N-linked glycosylation of the bone morphogenetic protein receptor type 2 (BMPR2) enhances ligand binding
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The bone morphogenetic protein (BMP) signaling pathway is essential for normal development and tissue homeostasis. BMP signal transduction occurs when ligands interact with a complex of type 1 and type 2 receptors to activate downstream transcription factors. It is well established that a single BMP receptor may bind multiple BMP ligands with varying affinity, and this has been largely attributed to conformation at the amino acid level. However, all three type 2 BMP receptors (BMPR2, ACVR2A/B) contain consensus N-glycosylation sites in their extracellular domains (ECDs), which could play a role in modulating interaction with ligand. Here, we show a differential pattern of N-glycosylation between BMPR2 and ACVR2A/B. Site-directed mutagenesis reveals that BMPR2 is uniquely glycosylated near its ligand binding domain and at a position that is mutated in patients with heritable pulmonary arterial hypertension. We further demonstrate using a cell-free pulldown assay that N-glycosylation of the BMPR2-ECD enhances its ability to bind BMP2 ligand but has no impact on binding by the closely-related ACVR2B. Our results illuminate a novel aspect of BMP signaling pathway mechanics and demonstrate a functional difference resulting from post-translational modification of type 2 BMP receptors. Additionally, since BMPR2 is required for several aspects of normal development and defects in its function are strongly implicated in human disease, our findings are likely to be relevant in several biological contexts in normal and abnormal human physiology.
KeywordsBone morphogenetic protein BMPR2 ACVR2A ACVR2B Activin Glycosylation Pulmonary hypertension Heritable pulmonary arterial hypertension
We gratefully acknowledge James West (Vanderbilt) for the hBMPR2 cDNA; Mark de Caestecker (Vanderbilt) for early discussions; Giuseppe Intini (HSDM) for assistance with Bmpr2 knockdown. J.W.L. and J.M.A. are recipients of the Harvard School of Dental Medicine Dean’s Scholar Award and Research Science Institute fellowship, respectively. This work was supported by the NIH/NIAMS grant R01AR055904 awarded to V.R.
Conflict of interests
The authors declare that they have no competing interests.
- 10.Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, Knowles JA (2000) Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet 67(3):737–744PubMedCentralPubMedCrossRefGoogle Scholar
- 11.Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA 3rd, Loyd JE, Nichols WC, Trembath RC, The International PPH Consortium (2000) Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension. Nat Genet 26(1):81–84PubMedCrossRefGoogle Scholar
- 13.Shore EM, Xu M, Feldman GJ, Fenstermacher DA, Cho TJ, Choi IH, Connor JM, Delai P, Glaser DL, LeMerrer M, Morhart R, Rogers JG, Smith R, Triffitt JT, Urtizberea JA, Zasloff M, Brown MA, Kaplan FS (2006) A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet 38(5):525–527PubMedCrossRefGoogle Scholar
- 15.Howe JR, Sayed MG, Ahmed AF, Ringold J, Larsen-Haidle J, Merg A, Mitros FA, Vaccaro CA, Petersen GM, Giardiello FM, Tinley ST, Aaltonen LA, Lynch HT (2004) The prevalence of MADH4 and BMPR1A mutations in juvenile polyposis and absence of BMPR2, BMPR1B, and ACVR1 mutations. J Med Genet 41(7):484–491PubMedCentralPubMedCrossRefGoogle Scholar
- 16.Lehmann K, Seemann P, Stricker S, Sammar M, Meyer B, Suring K, Majewski F, Tinschert S, Grzeschik KH, Muller D, Knaus P, Nurnberg P, Mundlos S (2003) Mutations in bone morphogenetic protein receptor 1B cause brachydactyly type A2. Proc Natl Acad Sci USA 100(21):12277–12282PubMedCentralPubMedCrossRefGoogle Scholar
- 22.Kotzsch A, Nickel J, Seher A, Heinecke K, van Geersdaele L, Herrmann T, Sebald W, Mueller TD (2008) Structure analysis of bone morphogenetic protein-2 type I receptor complexes reveals a mechanism of receptor inactivation in juvenile polyposis syndrome. J Biol Chem 283(9):5876–5887PubMedCrossRefGoogle Scholar
- 37.Sako D, Grinberg AV, Liu J, Davies MV, Castonguay R, Maniatis S, Andreucci AJ, Pobre EG, Tomkinson KN, Monnell TE, Ucran JA, Martinez-Hackert E, Pearsall RS, Underwood KW, Seehra J, Kumar R (2010) Characterization of the ligand binding functionality of the extracellular domain of activin receptor type IIb. J Biol Chem 285(27):21037–21048PubMedCentralPubMedCrossRefGoogle Scholar
- 39.Pfarr N, Szamalek-Hoegel J, Fischer C, Hinderhofer K, Nagel C, Ehlken N, Tiede H, Olschewski H, Reichenberger F, Ghofrani AH, Seeger W, Grunig E (2011) Hemodynamic and clinical onset in patients with hereditary pulmonary arterial hypertension and BMPR2 mutations. Respir Res 12:99PubMedCentralPubMedCrossRefGoogle Scholar
- 51.Gamer LW, Tsuji K, Cox K, Capelo LP, Lowery J, Beppu H, Rosen V (2011) BMPR-II is dispensable for formation of the limb skeleton. Genesis 49:719–724Google Scholar
- 55.Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739Google Scholar