The Activation of the Fibrodysplasia Ossificans Progressiva-Inducing ALK2-R206H Mutant Depends on the Distinct Homo-Oligomerization Patterns of ACVR2B and ACVR2A.

Mutations in activin-like kinase 2 (ALK2), e.g., ALK2-R206H, induce aberrant signaling to SMAD1/5/8, leading to Fibrodysplasia Ossificans Progressiva (FOP). In spite of extensive studies, the underlying mechanism is still unclear. Here, we quantified the homomeric and heteromeric interactions of ACVR2A, ACVR2B, ALK2-WT, and ALK2-R206H by combining IgG-mediated immobilization of one receptor with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of a co-expressed receptor. ACVR2B formed stable homomeric complexes that were enhanced by Activin A (ActA), while ACVR2A required ActA for homodimerization. ALK2-WT, but not ALK2-R206H, exhibited homomeric complexes unaffected by ActA. ACVR2B formed ActA-enhanced heterocomplexes with ALK2-R206H or ALK2-WT, while ACVR2A interacted mainly with ALK2-WT. The extent of the homomeric complex formation of ACVR2A or ACVR2B was reflected in their ability to induce the oligomerization of ALK2-R206H and ALK2-WT. Thus, ACVR2B, which forms dimers without ligand, induced ActA-independent ALK2-R206H clustering but required ActA for enhancing the oligomerization of the largely dimeric ALK2-WT. In contrast, ACVR2A, which undergoes homodimerization in response to ActA, required ActA to induce ALK2-R206H oligomerization. To investigate whether these interactions are translated into signaling, we studied signaling by the FOP-inducing hyperactive ALK2-R206H mutant, with ALK2-WT signaling as control. The activation of SMAD1/5/8 signaling in cells expressing ALK2-R206H alone or together with ACVR2A or ACVR2B was measured by blotting for pSMAD1/5/8 and by transcriptional activation assays using BRE-Luc reporter. In line with the biophysical studies, ACVR2B activated ALK2-R206H without ligand, while activation by ACVR2A was weaker and required ActA. We propose that the homodimerization of ACVR2B or ACVR2A dictates their ability to recruit ALK2-R206H into higher complexes, enabling the homomeric interactions of ALK2-R206H receptors and, subsequently, their activation.

Characterization of Fibrodysplasia Ossificans Progessiva relevant Acvr1/Acvr2 Activin receptors in medaka (Oryzias latipes).

Activin and Bone Morphogenetic Protein (BMP) signaling plays crucial roles in vertebrate organ formation, including osteo- and angiogenesis, and tissue homeostasis, such as neuronal maintenance. Activin and BMP signaling needs to be precisely controlled by restricted expression of shared receptors, stoichiometric composition of receptor-complexes and presence of regulatory proteins. A R206H mutation in the human (hs) BMP type I receptor hsACVR1, on the other hand, leads to excessive phosphorylation of Sons of mothers against decapentaplegic (SMAD) 1/5/8. This in turn causes increased inflammation and heterotopic ossification in soft tissues of patients suffering from Fibrodysplasia Ossificans Progressiva (FOP). Several animal models have been established to understand the spontaneous and progressive nature of FOP, but often have inherent limitations. The Japanese medaka (Oryzias latipes, ola) has recently emerged as popular model for bone research. To assess whether medaka is suitable as a potential FOP animal model, we determined the expression of Activin receptor type I (ACVR1) orthologs olaAcvr1 and olaAcvr1l with that of Activin type II receptors olaAcvr2ab, olaAcvr2ba and olaAcvr2bb in embryonic and adult medaka tissues by in situ hybridization. Further, we showed that Activin A binding properties are conserved in olaAcvr2, as are the mechanistic features in the GS-Box of both olaAcvr1 and olaAcvr1l. This consequently leads to FOP-typical elevated SMAD signaling when the medaka type I receptors carry the R206H equivalent FOP mutation. Together, this study therefore provides experimental groundwork needed to establish a unique medaka model to investigate mechanisms underlying FOP.

A versatile Halo- and SNAP-tagged BMP/TGFß receptor library for quantification of cell surface ligand binding.

TGFßs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFß receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFß/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFß1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways.

Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes.

BACKGROUND: Fluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions. RESULTS: We report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress. CONCLUSIONS: We identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions.

AMOT130 drives BMP-SMAD signaling at the apical membrane in polarized cells.

The large isoform of the transmembrane protein angiomotin (AMOT130) controls cell proliferation and migration of many cell types. AMOT130 associates to the actin cytoskeleton and regulates tight-junction maintenance and signaling often via endosomal uptake of polarity proteins at tight junctions. AMOT130 is highly polarized and present only at the apical side of polarized cells. Here we show that bone morphogenetic protein (BMP) growth factor signaling and AMOT function are interlinked in apical-basal polarized cells. BMP6 controls AMOT internalization and endosomal trafficking in epithelial cells. AMOT130 interacts with the BMP receptor BMPR2 and facilitates SMAD activation and target gene expression. We further demonstrate that this effect of AMOT on BMP-SMAD signaling is dependent on endocytosis and specific to the apical side of polarized epithelial and endothelial cells. Knockdown of AMOT reduces SMAD signaling only from the apical side of polarized cells, while basolateral BMP-SMAD signaling is unaffected. This allows for the first time interference with BMP signaling in a polarized manner and identifies AMOT130 as a novel BMP signaling regulator.