BACKGROUND
the transforming growth factor β  superfamily of secreted proteins comprises more than  <dig> structurally related, yet functionally distinct proteins that play critical roles in embryological tissue development and patterning, wound healing, and adult tissue maintenance .
the tgfβ superfamily can be divided into three subclasses: tgfβs, bone morphogenetic proteins , and activins/inhibins.
growth/differentiation factors  <dig> and  <dig>  are two closely related members of the activin/inhibin subclass that share ~90% sequence identity in their mature, c-terminal signaling domains and ~52% identity in their n-terminal prodomains.
gdf <dig> and gdf <dig> bind to similar receptors  and extracellular antagonists  <cit> , leading to the assumption that mature gdf <dig> and gdf <dig> ligands are functionally indistinguishable.
while it is clear that gdf <dig> and gdf <dig> share many commonalities, including overlapping or redundant roles in certain biological processes , there is accumulating evidence that the two ligands may be functionally distinct .
gdf <dig> is expressed postnatally by skeletal and cardiac muscle and therein negatively regulates skeletal muscle mass by suppressing both the number and size of individual muscle fibers  <cit> .
in contrast, gdf <dig> appears to act more broadly, regulating anterior/posterior patterning and development of multiple organs/tissues  <cit> .
many tissues express gdf <dig> postnatally, including the spleen, pancreas, kidney, and skeletal muscle .
however, determination of gdf11’s exact role in the adult has remained elusive due to the embryonic lethality of gdf11
-/- mice  <cit> .
in stark contrast, gdf8
-/- mice survive into adulthood and have a profound hypermuscular phenotype, which can be recapitulated in wild-type mice using natural occurring antagonists of gdf <dig>  such as follistatin , follistatin-like  <dig> , and growth/differentiation factor-associated serum protein  <dig>  .
interestingly, gdf8
-/-
/gdf11
-/- mice have exaggerated homeotic axial transformations compared to gdf11
-/- mice, suggesting that gdf <dig> and gdf <dig> have redundant functions in skeletal patterning  <cit> .
however, muscle-specific knockout of gdf <dig> does not result in significant increases in muscle mass and circulating gdf <dig> does not overcome the hypermuscular phenotype found in gdf8
-/- mice, suggesting that gdf <dig> and gdf <dig> do not serve redundant roles in regulating skeletal muscle mass  <cit> .
thus, while it is clear that loss of one ligand compared to the other yields drastically different phenotypes, it has been argued that these differences relate primarily to differential localization of ligand expression and do not reflect differences in ligand signaling.
similar to other tgfβ ligands, gdf <dig> and gdf <dig> are disulfide-linked dimers that are initially synthesized as precursors, which are cleaved by furin-like proteases to separate the n-terminal prodomain from the c-terminal mature domain  <cit> .
unlike most tgfβ ligands, mature gdf <dig> and gdf <dig> remain tightly bound to their prodomains, holding them in a latent state .
ligand activation requires additional cleavage of the prodomain by bmp1/tolloid  metalloproteinases .
the ligand dimer elicits signal transduction by symmetrically binding two type ii and two type i transmembrane serine/threonine kinase receptors .
ligand-induced receptor clustering leads to phosphorylation of smad <dig> and smad <dig>  transcription factors by the type i receptor.
subsequent accumulation of smad2/ <dig> in the nucleus results in activation or repression of gdf <dig> and gdf <dig> responsive genes  .
similar to other ligands in the activin/inhibin subclass, gdf <dig> and gdf <dig> predominantly signal through the type ii receptors, activin receptor kinase iia  and actriib  and the type i receptors, activin-like receptor kinase  <dig>  and alk <dig>  .
there is also evidence that gdf <dig> can signal using the type i receptor alk <dig>   <cit> .
furthermore, signaling by both gdf <dig> and gdf <dig> is controlled by extracellular protein antagonists, including fs  <cit> , fstl <dig>  <cit> , gasp <dig>  and gasp <dig> .fig.
<dig> gdf <dig> is a more potent ligand than gdf <dig>  a overview of the well-established canonical activin a, activin b, gdf <dig>  gdf <dig>  and tgfβ receptor utilization and downstream smad pathway.
b, c, d potency differences between gdf <dig> and gdf <dig>  luciferase reporter gene assay  <dig> promoter) following titration of gdf <dig>  and gdf <dig>  ligands in hek <dig>  and hepg <dig>  cells.
luciferase activity was assessed 18–24 h post ligand treatment.
the black arrows in  indicate the ligand concentrations utilized in panels e and f. in d, mouse gonadotrope  cells were treated with increasing doses of gdf <dig>  or gdf <dig> .
follicle-stimulating hormone  release was measured 24 h later, as previously described  <cit> .
refer to additional file 1: table s <dig> for a corresponding analysis of the activation curves.
e short exposure to gdf <dig> results in a significantly enhanced smad3-dependent response compared to gdf <dig>  the experimental design  is such that the ligand was added to hek <dig> cells stably transfected with the  <dig> promoter driving the luciferase gene for the indicated time followed by replacement of media without ligand.
activity was measured 24 h after initial treatment.
