Activin A functions in BMP signaling in two ways: it either engages ACVR1B to activate Smad2/3 signaling or binds ACVR1 to form a non-signaling complex (NSC). Although the former property has been studied extensively, the roles of the NSC remain unexplored. The genetic disorder fibrodysplasia ossificans progressiva (FOP) provides a unique window into ACVR1/Activin A signaling because in that disease Activin can either signal through FOP-mutant ACVR1 or form NSCs with wild-type ACVR1. To explore the role of the NSC, we generated ‘agonist-only’ Activin A muteins that activate ACVR1B but cannot form the NSC with ACVR1. Using one of these muteins, we demonstrate that failure to form the NSC in FOP results in more severe disease pathology. These results provide the first evidence for a biological role for the NSC in vivo and pave the way for further exploration of the NSC’s physiological role in corresponding knock-in mice.

TGFβ family ligands, which include the TGFβs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFβs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFβ and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFβ, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe ⁸⁴ ), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.

Activin ligands are formed from two disulfide-linked inhibin β (Inhβ) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodic yet cumulative heterotopic ossification (HO) of skeletal muscles, tendons, ligaments, and fascia. FOP arises from missense mutations in Activin Receptor type I (ACVR1), a type I bone morphogenetic protein (BMP) receptor. Although initial findings implicated constitutive activity of FOP-variant ACVR1 (ACVR1^FOP) and/or hyperactivation by BMPs, it was later shown that HO in FOP requires activation of ACVR1^FOP by Activin A. Inhibition of Activin A completely prevents HO in FOP mice, indicating that Activin A is an obligate driver of HO in FOP, and excluding a key role for BMPs in this process. This discovery led to the clinical development of garetosmab, an investigational antibody that blocks Activin A. In a phase 2 trial, garetosmab inhibited new heterotopic bone lesion formation in FOP patients. In contrast, antibodies to ACVR1 activate ACVR1^FOP and promote HO in FOP mice. Beyond their potential clinical relevance, these findings have enhanced our understanding of FOP’s pathophysiology, leading to the identification of fibroadipogenic progenitors as the cells that form HO, and the discovery of non-signaling complexes between Activin A and wild type ACVR1 and their role in tempering HO, and are also starting to inform biological processes beyond FOP.

Ligands in the transforming growth factor β (TGF-β) family [activins, Bone Morphogenetic Proteins (BMPs), and TGF-βs] signal by bringing together two type I and two type II receptors. Activin receptor-like kinase-2 (ALK2) is the only type I receptor among the seven TGF-β type I receptors that interacts with both activin and BMP ligands. With BMPs, ALK2 acts as a signaling receptor to activate small mothers against decapentaplegic 1 (SMAD1)/5/8 signaling. Alternatively, with activins, such as Activin A (ActA), ALK2 forms nonsignaling complexes that negatively regulate ALK2 and ActA signaling. To gain insight into how ALK2 interacts with two distinct classes of ligands, we resolved the cryoelectron microscopy structure of ALK2 in complex with the type II receptor, ActRIIB, and the ligand, BMP6, in parallel with the corresponding structure with ALK3 for direct comparison. These structures demonstrate that ALK2 and ALK3 utilize different mechanisms to interact with BMP6 at the wrist interface, with ALK2 relying on BMP6 glycosylation and ALK3 relying on a salt bridge. Modeling of ALK2:ActA reveals that binding relies on ActA’s fingertip region, mirroring the interaction of ActA with its other receptor, ALK4. Our results demonstrate that ALK2 is a “hybrid” receptor that incorporates features of BMP type I receptors such as ALK3 at the wrist interface and an activin type I receptor such as ALK4 at the fingertip.

BMP/TGFß family ligands act mainly as factors that differentially initiate Smad1/5/8 or Smad2/3 signaling via heterotetrameric complexes comprised of two type I and two type II receptors (IIR). ActA (ActA) stands out as it activates Smad2/3 signaling through type I receptor ACVR1B, whereas it generates non-signaling complexes (NSCs) with ACVR1. In fibrodysplasia ossificans progressiva (FOP), a genetic disorder caused by missense mutations in ACVR1 (ACVR1.FOP), ACVR1.FOP•ActA•IIR complexes activate Smad1/5 signaling, mimicking those formed with BMPs. As the NSCs that ActA forms with ACVR1 are stoichiometrically identical with signaling complexes formed with ACVR1.FOP, we explored how NSCs differ from their signaling counterparts. We show that ACVR1•ActA•IIR complexes rapidly traffic to lysosomes, where their constituent components are degraded, reducing the cell’s responsiveness to BMPs along with ActA’s availability. This property is specific to ActA as Activin B, AB, and AC do not form lysosomal trafficking complexes with ACVR1, but rather remain on the surface as NSCs. Lysosomal trafficking and degradation of ACVR1•ActA•IIR complexes is a novel regulatory mechanism of BMP/TGFß signaling whose physiological roles remain largely unexplored.