Activins and bone morphogenetic proteins (BMPs) are members of the transforming growth factor-␤ family of growth and differentiation factors that induce signaling in target cells by assembling type II and type I receptors at the cell surface. Ligand residues involved in type II binding are located predominantly in the C-terminal region that forms an extended ␤-sheet, whereas residues involved in type I binding are located in the ␣-helical and preceding loop central portion of the molecule. To test whether the central residues are sufficient to determine specificity toward type I receptors, activin A/BMP chimeras were constructed in which the central residues (45-79) of activin A were replaced with corresponding residues of BMP2 and BMP7. The chimeras were assessed for activin type II receptor (Act RII) binding, activin-like bioactivity, and BMP-like activity as well as antagonistic properties toward activin A and myostatin. ActA/BMP7 chimera retained Act RII binding affinity comparable with wild type activin A, whereas ActA/BMP2 chimera showed a slightly reduced affinity toward Act RII. Both the chimeras were devoid of significant activin bioactivity in 293T cells in the A3 Lux reporter assay up to concentrations 10-fold higher than the minimal effective activin A concentration (ϳ4 nM). In contrast, these chimeras showed BMP-like activity in a BRE-Luc assay in HepG2 cells as well as induced osteoblast-like phenotype in C2C12 cells expressing alkaline phosphatase. Furthermore, both the chimeras activated Smad1 but not Smad2 in C2C12 cells. Also, both the chimeras antagonized ligands that signal via activin type II receptor, such as activin A and myostatin. These data indicate that activin residues in the central region determine its specificity toward type I receptors. ActA/BMP chimeras can be useful in the study of receptor specificities and modulation of transforming growth factor-␤ members, activins, and BMPs.

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...

Activins are pluripotent hormones/growth factors that belong to the TGF-β superfamily of growth and differentiation factors. They play a role in cell growth, differentiation and apoptosis, endocrine function, metabolism, wound repair, immune responses, homeostasis, mesoderm induction, bone growth and many other biological processes. Activins and the related bone morphogenic proteins (BMPs) transduce their signal through two classes of single transmembrane receptors. The receptors possess intracellular serine/threonine kinase domains. Signaling occurs when the constitutively active type II kinase domain phosphorylates the type I receptor, which upon activation phosphorylates intracellular signaling molecules. To generate antagonistic ligands, we generated chimeric molecules that disrupt the receptor interactions and thereby the phosphorylation events. The chimeras were designed based on available structural data to maintain high affinity binding to type II receptors. The predicted type I receptor interaction region was replaced by residues present in inactive homologs or in related ligands with different type I receptor affinities.