cells were treated with gdf <dig> or gdf <dig> at a ligand concentration of  <dig> pm  and  <dig> pm .
f time-dependent differences in the smad <dig> activation by gdf <dig> and gdf <dig>  similar experimental design  as in e, but instead cells were lysed and assessed for luciferase activity at the indicated time of ligand treatment.
cells were treated with gdf <dig> or gdf <dig> at a ligand concentration of  <dig> pm  and  <dig> pm .
data information: in b, c and e, f, data are presented as fold activation above background .
each concentration was performed in triplicate and shown as the mean ± standard error of the mean  of three independent experiments.
in d, data are presented as fold fsh release above background .
each concentration was performed in triplicate and shown as the mean ± sem of one experiment; data shown are representative of  <dig> independent experiments.
in e, f, curves were compared using two-way anova with bonferroni correction .
ligand sources: for b, e, f, gift from acceleron pharma; for c, d, purchased from r&d systems; cat.
no.
788-g8-cf and cat.
no.
1958-gd-010-cf)
there have been conflicting reports comparing the biological functions of gdf <dig> and gdf <dig>  with some question as to whether the two ligands are functionally equivalent .
specifically, gdf <dig> was identified as a circulating factor that declines with age, and exogenous delivery of the mature ligand reversed many pathologies associated with aging  <cit> , including reducing age-related cardiac hypertrophy  <cit> , improving skeletal muscle performance and repair  <cit> , improving olfaction  <cit> , and increasing neurogenesis  <cit> .
conversely, other reports have challenged these claims, arguing that gdf <dig> and gdf <dig> are functionally and biologically equivalent  <cit> .
however, it is difficult to compare the results, given that many of these studies did not utilize the same experimental design, sources of recombinant proteins, or strategies for detection of gdf <dig> and gdf <dig> proteins .
therefore, the extent of functional overlap between mature gdf <dig> and gdf <dig> without the prodomain remains to be determined.
furthermore, a rigorous side-by-side biochemical and biological comparison of mature gdf <dig> and gdf <dig> has yet to be performed to determine if distinct features exist between these growth factors.
in this study, using a variety of in vitro and in vivo systems, we show that gdf <dig> is significantly more potent than gdf <dig> and that the enhanced activity is due to differences in type i receptor utilization.
in addition, we present three new x-ray crystal structures of apo-gdf <dig>  apo-gdf <dig>  and gdf <dig> in complex with follistatin  <dig> .
these structures, including an additional recently solved apo-gdf <dig> crystal structure  <cit> , reveal that differences between gdf <dig> and gdf <dig> cluster in the type i receptor binding epitope.
intriguingly, our structural analysis has revealed unique and alternate conformations of both gdf <dig> and gdf <dig>  suggesting that both ligands are inherently flexible.
gdf8/gdf <dig> chimeras, in which particular gdf <dig> residues are substituted into the gdf <dig> sequence, show that increased potency can be conferred to gdf <dig>  taken together, our results demonstrate that, despite the high sequence identity between mature gdf <dig> and gdf <dig>  the ligands indeed possess different signaling properties.
RESULTS
gdf <dig> is a more potent ligand than gdf8
recent reports have attributed unique in vivo properties to gdf <dig> relative to gdf <dig> .
therefore, we first aimed to determine if signaling differences exist between these two growth factors using hek <dig>  and hepg <dig>  cells stably or transiently transfected, respectively, with the smad3-responsive  <dig> luciferase reporter .
side-by-side titration of mammalian-derived  ligands revealed that gdf <dig> was more potent than gdf <dig> in both cell lines .
remarkably, we determined that the half-maximal effective concentration  <dig>  values for gdf <dig> in hek <dig> and hepg <dig> cells were  <dig>  nm and  <dig>  nm, respectively, compared to  <dig>  nm and  <dig>  nm for gdf <dig>  respectively .
interestingly, the maximal smad <dig> response achieved by gdf <dig> was ~ fourfold higher compared to gdf <dig> in hepg <dig> cells , suggesting that there may be differences in ligand potency and/or in the repertoire of receptors and their relative utilization.
furthermore, we found that gdf <dig>  more potently stimulated the release of follicle-stimulating hormone  than gdf <dig>  in murine lβt <dig> pituitary gonadotrope cells .
finally, gdf <dig> activated smad <dig> with ~ a sevenfold lower ec <dig> compared to gdf <dig>  in a <dig> cells transfected with the  <dig> luciferase reporter .
these results are consistent with previous reports, which likewise indicated that gdf <dig> is a more potent activator of smad3-dependent signaling than gdf <dig>  <cit> .
note that ligands from different sources were utilized and were directly compared using the smad3-responsive  <dig> luciferase reporter assay .
for clarity, we have indicated the ligand sources utilized for each study in the figure legends.
given that gdf <dig> stimulated greater smad <dig> activation in a concentration-dependent fashion compared to gdf <dig>  we wanted to determine if this difference was maintained when we performed a pulse-chase exposure to each ligand .
to examine this, we performed two separate experiments.
in the first, we treated hek <dig> cells with gdf <dig> or gdf <dig> at three different concentrations  for the indicated times , after which the culture medium was removed and replaced with ligand-free culture medium for the duration of the experiment.
