Pathogenic ACVR1^R206H activation by Activin A‐induced receptor clustering and autophosphorylation

Abstract

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF-β family type I receptor. ACVR1 R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A-mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild-type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1 R206H activation does not require upstream kinases, but is predominantly activated via Activin A-dependent receptor clustering, which induces its auto-activation. We use optogenetics and live-imaging approaches to demonstrate Activin A-induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling.

Therapeutic Targets in Diffuse Midline Gliomas—An Emerging Landscape

“…However, it should be noted that whilst statistically significant the increase in survival was only modest, with both drugs increasing survival by 15 days [ 151 ]. The in vitro efficacy of LDN-193189 in DMGs has been observed by others, with some evidence of improved efficiency in ACVR1 -mutant cells above those of the wild type; in contrast, another study into LDN-214117 found no significant effect on DMGs in vitro [ 107 , 153 , 155 ]. However, several analogues of LDN-214117 have been developed which exhibit a profile with increased potency, selectivity and BBB permeability.…”

“…ACVR1 mutations result in hyperactivity in response to BMP ligands and constitutive BMP signalling, independent of ligand binding [ 148 , 149 , 150 ]. Furthermore, activin A promotes SMAD1/5/8 signalling in ACVR1 mutated cells at even low ligand concentrations as a result of activin A forming ACVR2A/B–ACVR1R206H complexes [ 151 , 152 , 153 , 154 ]. ACVR1 mutations in DMGs are associated with upregulated SMAD1/5/8 signalling and increases in cell proliferation, indicating an oncogenic role for the BMP pathway in this context [ 107 , 151 , 155 ].…”

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

“…Here, we aimed to visualize receptor dependent Activin A binding on living transfected COS-7 cells in a fluorescence-based approach. Fluorescently labeled Activin A was recently used to show ACVR2B clustering (Ramachandran et al, 2021). Accordingly, we have labeled Activin A with Cy5 and established an analysis pipeline for assessment of Activin A-Cy5 binding on COS-7 cells dependent on the expression of various Halo-tagged receptors ( Fig.…”

“…Recent examples include the determination of SNAP-mGluR4 co-localization (with Ca v 2.1 and Bassoon) by two-color dSTORM microscopy (Siddig et al, 2020), the compositional diversity of differently tagged-kainate receptor assembly by three-color single molecule TIRF microscopy (Selvakumar et al, 2021), and the endocytosis and turnover of Halo-TfR by widefield imaging (Jonker et al, 2020). Alternatively, visualization of cell surface ligand binding, endo- and exocytosis, as well as receptor clustering was achieved using ligands covalently linked to fluorophores (Alborzinia et al, 2013; Ast et al, 2020; Nanda & Lorsch, 2014; Paarmann et al, 2016; Ramachandran et al, 2021; Trujillo et al, 2020). For this, ligands were either directly labeled at deprotonated primary amines using NHS-activated fluorophores (Alborzinia et al, 2013; Nanda & Lorsch, 2014), or through introduction of an N- or C-terminal cysteine residue allowing for site-directed maleimide coupling of the dyes (Ast et al, 2020; Paarmann et al, 2016).…”

“…Mature Activin A was prepared by refolding from inclusion bodies as described before for Atto-647 and CF640R-labelled activin A (Ramachandran et al, 2021) with the exception that mature activin A was untagged and the label used here was NHS-activated Cy5 (Lumiprobe, cat. no 13020).…”