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  1. Evaluation Summary:

    This study was designed to examine the orphan ligand ActivinC. The authors show that ActivinC signals through ALK7. The data presented are strong and convincing. Indeed, the use of purified proteins for the interaction assays and in vivo analysis of adipocytes provide considerable rigor to the analysis. The work will be of interest to the areas of signal transduction and drug design.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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  2. Reviewer #2 (Public Review):

    Activin isoforms, ActA, ActB, ActC, ActAC, ActE, have important roles regulating metabolism, and as well as endocrine function and cell differentiation in animals, and thus understanding how the isoforms function and how they have diverged from one another to carry out their unique roles in vivo is important.

    In previous studies, the receptor binding and signaling properties of ActA and ActB, as well as two other activin class ligands, GDF8 and GDF11, have been fairly thoroughly characterized with respect to their signaling activity and receptor binding properties (and the underlying structural basis for this). In contrast, ActC, which is expressed at high levels in both the liver and in mature adipocytes, remains poorly characterized, with conflicting reports regarding its signaling activity and antagonistic activity against other activin isoforms.

    In this study, the authors employ purified activins, and purified activin class type I and type II receptors (Alk4, Alk5, and Alk7 and ActRII and ActRIIB, respectively), together with cell-based assays and SPR binding studies to characterize the receptor binding and signaling properties of ActC and ActAC, relative to the much more well-characterized ActA and ActB.

    Consistent results obtained using orthogonal approaches convincingly demonstrate that the inhibin betaC monomer (in the context of a dimeric activin) does not bind Alk4, but it does bind Alk7 weakly and it also binds ActRII and ActRIIB weakly (with a preference for ActRII over ActRIIB). In this light, ActC is clearly unique compared to ActA and ActB in that it has markedly reduced type II receptor binding affinity, but it does uniquely bind Alk7. Importantly, the combination of ActRII/ActRIIB together with over expressed Alk7 in HEK293 is shown to confer ActC with robust signaling activity. It is also shown that ActAC can bind Alk7 and signal, though because of the inhibin bC subunit (with lower type II receptor binding affinity and a preference for weakly binding Alk7 over Alk4 and Alk5) its signaling activity is reduced relative to ActA. In short, the authors conclude from this data that the competition previously reported for ActC against ActA and ActB likely arises not from binding competition (akin to the way inhibin A antagonizes alctivins), but by promoting the formation of ActAC, which is a less potent signaling ligand than ActA.

    In this study, the authors also use the tools described above to interrogate the effects of known activin antagonists, follistatin (specifically Fst288 and FstL3) and inhibin A, and show that while ActC is sensitive to antagonism by InhA, it is not antagonized by Fst288 or FstL3.

    One very important strength of the studies reported here is their reliance on highly purified activins and activin class receptor extracellular domains, which allow the authors to characterize in detail the binding properties and in turn relate this to signaling activity in their well-controlled HEK-293 CAGA-Luc reporter cell line. Overall, these results shed significant new light on the underlying and unique behavior of ActC (and ActAC) compared to ActA and ActB and are highly robust.

    In order to relate the results described above to role of ActC in vivo, the authors show that while ActC is not capable of signaling in primary non-differentiated adipocytes isolated from mice where Alk7 expression is limited, but they are capable of signaling once the adipocytes mature and differentiate and express Alk7 at a much higher level.

    Finally, to understand the underlying molecular basis for ActC receptor binding properties, the authors noted that one of the conserved Ala residues in the core of the type II receptor binding interface was substituted with glutamine; substitution of this in the context of ActC conferred enhanced type II receptor binding affinity, consistent with the idea that glutamine at this conserved position sterically impairs type II receptor binding.

    Overall, the results presented strongly support the author's claims and clearly demonstrate that ActC is unique with respect to both type I and type II receptor, as well as follistatin binding, compared to ActA and ActB. In terms of the larger picture, these differences likely engender ActC with the ability to regulate activin signaling in a manner that is largely independent of the more widely expressed ActA and ActB.

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  3. Reviewer #1 (Public Review):

    The Activin isoform ActC is expressed at high levels in liver and adipose tissue where it plays important physiological roles. However, little is known about ActC signaling mechanisms. Here the authors use purified ActC and purified receptors to demonstrate that ActC interacts with ALK7. An important test of the authors' hypothesis that ALK7 mediates the effect of ActC is the demonstration that pre-adipocytes from ALK7-impaired fail to respond to ActC, but responsiveness is restored by differentiation associated with increased ALK7 expression. Collectively, these data provide strong evidence that ALK7 mediates the actions of ActC. This represents a significant advance in knowledge.

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