at 24 h , the cells were lysed and assayed for smad3-dependent luciferase activity .
gdf <dig> elicited a stronger response compared to gdf <dig> in both a concentration- and a time-dependent manner.
in fact, at lower concentrations and exposure times, treatment with gdf <dig>  but not gdf <dig>  resulted in productive signaling .
a second experiment was performed where the cells were treated for the indicated time  and immediately assayed for activity .
interestingly, at the lower concentration , we observed a significant difference in smad <dig> activation compared to gdf <dig> at most time points.
these differences were less pronounced or non-existent at higher concentrations .
together, these results indicate that the cellular response to mature gdf <dig> and gdf <dig> can be significantly different depending on the concentration and duration of exposure.
modulation of gdf <dig> and gdf <dig> activity by known extracellular antagonists
like many tgfβ ligands, extracellular antagonists modulate gdf <dig> and gdf <dig> signaling.
here, we determined if there were differences in the effectiveness of the known antagonists fs <dig>  fstl <dig>  gasp <dig>  and gasp <dig> to inhibit gdf <dig> or gdf <dig> using our hek <dig>  <dig> luciferase assay.
fs <dig> and gasp <dig> similarly inhibited gdf <dig> or gdf <dig> .
however, fstl <dig> and gasp <dig>  more potently inhibited the actions of gdf <dig> relative to gdf <dig> .
curiously, inhibition by fstl <dig> for gdf <dig> revealed a more negative hill slope compared to gdf <dig>  suggesting that, while they are both potently inhibited, the binding interactions between the ligands and antagonists may not be identical.fig.
<dig> inhibition of gdf <dig> and gdf <dig> by known antagonists.
a, b, c, d luciferase reporter assays showing the inhibitory activity following titration of fs <dig> , fstl <dig> , gasp <dig> , and gasp <dig>  against a constant concentration  of gdf <dig>  or gdf <dig>  in hek <dig>  <dig> cells.
refer to table  <dig> for a corresponding analysis of the inhibition curves.
data information: in a–d, data are presented as fraction activation .
each concentration was performed in triplicate and shown as the mean ± sem of two to three independent experiments.
data from independent experiments were combined and fit to non-linear regression with a variable slope.
ligand source: gift from acceleron pharma
a
ci confidence interval
b
sem standard error of the mean
structure of gdf <dig> bound to fs288
the complex of the gdf <dig> dimer bound to two molecules of fs <dig> was resolved using x-ray crystallography to  <dig>  Å .
this is the first structure of gdf <dig> bound to a known antagonist.
similar to previous ligand:follistatin structures , two molecules of fs <dig> bind symmetrically to wrap around the gdf <dig> dimer occluding both type ii and type i receptor binding sites.
as expected, follistatin domains  <dig>  and d <dig> overlap with the type ii binding epitope, whereas the follistatin n-terminal domain  occupies the type i binding slot.
the overall structure of gdf11:fs <dig> is highly similar to that of the gdf8:fs <dig> complex  =  <dig>  Å).
nonetheless, the structure of gdf11:fs <dig> reveals minor changes in the positioning of residues in the helix of nd .
for example, f <dig> of fs <dig> is pushed inward to accommodate the larger side chain of m <dig> in gdf <dig> versus v <dig> in gdf <dig> .
this is consistent with other ligand:fs structures, showing that the nd helix is quite plastic in its “molding” to accommodate ligand differences at the type i interface .
as observed in previous ligand:fs structures , the nd from one molecule interacts with d <dig> from the other follistatin in a head-to-tail fashion.
similar to gdf8:fs <dig>  <cit> , the electrostatic surface potential of gdf11:fs <dig> depicts a large, continuous electropositive surface on one side of the complex.
this is formed by the combination of the heparin binding motif of the two fs molecules with positively charged residues of gdf <dig>   <cit> .
as such, the gdf11:fs <dig> complex has increased affinity for heparin as compared to fs <dig> alone, and slightly stronger affinity than gdf8:fs <dig>  .fig.
<dig> the structure of the gdf11:fs <dig> complex.
a comparison of the gdf11:fs <dig>  and gdf8:fs <dig>  3hh2;  <cit> ) structures.
the inset shows the differences between the interaction of the fsnd helix and the ligand type i receptor epitope.
recombinant human gdf <dig> was obtained from acceleron pharma for resolution of this crystal structure.
b isoelectric surface representation of the gdf11:fs <dig>  and gdf8:fs <dig>  structures.
surfaces are colored blue  and red  on a scale of – <dig> to  <dig> kbt/ec using the apbs plugin  <cit>  for pymol.
c heparin affinity analysis of fs <dig> alone, gdf11:fs <dig>  and gdf8:fs <dig> complexes.
gdf11:fs <dig> complex has higher affinity for heparin than fs <dig> alone indicated by elution at a higher ionic strength.
purified proteins and complexes  were applied to a heparin column and eluted with a linear sodium chloride  gradient.
the approximate nacl concentration for protein elution is shown at the peak maxima.
