Abstract
Activins control many physiologic and pathophysiologic processes in multiple tissues and, like other TGF-beta superfamily members, signal via type II (ActRII/IIB) and type I (ALK4) receptor serine kinases. ActRII/IIB are promiscuous receptors known to bind at least a dozen TGF-beta superfamily ligands including activins, myostatin, several BMPs, and nodal. Here we utilize a new screening procedure to rapidly identify activin-A mutants with loss of signaling activity. Our goal was to identify activin-A mutants able to bind ActRII but unable to bind ALK4 and which would be, therefore, candidate type II activin receptor antagonists. Using the structure of BMP-2 bound to its type I receptor (ALK3) as a guide, we introduced mutations in the context of the inhibin betaA cDNA and assessed the signaling activity of the resulting mutant proteins. We identified several mutants in the finger (M91E, I105E, M108A) and wrist (activin A/activin C chimera, S60P, I63P) regions of activin-A with reduced signaling activity. Of these the M108A mutant displayed the lowest signaling activity while retaining wild-type-like affinity for ActRII. Unlike wild-type activin-A, the M108A mutant was unable to form a cross-linked complex with ALK4 in the presence of ActRII indicating that its ability to bind ALK4 was disrupted. This data suggested that the M108A mutant might be capable of modulating signaling of activin and related ligands. Indeed, the M108A mutant antagonized activin-A and myostatin, but not TGF-beta, signaling in 293T cells, indicating it may be generally capable of blocking ligands that signal via ActRII/IIB.
Highlights
The transforming growth factor  (TGF-)1 superfamily comprises 42 members in human [1] that control cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses, wound repair, endocrine function, and many other physiologic processes [2,3,4,5,6]
The M108A mutant antagonized activin-A and myostatin, but not TGF-, signaling in 293T cells, indicating it may be generally capable of blocking ligands that signal via ActRII/IIB
Consistent with this model, an allosteric conformational change was observed in the “wrist” region of BMP7 following binding to the ActRII-ECD, and this may allow for the cooperative type I/type II receptor assembly induced by TGF- superfamily members [17]
Summary
The transforming growth factor  (TGF-) superfamily comprises 42 members in human [1] that control cell proliferation, cell death, metabolism, homeostasis, differentiation, immune responses, wound repair, endocrine function, and many other physiologic processes [2,3,4,5,6]. In the crystal structure of the ActRII-ECD bound to BMP7 [17], as well as that of activin bound to ActRIIB [16], the activin type II receptor ECDs make contact with the convex outer face or “knuckle” of the finger region of each ligand. Consistent with this model, an allosteric conformational change was observed in the “wrist” region of BMP7 following binding to the ActRII-ECD, and this may allow for the cooperative type I/type II receptor assembly induced by TGF- superfamily members [17]. To test whether residues in the wrist region of activin-A are important for binding to ALK4, we have subjected activin-A to both alanine and homology scanning mutagenesis Using these approaches we have shown that substantial mutations in the ␣-helix region are required to have any significant effects on activin-A activity. Mutation of these residues generated activin mutants with disrupted ALK4 binding, dramatically reduced signaling and antagonist/partial agonist activity
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