N-linked glycosylation of the bone morphogenetic protein receptor type 2 (BMPR2) enhances ligand binding

Abstract

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.

Fig. 1

Type 2 BMP receptors are predicted to be glycoproteins. a Prediction of N-linked glycosylation sites in hBMPR2, hACVR2A, and hACVR2B using NetNGlyc. b Alignment comparison of type 2 BMP receptors. Human isoforms of BMPR2 (NP_031587.1), ACVR2B (NP_001097.2), and ACVR2A (NP_001067.1) were aligned using MUSCLE [54] in MEGA5 [55]. Secondary structures are annotated after Mace et al. [46] and Yin et al. [26]. Asparagine residues predicted to be glycosylated are indicated by red boxes in hBMPR2 and blue boxes in ACVR2A/B. Residue numbers indicate the position in each respective RefSeq sequence without gaps. c, d Structural comparison of BMPR2 to ACVR2A/B, highlighting the unique glycosylation sites in BMPR2. Views in (c) are the interior, ligand-binding faces of the extra-cellular domains. Select β-strands are annotated after Mace et al. [46] and Yin et al. [26]. Views in (d) are rotated 90° in the forward orientation (toward the reader) from (c). Interior Surface and Exterior Surface refer to the faces of the molecules with respect to the ternary complex

Fig. 2

Determination of N-linked glycosylation sites in BMPR2. a Western blot for glycosylation status of endogenous BMPR2 in W20 cells compared to HSP90 loading control. Lysates were treated with vehicle or PNGase F. Specificity of anti-BMPR2 antibody is confirmed by lack of signal after Bmpr2 knockdown (Bmpr2-KD); shRNA against Gfp serves as negative control (Gfp-KD). Image is representative of two independent experiments. b Western blot for V5-tagged wild-type and triple or double mutant versions of hBMPR2 in W20 cell lysates treated with vehicle or PNGase F. c Expression analysis of V5-tagged wild-type and single glycosylation mutant versions of hBMPR2 in W20 cells. d Expression analysis of V5-tagged wild-type and mutant versions of hBMPR2 in HEK293T cells. Blots in b–d were performed using anti-V5 antibody; specificity of anti-V5 antibody is confirmed by lack of signal in lysates from cells transfected with empty plasmid control. Triple mutant is N55Q; N11Q; N126Q. N55 Only mutant is N110Q; N126Q. N110 Only mutant is N55Q; N126Q. N126 Only mutant is N55Q; N110Q

Fig. 3

N-linked glycosylation enhances ligand binding to BMPR2-ECD. a Western blot for glycosylation status of the human His-tagged BMPR2 extracellular domain (hBMPR2-ECD)/Fc fusion protein using anti-His antibody. b, c Pulldown of BMP2 using native or de-glycosylated hBMPR2-ECD. Western blot images in (b) for His-tagged hBMPR2-ECD and BMP2 are from a single blot with intervening lane removed (vertical bars) and are representative of five independent experiments (quantified in c). Data in (c) are BMP2/BMPR2-ECD ratio (mean ± SEM) expressed as percent relative to native. *p < 0.05 by unpaired t test against pulldown using native hBMPR2-ECD

Fig. 4

Ligand binding to ACVR2B is unaffected by N-linked glycosylation status. a Western blot for glycosylation status of the human His-tagged ACVR2B extracellular domain (hACVR2B-ECD)/Fc fusion protein using anti-His antibody. b, c Pulldown of BMP2 using native or de-glycosylated ACVR2B-ECD. Western blot images in (b) for ACVR2B-ECD and BMP2 are representative of four independent experiments (quantified in c). Data in (c) are BMP2/ACVR2B-ECD ratio (mean ± SEM) expressed as percent relative to native