ligand source: gift from acceleron pharma
r
merge
r
pim
mn b
cc1/2
c
r
work , r
free
clashscoree
avalues in parentheses are for highest resolution shell
bmn is defined as < merged < ih >/sd  > ≈ signal/noise
ccc1/ <dig> for highest resolution shell
d
r
free calculated from 5% of initial total number of reflections
edetermined by molprobity
gdf <dig> and gdf <dig> structural differences: follistatin-bound versus apo states
initial investigation of the sequence alignment between gdf <dig> and gdf <dig> mature domains did not provide a clear explanation for why gdf <dig> is more potent than gdf <dig> .
in other words, there did not appear to be major amino acid differences  between the two ligands that would indicate significant differences in how they would interact with binding partners or receptors.
however, when the differences between gdf <dig> and gdf <dig> were plotted on the structure, we saw that the majority are found near the type i receptor interface .
therefore, to determine if there were any unique structural consequences of these amino acid differences, we made a more thorough examination of the x-ray crystal structures of mature gdf <dig> and gdf <dig> in the follistatin-bound and apo states.fig.
<dig> sequence alignment and structural comparison of gdf <dig> and gdf <dig> in their fs288-bound state.
a sequence alignment of mature gdf <dig> and gdf <dig>  secondary structure for each ligand in the fs288-bound form is shown above and below the gdf <dig> and gdf <dig> sequences, respectively, where arrows represent β-sheet and cylinders represent α-helix.
cysteines are highlighted in yellow, and residues that are different between gdf <dig> and gdf <dig> are highlighted in blue.
solid black lines joining two cysteines indicate intramolecular disulfide bonds.
the dotted black line indicates the cysteine responsible for the intermolecular disulfide bond.
b distribution of the amino acid differences between gdf <dig> and gdf <dig>  gdf <dig> is shown as ribbon, and non-identical amino acids are shown as orange spheres.
symmetrical type i and type ii interfaces are depicted with blue and yellow lines .
insets represent zoomed-in views depicting the molecular differences between fs288-bound gdf <dig>  and fs288-bound gdf <dig>  in the vicinity of the type i receptor binding interface.
the arrow points to the hydrogen bond  in the fs288-bound gdf <dig> between q <dig> and carbonyl oxygen of y <dig>
superposition of the mature ligands from their respective fs <dig> complex structures did not reveal any major structural differences in the type ii interface  of the ligand.
however, examination of the type i interface revealed two unique interactions in the wrist of gdf <dig> compared to gdf <dig> .
the wrist region is known to be critically important for dictating ligand type i receptor affinity and specificity  <cit> .
first, superposition of the ligands showed that y55a of gdf <dig> was shifted by ~ <dig>  Å inward toward the n-terminal side of the wrist helix on the adjacent monomer.
this was facilitated by the lack of a side chain in g100b of gdf <dig> .
in gdf <dig>  the corresponding residue is a100b where the methyl side chain sterically occludes y55a.
this difference allows y55a to more intimately interact with the opposing chain and facilitates additional hydrophobic interactions with m79b and the aliphatic side chain of k54a.
the second unique feature of gdf <dig> is the formation of an additional hydrogen bond between the backbone  of the pre-helix loop and q62a located on the c-terminal side of the wrist helix .
in gdf <dig>  the corresponding residue is h62a and does not interact with the pre-helix loop in the fs288-bound gdf <dig> crystal structure .
in fact, h62a does not interact with the pre-helix loop in additional crystal structures of gdf <dig> bound to either fstl <dig>  <cit>  or a neutralizing antibody   <cit> .
thus, amino acid differences between gdf <dig> and gdf <dig> impose unique intramolecular contacts in gdf <dig> that are not present in gdf <dig>  given that these changes occur at the type i interface, it is intriguing to speculate that they could contribute to differences in ligand activity.
however, we cannot exclude the possibility that these newly identified interactions are a result of binding to fs <dig>  to explore this possibility, we examined the x-ray crystal structures of apo-gdf <dig> and apo-gdf <dig>  <cit> .fig.
<dig> structural comparison of gdf <dig> and gdf <dig> in fs288-bound and apo-conformations reveal ligand flexibility.
cartoon diagrams of the bound and apo states of gdf <dig>  and gdf <dig> .
each monomer is colored independently to highlight the differences in conformation.
superposition of each conformation using monomerb  is shown on the left.
protein data bank  ids for bound ligands  and apo ligands .
resolution of apo-gdf <dig> was determined from recombinant human gdf <dig> produced and purified as previously described  <cit> .
resolution of fs288-bound and apo-gdf <dig> was determined from recombinant human gdf <dig> produced and purified by acceleron pharma
we resolved the structure of apo-gdf <dig> to  <dig>  Å  and the apo-gdf <dig> structure to  <dig>  Å  using x-ray crystallography, thus allowing a direct comparison to the recently published apo-gdf <dig> structure   <cit> .
unexpectedly, resolution of the apo-gdf <dig> structure revealed a unique conformation such that the fingertips of each monomer are positioned proximally in a “closed” conformation compared to the classical distal positioning or “open” conformation observed for many ligands, including structures of fs-bound gdf <dig>   <cit> .
