An Activin-A/C Chimera Exhibits Activin and Myostatin Antagonistic Properties

Abstract

Activins are involved in many physiological and pathological processes and, like other members of the transforming growth factor-␤ superfamily, signal via type II and I receptor serine kinases. Ligand residues involved in type II receptor binding are located in the two anti-parallel ␤ strands of the TGF-␤ proteins, also known as the fingers. Activin-A mutants able to bind ActRII but unable to bind the activin type I receptor ALK4 define ligand residues involved in ALK4 binding and could potentially act as antagonists. Therefore, a series of FLAG-tagged activin-A/C chimeras were constructed, in each of which eight residues in the wrist loop and helix region (A/C 46 -53, 54 -61, 62-69, and 70 -78) were replaced. Additionally, a chimera was generated in which the entire wrist region (A/C 46 -78) was changed from activin-A to activin-C. The chimeras were assessed for ActRII binding, activin bioactivity, as well as antagonistic properties. All five chimeras retained high affinity for mouse ActRII. Activins belong to the TGF-␤ 2 superfamily of growth factors, which control a variety of physiological functions such as cell growth, differentiation and apoptosis, endocrine function, metabolism, wound repair, immune responses, homeostasis, mesoderm induction, bone growth, and many others (1-5). The TGF-␤ family comprises at least 42 members in human (6) including activin, TGF-␤, bone morphogenetic protein, growth and differentiation factor, and nodal proteins, which are all characterized by a distinct structural feature, namely a cysteine knot scaffold (7).Activins are homo-or heterodimers consisting of two ␤ subunits, which are linked by a single covalent disulfide bond. In each monomer, two pairs of antiparallel ␤ strands form short and long "fingers" that stretch out from the cysteine core of the dimer. The base of the fingers is formed by an ␣-helix together with a preceding loop and is also known as the "wrist" epitope (8, 9). In human, genes encoding for four different ␤ subunits have been identified (␤A, ␤B, ␤C, and ␤E), which, in theory, offers a variety of different activin dimers. However, only activin-A (␤A␤A), activin-B (␤B␤B), and activin-AB (␤A␤B) have been proven to be biologically active (10). The expression of activins C (␤C␤C), E (␤E␤E), AC (␤A␤C), AE (␤A␤E), CE (␤C␤E) (11), as well as BC (␤B␤C) (12) has been demonstrated, but little is known on their function and stability. The ␤C subunit appears to be able to attenuate activin activity by forming a heterodimer with a ␤A subunit and thereby decreasing the source for the formation of biologically active activins (13). Results on the functional activity of activin-C were inconsistent. Lau et al. (14) found that genes encoding for activin ␤C and ␤E were not essential for mouse liver growth, differentiation, and regeneration. Chabicovsky et al. (15) observed that the overexpression of activin ␤C or activin ␤E in the mouse liver inhibited regenerative DNA synthesis of hepatic cells, and Vejda et al. (16) demonstrated that the expression of activins C and...

Activin A/Bone Morphogenetic Protein (BMP) Chimeras Exhibit BMP-like Activity and Antagonize Activin and Myostatin

“…As predicted, ActA/ BMP2 and ActA/BMP7 chimeras possessing affinity for Act RII comparable with wt activin A and at the same time devoid of significant wt activin-like bioactivity turned out to be antagonist for ligands signaling via Act RII and ALK4. These findings are in line with our previous reports on mutants of activin such as the M108A point mutant (32) and the activin A/C chimera (31). The antagonistic effect of ActA/BMP2 (IC 50 1-8 nM) or ActA/BMP7 (IC 50 1-10 nM) toward 100 pM activin A and 500 pM myostatin (IC 50 for ActA/BMP2, 1-5 nM; IC 50 for ActA/BMP7, 1-5 nM) appears similar to that reported for the activin A/C chimera (IC 50 , 1-10 nM) (31) and the activin (M108A) point mutant (32).…”

“…Recently, we showed that exchanging the entire wrist region of activin A with biologically inactive activin C blocks its type I receptor binding while retaining its Act RII binding (31). Concurrently, this activin A/C chimera acted as an antagonist to molecules that signal via the Act RII receptor.…”

“…For example, an activin A/C chimera retaining its type II receptor binding was shown to be devoid of activinlike activity and, consequently, possessed activin and myostatin antagonistic properties (31). However, a point mutation in the finger region (M108A) of activin yielded a ligand that binds the type II receptor and has a biological activity 3 orders of magnitude lower than wt and antagonized activin A in 293T cells (32).…”

The structural basis of TGF-β, bone morphogenetic protein, and activin ligand binding

“…Mutating one of the conserved tryptophan residues on activin produced a true activin antagonist able to bind only the type II receptor, and further mutations in this region have implicated a number of amino acids giving rise to a large hydrophobic patch on the concave finger region of activin (Cook et al 2005). Also contributing to the type I interface is the wrist helical region; however, only large domain mutations in the wrist helix have led to the disrupution of signaling, suggesting that ALK4 contacts both subunits of ligand concurrently (Muenster et al 2005). Different suggestions have been put forward to explain the two-step assembly of a functional signaling holocomplex for TGF-b ligands.…”

Activins and activin antagonists in hepatocellular carcinoma

“…Receptors for activins containing βC or βE subunits have not been identified so far. Activin C, however, did not compete with activin A for receptor binding [29] and a chimeric activin construct in which the receptor binding sequence (amino acids 46-78) of βA was replaced by the corresponding region of βC retained type Ⅱ receptor binding but was unable to recruit the typeⅠreceptor ALK 4 [30] .…”