity represents the efficiency of translation readthrough. (C) Cells transfecting with the reporter showed an increased gal-luc ratio followinggentamicin treatment. (D) An outline of the single reporter-based assay (pTN147; see Fig. 2 and Supplementary Material for details) harbouring PTC introducedby BMPR2 frameshift mutation. Translation termination generates a beta-gal protein, which is extended in the C-terminal region by translation readthrough. (E)Gentamicin treatment markedly increased beta-gal activity. An independent luciferase reporter was used as reference standard.


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mutations found in PAH. Aminoglycosides such as gentamicinhave been shown to enhance translational readthrough inmammalian cells (29,30) and in animal models of nonsensemutation diseases including Duchenne muscular dystrophyand cystic fibrosis (CF) (31,36). Indeed, gentamicin-dependentcorrection of CF transmembrane conductance regulator(CFTR) function has been reported in early clinical transla-tional studies (37). In our work, we observed that treatmentof cells, to which we had introduced a BMPR2 reporter con-struct harbouring a nonsense codon, promoted reporter activitywithout affecting NMD pathway. These observations togetherwith increasing interest in the development of chemicals thatspecifically target nonsense mutations provide a strong plat-form for further studies that should examine the clinical poten-tial for the treatment of nonsense-associated PAH mutations.

MATERIALS AND METHODS

The details of the plasmid construction and list of the con-structs (Supplementary Material, Table S1) used in thisstudy are available upon request.

The effect of mutation on BMPR2 transcript integrity wasanalysed by GENSCAN (38) (http://genes.mit.edu/GENSCAN.html) algorithm. Gene transfer, cell culture, repor-ter assays, fluorescence microscopy, co-immunoprecipitation,treatments with chemicals and siRNAs were performed asdescribed elsewhere (12,20,25,26,28,29,39). The amount ofplasmid DNA transfected into the HEK293 cell line variedfrom (from 30 ng to 1 mg) assay to assay.

Quantitative PCR for determining BMPR2 transcripts wasperformed using TaqMan Gene Expression Assay (AppliedBiosystems) on 7900HT Fast Real-Time PCR system(Applied Biosystems) according to manufacturer’s protocol.The detailed protocols for immunoprecipitation, immunoblotand kinase assays are described in the supplemented methodsection.

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online.

ACKNOWLEDGEMENTS

The authors express sincere thanks to R. Machado for criti-cally reading through the manuscript.

Conflict of Interest statement. None declared.

FUNDING

The research is supported by a Programme Grant (RG/03/005)from the British Heart Foundation awarded to NWM and RCT.This study also received financial support from the EuropeanCommission under the 6th Framework Programme (ContractNo LSHM-CT-2005-018725, PULMOTENSION).

REFERENCES

1. Hogan, B.L. (1996) Bone morphogenetic proteins in development. Curr.Opin. Genet. Dev., 6, 432–438.

2. Runo, J.R. and Loyd, J.E. (2003) Primary pulmonary hypertension.Lancet, 361, 1533–1544.

3. Massague, J. and Wotton, D. (2000) Transcriptional control by theTGF-beta/Smad signaling system. EMBO J., 19, 1745–1754.

4. Yang, X., Long, L., Southwood, M., Rudarakanchana, N., Upton, P.D.,Jeffery, T.K., Atkinson, C., Chen, H., Trembath, R.C. and Morrell, N.W.(2005) Dysfunctional Smad signaling contributes to abnormal smoothmuscle cell proliferation in familial pulmonary arterial hypertension. Circ.Res., 96, 1053–1063.

5. Raida, M., Clement, J.H., Ameri, K., Han, C., Leek, R.D. and Harris, A.L.(2005) Expression of bone morphogenetic protein 2 in breast cancer cellsinhibits hypoxic cell death. Int. J. Oncol., 26, 1465–1470.

6. Raida, M., Clement, J.H., Leek, R.D., Ameri, K., Bicknell, R.,Niederwieser, D. and Harris, A.L. (2005) Bone morphogenetic protein 2(BMP-2) and induction of tumor angiogenesis. J. Cancer Res. Clin.Oncol., 131, 741–750.

7. Lane, K.B., Machado, R.D., Pauciulo, M.W., Thomson, J.R., Phillips,J.A., 3rd, Loyd, J.E., Nichols, W.C. and Trembath, R.C. (2000)Heterozygous germline mutations in BMPR2, encoding a TGF-betareceptor, cause familial primary pulmonary hypertension. TheInternational PPH Consortium. Nat. Genet., 26, 81–84.

8. Thomson, J.R. and Trembath, R.C. (2000) Primary pulmonaryhypertension: the pressure rises for a gene. J. Clin. Pathol., 53, 899–903.