while the structure of apo-gdf <dig> adopted an unexpected “closed” conformation, a similar conformation has been observed for activin a when in complex with actriib  <cit> .
in both cases, the wrist region is disordered and not resolved in the crystal structure .
our new apo-gdf <dig> structure, solved under similar crystallization conditions as apo-gdf <dig>  adopted an exaggerated “open” conformation such that the fingers extend past the horizontal plane .
this conformation shares a similar exaggerated “open” conformation to the crystal structure of gdf <dig> bound to a neutralizing antibody   <cit> .
however, the wrist region in our apo-gdf <dig> structure is also disordered and not resolved in the crystal structure, similar to apo-gdf <dig> .
the previously published apo-gdf <dig> crystal structure adopts the classic “open” conformation with an ordered wrist helix that includes the additional interchain hydrogen bond between q62a and the pre-helix loop observed in the fs288-bound gdf <dig> crystal structure.
taken together, crystallization of both gdf <dig> and gdf <dig> in their apo states reveals intrinsic structural flexibility in these ligands.
gdf <dig> signals more effectively through type i receptors than gdf8
to better understand the potency differences between gdf <dig> and gdf <dig>  we compared their receptor binding affinities and utilization.
given that the structural differences between gdf <dig> and gdf <dig> are positioned at the type i receptor interface, we hypothesized that potency differences are likely through differences in type i interactions.
in order to rule out potential differences in type ii receptor binding, we first compared the inhibitory potential  of the soluble receptors, actriib extracellular domain  and fc-actriib-ecd using our hek <dig>  <dig> luciferase assay .
we found no difference in the ic <dig> value for actriib-ecd between gdf <dig> and gdf <dig> , whereas fc-actriib-ecd was ~ threefold more potent in inhibiting gdf <dig>  than gdf <dig> .
while the calculated hill slope for the actriib-ecd was similar for gdf <dig> and gdf <dig>  we observed a more negative hill slope for the inhibition of gdf <dig> by the fc-actriib-ecd compared to gdf <dig>  similar to our earlier results showing a more negative hill slope when gdf <dig> was antagonized by fs <dig>  fstl <dig>  and gasp <dig> .fig.
<dig> enhanced type i receptor utilization by gdf <dig> compared to gdf <dig>  a, b, luciferase reporter assays showing the inhibitory activity following titration of actriib-ecd , and fc-actriib-ecd  against a constant concentration  of gdf <dig>  or gdf <dig>  in hek <dig>  <dig> cells.
refer to table  <dig> for a corresponding analysis of the inhibition curves.
c differences in type i receptor utilization by gdf <dig> following transient transfection of a single type i receptor  or a combination of type i receptors .
rib l <dig> cells were co-transfected with  <dig>  ng of the individual receptor dna alone or various receptor dna combinations  and  <dig>  ng  <dig> promoter plasmid driving the luciferase gene.
purified recombinant ligands were added 12 h post transfection.
cells were then lysed and assessed for luciferase activity 8 h post ligand treatment.
d treatment of rib l <dig> cells with dmso or the type i receptor small molecule inhibitor sb <dig> following transfection of empty vector  or alk <dig> s270t.
rib l <dig> cells were co-transfected with  <dig>  ng of the individual receptor dna and  <dig>  ng  <dig> promoter plasmid driving the luciferase gene.
purified recombinant ligands were added 12 h post transfection.
cells were then lysed and assessed for luciferase activity 8 h post ligand treatment.
data information: in a, b, data are presented as fraction activation .
each concentration was performed in triplicate and shown as the mean ± sem of two independent experiments.
data from independent experiments were combined and fit to non-linear regression with a variable slope.
in c, d, data are presented as fold activation defined as the total activation from each ligand compared to cells only transfected with the  <dig> reporter construct.
each bar is the mean ± sem.
a representative experiment is shown of at least two independent experiments in which each concentration was performed in duplicate or triplicate.
only comparisons between gdf <dig> and gdf <dig> were made.
*p ≤  <dig>  and **p ≤  <dig>  .
ligand sources: gdf <dig> and gdf <dig>  gift from acceleron pharma; activin a, activin b, and tgfβ <dig>  produced and purified as described in “methods”
a
ci confidence interval
b
sem standard error of the mean
c
ecd extracellular domain
next, we determined if gdf <dig> and gdf <dig> signal through similar type i receptors.
numerous studies have shown that gdf <dig> and gdf <dig> utilize the type i receptors alk <dig> and alk <dig> .
in addition, gdf <dig> can signal through alk <dig>  <cit> , although it is unclear if gdf <dig> also utilizes this receptor.
to our knowledge, a study directly comparing signaling by both ligands via the three receptors has not been reported.
therefore, we assessed the relative  <dig> luciferase reporter response to gdf <dig> or gdf <dig> following transient expression of individual type i receptors in rib l <dig> cells .
this cell line lacks alk <dig> expression and expresses low levels of alk <dig>  <cit> .
the level of alk <dig> expression in these cells has not been reported.
as expected, activin a robustly signaled in cells transfected with alk <dig>  <cit> , activin b signaled through both alk <dig> and alk <dig>  <cit> , and tgfβ <dig> specifically signaled through alk <dig>   <cit> .
