Reconstitution and Analysis of Soluble Inhibin and Activin Receptor Complexes in a Cell-free System

Abstract

Activins and inhibins compose a heterogeneous subfamily within the transforming growth factor-␤ (TGF-␤) superfamily of growth and differentiation factors with critical biological activities in embryos and adults. They signal through a heteromeric complex of type II, type I, and for inhibin, type III receptors. To characterize the affinity, specificity, and activity of these receptors (alone and in combination) for the inhibin/activin subfamily, we developed a cell-free assay system using soluble receptor-Fc fusion proteins. The soluble activin type II receptor (sActRII)-Fc fusion protein had a 7-fold higher affinity for activin A compared with sActRIIB-Fc, whereas both receptors had a marked preference for activin A over activin B. Although inhibin A and B binding was 20-fold lower compared with activin binding to either type II receptor alone, the mixture of either type II receptor with soluble TGF-␤ type III receptor (T␤RIII; betaglycan)-Fc reconstituted a soluble high affinity inhibin receptor. In contrast, mixing either soluble activin type II receptor with soluble activin type I receptors did not substantially enhance activin binding. Our results support a cooperative model of binding for the inhibin receptor (ActRII⅐sT␤RIII complex) but not for activin receptors (type II ؉ type I) and demonstrate that a complex composed of activin type II receptors and T␤RIII is both necessary and sufficient for high affinity inhibin binding. This study also illustrates the utility of this cell-free system for investigating hypotheses of receptor complex mechanisms resulting from crystal structure analyses.

Characterization of the Ligand Binding Functionality of the Extracellular Domain of Activin Receptor Type IIB

“…Previous studies have determined affinities of ActRIIB for different ligands (30,33,34); however, these studies did not result in any consensus because data varied greatly, depending on the assay design, experimental conditions, source of ligands and receptors, etc. To determine the binding parameters of different ligands and ActRIIB, we used SPR.…”

Single-Dose, Randomized, Double-Blind, Placebo-Controlled Study of ACE-011 (ActRIIA-IgG1) in Postmenopausal Women

“…In comparison, FSH was unchanged in the placebo group on day 8 (1.1% increase; p > 0.05). In addition, subjects receiving ACE-011 showed significant reductions from baseline in comparison with those receiving placebo at most time points (p 0.01 at days 3, 8, 15, 29, and 57 in the 1.0-mg/kg IV group; p 0.01 at days 3,8,15,29,57, and 85 in the 3.0-mg/kg IV group). In these two dose groups, the greatest change in placebo-corrected mean percent change from baseline in FSH was 39.1% and 50.1% for the 1.0-and 3.0-mg/kg IV groups at day 15, respectively.…”

“…Serum concentrations of FSH were assessed as a readout for biological activity of ACE-011. Bone-specific alkaline phosphatase (BSALP), procollagen type I N-terminal propeptide (PINP), procollagen type I C terminal propeptide (PICP), and total osteocalcin were assessed regularly to look at changes in markers of bone formation, and serum C-terminal type 1 collagen telopeptide (CTX) and TRACP-5b were assessed at baseline and study days 3,8,15,29,57,85, and 120 to look at changes in markers of bone resorption. Bone biomarkers were measured by immunoassay (Synarc, Lyon, France).…”

“…(15) ACE-011 (ActRIIA-IgG1) is a fully human glycosylated dimeric fusion protein consisting of two extracellular domains (ECDs) of ActRIIA linked to the human IgG1 Fc domain including the hinge, CH 2 , and CH 3 domains. By binding activin, ACE-011 prevents activin from binding endogenous receptors and thus acts as a decoy.…”

Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva

“…These studies revealed that mutated FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-β signaling but not BMP signaling. This neofunction of Activin-A raised the possibility that the inhibition of Activin-A signaling by Activin-A-specific-neutralizing antibody or by broad-acting BMP and Activin blockers like ACVR2A-Fc and ACVR2B-Fc (38)(39)(40)(41)(42) could be a new treatment strategy for FOP. Although Activin-A evoked enhanced chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via BMP and TGF-β signaling (38,43), the detailed mechanism, especially downstream effectors of Activin-A/FOP-ACVR1, is largely unexplained.…”