Figure 8. A model for dysfunctional BMPR-II signalling in PAH. (i) A recep-tor complex consisting of ligand, type II and type I receptors with correct stoi-chiometry is required for optimal signalling. (ii) Majority of the transcriptderived from a nonsense or frameshift allele will be decayed by NMDleading to haploinsufficiency and a reduced contribution to the receptorcomplex. (iii) Missense mutations defective in type I receptor interactionsmay contribute to stoichiometric imbalance. (iv) Agents capable of restoringthe correct stoichiometry might provide protection or attenuate the diseaseprogression.


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ic.oup.com/hm


9. Trembath, R.C. and Harrison, R. (2003) Insights into the genetic andmolecular basis of primary pulmonary hypertension. Pediatr. Res., 53,883–888.

10. Maquat, L.E. (2004) Nonsense-mediated mRNA decay: splicing,translation and mRNP dynamics. Nat. Rev. Mol. Cell. Biol., 5, 89–99.

11. Disset, A., Bourgeois, C.F., Benmalek, N., Claustres, M., Stevenin, J. andTuffery-Giraud, S. (2006) An exon skipping-associated nonsense mutationin the dystrophin gene uncovers a complex interplay between multipleantagonistic splicing elements. Hum. Mol. Genet., 15, 999–1013.

12. Fackenthal, J.D., Cartegni, L., Krainer, A.R. and Olopade, O.I. (2002)BRCA2 T2722R is a deleterious allele that causes exon skipping.Am. J. Hum. Genet., 71, 625–631.

13. Nystrom-Lahti, M., Holmberg, M., Fidalgo, P., Salovaara, R., de laChapelle, A., Jiricny, J. and Peltomaki, P. (1999) Missense and nonsensemutations in codon 659 of MLH1 cause aberrant splicing of messengerRNA in HNPCC kindreds. Genes Chromosomes Cancer, 26, 372–375.

14. Cartegni, L., Chew, S.L. and Krainer, A.R. (2002) Listening to silence andunderstanding nonsense: exonic mutations that affect splicing. Nat. Rev.Genet., 3, 285–298.

15. Rudarakanchana, N., Flanagan, J.A., Chen, H., Upton, P.D., Machado, R.,Patel, D., Trembath, R.C. and Morrell, N.W. (2002) Functional analysis ofbone morphogenetic protein type II receptor mutations underlyingprimary pulmonary hypertension. Hum. Mol. Genet., 11, 1517–1525.

16. Roberts, K.E., McElroy, J.J., Wong, W.P., Yen, E., Widlitz, A., Barst,R.J., Knowles, J.A. and Morse, J.H. (2004) BMPR2 mutations inpulmonary arterial hypertension with congenital heart disease. Eur.Respir. J., 24, 371–374.

17. Humbert, M., Deng, Z., Simonneau, G., Barst, R.J., Sitbon, O., Wolf, M.,Cuervo, N., Moore, K.J., Hodge, S.E., Knowles, J.A. et al. (2002) BMPR2germline mutations in pulmonary hypertension associated withfenfluramine derivatives. Eur. Respir. J., 20, 518–523.

18. Abramowicz, M.J., Van Haecke, P., Demedts, M. and Delcroix, M. (2003)Primary pulmonary hypertension after amfepramone (diethylpropion)with BMPR2 mutation. Eur. Respir. J., 22, 560–562.

19. Nishihara, A., Watabe, T., Imamura, T. and Miyazono, K. (2002)Functional heterogeneity of bone morphogenetic protein receptor-IImutants found in patients with primary pulmonary hypertension. Mol.Biol. Cell, 13, 3055–3063.

20. Paillusson, A., Hirschi, N., Vallan, C., Azzalin, C.M. and Muhlemann, O.(2005) A GFP-based reporter system to monitor nonsense-mediatedmRNA decay. Nucleic Acids Res., 33, e54.

21. Long, L., MacLean, M.R., Jeffery, T.K., Morecroft, I., Yang, X.,Rudarakanchana, N., Southwood, M., James, V., Trembath, R.C. andMorrell, N.W. (2006) Serotonin increases susceptibility to pulmonaryhypertension in BMPR2-deficient mice. Circ. Res., 98, 818–827.

22. Beppu, H., Kawabata, M., Hamamoto, T., Chytil, A., Minowa, O., Noda,T. and Miyazono, K. (2000) BMP type II receptor is required forgastrulation and early development of mouse embryos. Dev. Biol., 221,249–258.

23. Sharp, A., Pichert, G., Lucassen, A. and Eccles, D. (2004) RNA analysisreveals splicing mutations and loss of expression defects in MLH1 andBRCA1. Hum. Mutat., 24, 272.