consistent with previous reports, gdf <dig> and gdf <dig> readily signaled through alk <dig> and alk <dig> .
additionally, we confirmed that gdf <dig> signaled through alk <dig>   <cit> .
strikingly, gdf <dig> induced greater reporter responses than gdf <dig> via alk <dig>  alk <dig>  or alk <dig> .
we should note that activin a, activin b, gdf <dig>  and to a lesser extent gdf <dig>  stimulated some reporter activity in the absence of exogenous receptors, suggesting low levels of endogenous alk <dig> and alk <dig> expression by these cells .
consistent with this idea, activin b, gdf <dig>  and gdf <dig> induction of reporter activity was completely blocked by addition of the alk4/5/ <dig> small molecule inhibitor sb <dig>  .
importantly, signaling could be rescued by transfection of an sb431542-resistant form  of human alk <dig> , confirming that all three ligands can signal via alk <dig> with relative potencies of activin b > gdf11 > gdf <dig>  collectively, our results indicate that gdf <dig> can generate a greater smad3-dependent signal compared to gdf <dig> through all three type i receptors.
given that type i receptor expression is not ubiquitous in all cells, we next wanted to determine if differences in alk4/5/ <dig> combinations would further distinguish or normalize response differences between gdf <dig> and gdf <dig>  different receptor combinations modestly enhanced signaling relative to individual receptors .
interestingly, when alk <dig> and alk <dig> were co-transfected, there was no statistically significant difference in response to gdf <dig> and gdf <dig> , in contrast to when either receptor was expressed alone .
co-transfection of alk <dig> and alk <dig> resulted in a more robust response to gdf <dig> over gdf <dig>  taken together, these results further suggest that ligand sensitivity can be driven, in part, by the receptor expressed on a particular cell type, thereby providing a possible avenue by which cells may discriminate between highly similar ligands, such as gdf <dig> and gdf <dig>
structural studies describing the interaction between the type i receptors and the activin/inhibin subclass are lacking, predominantly due to the inability to generate recombinant receptors whose folding and biological activity can be confirmed unequivocally.
while this barrier remains unresolved for alk <dig> and alk <dig>  we can reliably study the interactions between ligands and the alk5-ecd  <cit> .
we tested binding of gdf <dig> and gdf <dig> to recombinant alk5-ecd in the absence or presence of actriib-ecd using native-page and surface plasmon resonance .
neither mature gdf <dig> nor gdf <dig> alone were readily resolved using native-page .
however, addition of actriib-ecd to either ligand resulted in a mobility shift compared to actriib-ecd alone, indicative of complex formation between the ligand and type ii receptor .
mixing the ligands with alk5-ecd alone did not result in a detectable alk5-ecd-ligand complex .
however, when we co-incubated either ligand with a constant amount of actriib-ecd followed by titration with alk5-ecd, we observed a noticeable shift in the migration pattern for gdf <dig>  especially apparent at excess molar ratios of alk5-ecd.
under similar conditions, little to no change was observed with gdf <dig> .
although high concentrations were required, these results suggest that a low-affinity ternary complex composed of gdf11:actriib:alk <dig> was more readily formed than the corresponding complex with gdf <dig> fig.
<dig> binding of gdf <dig> to the type i receptor alk <dig>  a basic native-page  of gdf <dig>  and gdf <dig>  ligand, receptor, and ligand:receptor complexes.
to test for ligand-receptor complex formation, receptors were first diluted into 20 mm hepes ph  <dig>  followed by addition of the ligand.
the ligand:receptor mixtures were held at room temperature  for 15 min before being loaded and run in the gel.
amount loaded for each protein: ligand , alk5-ecd , ligand + alk5-ecd , actriib-ecd  = 3 μg each; ligand  =  <dig>  μg; ligand + actriib-ecd  =  <dig>  μg ligand and 3 μg actriib-ecd; for alk5-ecd titration  ligand =  <dig>  μg, actriib-ecd = 3 μg, and  <dig>   <dig>   <dig>  or 8 μg alk5-ecd.
proteins were visualized by colloidal coomassie stain.
b surface plasmon resonance  sensorgrams of gdf <dig>  and gdf <dig>  binding to fc-alk5-ecd.
fc-alk5-ecd was captured by protein a that was amine coupled to a cm <dig> chip.
serial dilutions  of each ligand alone  or in the presence  of a twofold molar excess of actriib-ecd were allowed to associate for 120 s at a 70 μl/min flow rate followed by a 280 s dissociation at 37 °c.
sensorgrams were double referenced using an average of two  <dig> nm ligand injections.
data information: ligand sources: gdf <dig> and gdf <dig>  gift from acceleron pharma; activin a, activin b, and tgfβ <dig>  produced and purified as described in “methods”
to clarify these results, we attempted to detect direct binding of either ligand to alk5-ecd, fc-alk5-ecd, and fc-actriib using spr .
in this experiment, the receptor was immobilized on the spr biosensor chip with standard amine coupling or captured using protein a. as expected, both ligands readily bound the fc-actriib-ecd  .
qualitatively, gdf <dig> interacted with both alk5-ecd and fc-alk5-ecd in a dose-dependent manner and exhibited a fast association and rapid dissociation .
in contrast, gdf <dig> did not bind to alk5-ecd or fc-alk5-ecd in a dose-dependent fashion .
given that alk <dig> binding to tgfβ is significantly enhanced through a cooperative interaction with tβrii  <cit> , we speculated that actriib might improve gdf <dig> or gdf <dig> binding to alk5-ecd.
interestingly, co-injection of actriib-ecd with gdf <dig> did not appear to alter the association or dissociation with alk5-ecd compared to gdf <dig> alone .
however, we did observe a detectable improvement in binding of gdf <dig> to alk5-ecd in the presence of actriib-ecd .
these results suggest that gdf <dig> binding to alk <dig> is likely more favorable compared to gdf <dig>  however, it is clear that additional experiments will be necessary to fully resolve the interactions between these ligands and their cognate type i receptors.
substitution of gdf11-like residues into gdf <dig> enhances ligand activity
to determine which residues are important for conferring enhanced activity to gdf <dig>  we generated a number of gdf8/gdf <dig> chimera constructs.
site-directed mutagenesis was performed on the full-length human gdf <dig> to introduce gdf11-like substitutions .
we compared the activity of the chimeras to wild-type  gdf <dig> by transient co-transfection of the constructs with tolloid-like  <dig>  and furin using hek <dig>  <dig> luciferase cells .
we focused our efforts on the regions showing the most divergence between gdf <dig> and gdf <dig>  specifically the type i interface consisting of the pre-helix loop and wrist .
interestingly, transient expression of a number of chimeric mutants revealed a significant gain in activity .
the increased activity was observed at nearly every concentration  tested , with differences between the chimeric mutants and wt gdf <dig> more pronounced at lower concentrations .
thus, incorporation of gdf11-like residues into gdf <dig> can increase ligand potency.
consistent with our overall hypothesis, we found that full substitution  of all different residues between mature gdf <dig> and gdf <dig> resulted in a very potent ligand compared to wt gdf <dig>  particularly at lower concentrations .
remarkably, a significant gain in activity was observed in the chimeras that incorporated the gdf <dig> residues, q <dig> and g <dig> , which were shown in the structure to form additional, stabilizing interactions at the ligand dimer interface .fig.
<dig> generation of gdf8/gdf <dig> chimeric ligand confers potency to gdf <dig>  a luciferase reporter gene assay  <dig> promoter) following transient co-transfection of hek <dig> cells with gdf8/gdf <dig> chimera constructs , furin , and tll <dig> .
all constructs contain human wild-type gdf <dig> prodomain followed by human wild-type or mutated mature gdf <dig>  18–24 h post transfection, the culture media were replaced with serum-free media and allowed to incubate for an additional 24 h, at which point the cells were lysed and measured for luciferase activity.
wt = gdf8; point mutations are indicated by number.
for chimera number  <dig> , each non-identical residue between gdf <dig> and gdf <dig> was mutated to generate the equivalent wt gdf <dig> ligand.
a surface representation of gdf <dig> dimer is shown.
each monomer is colored independently .
non-identical residues are shown in orange.
b, c luciferase reporter gene assay  <dig> promoter) following exogenous addition of purified empty vector, wt, or gdf8/gdf <dig> chimeric latent protein complexes .
the latent complexes  were activated by treatment with acid , or the hek <dig> cells were transiently transfected with tll <dig> prior to protein addition .
aa acid activated, not aa no acid activation, not aa + gasp <dig> no acid activation but complexes added to cells in the presence of  <dig> nm gasp <dig>  aa + gasp <dig> acid activation in the presence of  <dig> nm gasp <dig>  ev empty vector transfected, ev + gasp <dig> empty vector transfected, but complexes added in the presence of  <dig> nm gasp <dig>  tll <dig> transfected with tll <dig>  tll2 + gasp <dig> cells transfected with tll <dig>  complexes added in the presence of  <dig> nm gasp <dig>  data information: in a, b, c, data are presented as a ratio of the fold activation  where each was normalized to the response of empty vector control.
each concentration was performed in duplicate or triplicate, and each bar is shown as the mean ± sem from two to three independent experiments.
a
p ≤  <dig> , b
p ≤  <dig> , c
p ≤  <dig> , and ns = not significant .
ligand sources: produced and purified as described in “methods”
to rule out that activity differences were attributed to differences in the relative amounts of protein expression, we produced and purified wt  and selected mutant latent complexes .
to determine their specific activity relative to wt gdf <dig> , we used two methods known to activate the latent complex: acid activation  and activation with a member of the tld family, such as tolloid-like  <dig>   <cit> .
overall, our results from treatment with purified protein were consistent with the results from transient transfection .
as expected, little to no luciferase activity was detected in cells treated with the latent complexes unless they were acid activated  or applied to cells transfected with tll <dig> .
in addition, we performed the experiments in the presence of gasp <dig>  a specific antagonist of gdf <dig> and gdf <dig>  <cit> , to confirm that the signal was specific to wt gdf <dig> or gdf8/gdf <dig> chimeras .
both activation methods revealed similar findings with the exception of the acid-activated empty vector sample .