24. Aretz, S., Uhlhaas, S., Sun, Y., Pagenstecher, C., Mangold, E., Caspari,R., Moslein, G., Schulmann, K., Propping, P. and Friedl, W. (2004)Familial adenomatous polyposis: aberrant splicing due to missense orsilent mutations in the APC gene. Hum. Mutat., 24, 370–380.

25. Nasim, M.T., Chowdhury, H.M. and Eperon, I.C. (2002) A doublereporter assay for detecting changes in the ratio of spliced and unsplicedmRNA in mammalian cells. Nucleic Acids Res., 30, e109.

26. Nasim, M.T., Chernova, T.K., Chowdhury, H.M., Yue, B.G. and Eperon,I.C. (2003) HnRNP G and Tra2beta: opposite effects on splicing matchedby antagonism in RNA binding. Hum. Mol. Genet., 12, 1337–1348.

27. Ishida, W., Hamamoto, T., Kusanagi, K., Yagi, K., Kawabata, M.,Takehara, K., Sampath, T.K., Kato, M. and Miyazono, K. (2000) Smad6 isa Smad1/5-induced smad inhibitor. Characterization of bonemorphogenetic protein-responsive element in the mouse Smad6 promoter.J. Biol. Chem., 275, 6075–6079.

28. Nasim, M.T. and Trembath, R.C. (2005) A dual-light reporter system todetermine the efficiency of protein–protein interactions in mammaliancells. Nucleic Acids Res., 33, e66.

29. Manuvakhova, M., Keeling, K. and Bedwell, D.M. (2000)Aminoglycoside antibiotics mediate context-dependent suppression oftermination codons in a mammalian translation system. RNA, 6, 1044–1055.

30. Murphy, G.J., Mostoslavsky, G., Kotton, D.N. and Mulligan, R.C. (2006)Exogenous control of mammalian gene expression via modulation oftranslational termination. Nat. Med., 12, 1093–1099.

31. Howard, M.T., Shirts, B.H., Petros, L.M., Flanigan, K.M., Gesteland, R.F.and Atkins, J.F. (2000) Sequence specificity of aminoglycoside-inducedstop codon readthrough: potential implications for treatment of Duchennemuscular dystrophy. Ann. Neurol., 48, 164–169.

32. Machado, R.D., Pauciulo, M.W., Thomson, J.R., Lane, K.B., Morgan,N.V., Wheeler, L., Phillips, J.A., 3rd, Newman, J., Williams, D., Galie, N.et al. (2001) BMPR2 haploinsufficiency as the inherited molecularmechanism for primary pulmonary hypertension. Am. J. Hum. Genet., 68,92–102.

33. Machado, R.D., Aldred, M.A., James, V., Harrison, R.E., Patel, B.,Schwalbe, E.C., Gruenig, E., Janssen, B., Koehler, R., Seeger, W. et al.

(2006) Mutations of the TGF-beta type II receptor BMPR2 in pulmonaryarterial hypertension. Hum. Mutat., 27, 121–132.

34. Yu, P.B., Beppu, H., Kawai, N., Li, E. and Bloch, K.D. (2005) Bonemorphogenetic protein (BMP) type II receptor deletion reveals BMPligand-specific gain of signaling in pulmonary artery smooth muscle cells.J. Biol. Chem., 280, 24443–24450.

35. Nohe, A., Hassel, S., Ehrlich, M., Neubauer, F., Sebald, W., Henis, Y.I.and Knaus, P. (2002) The mode of bone morphogenetic protein (BMP)receptor oligomerization determines different BMP-2 signaling pathways.J. Biol. Chem., 277, 5330–5338.

36. Barton-Davis, E.R., Cordier, L., Shoturma, D.I., Leland, S.E. andSweeney, H.L. (1999) Aminoglycoside antibiotics restore dystrophinfunction to skeletal muscles of mdx mice. J. Clin. Invest., 104, 375–381.

37. Linde, L., Boelz, S., Nissim-Rafinia, M., Oren, Y.S., Wilschanski, M.,Yaacov, Y., Virgilis, D., Neu-Yilik, G., Kulozik, A.E., Kerem, E. et al.

(2007) Nonsense-mediated mRNA decay affects nonsense transcriptlevels and governs response of cystic fibrosis patients to gentamicin.J. Clin. Invest., 117, 683–692.

38. Burge, C. and Karlin, S. (1997) Prediction of complete gene structures inhuman genomic DNA. J. Mol. Biol., 268, 78–94.

39. Nasim, M.T. and Eperon, I.C. (2006) A double-reporter splicing assay fordetermining splicing efficiency in mammalian cells. Nature Protocols, 1,1022–1028.


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