the acid-activated ev sample showed a significant response that could not be attenuated with gasp <dig> , indicating possible contamination by additional ligands or ph-dependent artifacts.
despite this possibility, most of the signal with gdf <dig> or gdf8/gdf <dig> chimeras was attenuated by gasp <dig>  suggesting that the majority of the signal was specific to the ligands added.
moreover, the higher background signal was not observed in the tll <dig> experiment .
together, these data support the conclusions that mature gdf <dig> is more active than gdf <dig> and that gdf <dig> activity can be enhanced through substitution of specific residues from gdf <dig>
gdf <dig> is a more potent inducer than gdf <dig> of phosphorylated smad2/ <dig> in mouse primary skeletal myoblasts in vitro and the mouse myocardium in vivo
since we have shown that gdf <dig> is more potent than gdf <dig> in multiple cell lines, we next sought to determine if similar differences in potency could be seen in primary cells.
consistent with a previous report  <cit> , western analysis of cultured myoblasts treated with mature gdf <dig> or gdf <dig> at a range of concentrations showed robust, dose-dependent induction of phosphorylated smad2/ <dig>  compared to untreated cultures .
further, gdf <dig> treatment elicited a significantly greater induction of psmad2/ <dig> compared to equivalent concentrations of gdf <dig> .fig.
<dig> exogenous treatment of gdf <dig> potently activates smad2/ <dig> in cultured primary skeletal muscle myoblasts.
a, b western blot  and quantification  showing phosphorylated smad2/ <dig> , total smad2/ <dig> , and gapdh of cultured primary skeletal muscle myoblasts following treatment with gdf <dig> or gdf <dig>  cells were treated with a range of ligand concentrations  for 1 h, lysed, and probed for the indicated proteins.
20 μg total protein loaded.
primary skeletal myoblasts were obtained from three different animals .
data information: in b, data are presented as a ratio of psmad2/ <dig> to total smad2/ <dig>  to obtain this ratio, psmad2/ <dig> or smad2/ <dig> was first normalized to gapdh.
the ratio of this quotient was then graphed.
data are presented as the mean ± sem.
*p ≤  <dig>  and **p ≤  <dig>  .
ligand sources: peprotech cat.
no.
120- <dig> and cat.
no.
120-11
to determine if similar potency differences between gdf <dig> and gdf <dig> may also occur in vivo, we next examined smad <dig> phosphorylation  in the mouse myocardium following intravenous  injection of various doses of gdf <dig> and gdf <dig> .
psmad <dig> levels were significantly increased in the myocardium 1 h after injection of  <dig>  mg/kg of gdf <dig> or gdf <dig> .
at equivalent doses, gdf <dig> stimulated significantly more psmad <dig> than gdf <dig> .
in fact, nearly eightfold more gdf <dig> than gdf <dig> was required to achieve similar levels of psmad <dig> .
these data suggest that, depending on dose, intravenous gdf <dig> or gdf <dig> can result in a significantly different smad2/3-dependent response in the myocardium.fig.
<dig> intravenous injection of gdf <dig>  but not gdf <dig>  elicits robust phosphorylation of smad2/ <dig> in the mouse myocardium.
a, b, c, d representative western blots  showing phosphorylated smad <dig> , total smad2/ <dig> , and gapdh and corresponding quantification  in the mouse myocardium 1 h post tail-vein injection of saline, gdf <dig>  or gdf <dig>  a, c show a direct comparison in the level of psmad <dig> in the myocardium following injection of  <dig>  mg/kg gdf <dig> or gdf <dig> .
b, d show that nearly eightfold more gdf <dig> than gdf <dig> is required to achieve a similar psmad <dig> response .
data information: in c, d, data are presented as a ratio of psmad2/total smad2/ <dig>  to obtain this ratio, psmad <dig> or smad2/ <dig> was first normalized to gapdh.
the ratio of this quotient was then graphed.
data are presented as the mean ± sem.
*p ≤  <dig>  and ***p ≤  <dig>  .
ligand sources: peprotech cat.
no.
120- <dig> and cat.
no.
120-11
discussion
previous studies indicate that gdf <dig> and gdf <dig> have both divergent and overlapping roles in developmental processes and tissue maintenance in adult animals .
while the role of gdf <dig> during development is well defined, its role in the adult remains poorly understood.
however, recent evidence suggests that gdf <dig> is an important circulating factor that declines with age and exogenous treatment with gdf <dig> can reverse age-related effects including reducing cardiac hypertrophy  <cit> , improving skeletal muscle regeneration  <cit> , and promoting neurogenesis  <cit> .
however, other studies have contradicted certain aspects of these claims, fueling significant controversy on the functional similarities and differences between gdf <dig> and gdf <dig>  <cit> .
controversy notwithstanding, in this study, we investigated the signaling activity and structural features of the mature gdf <dig> and gdf <dig> ligands.
at the outset of our studies, we made the unexpected observation that gdf <dig> is a more potent signaling ligand than gdf <dig>  this observation was consistently and reproducibly observed across multiple laboratories, using different ligand preparations, in multiple cell lines and cultured primary cells, and recapitulated in vivo in the mouse myocardium.
the current dogma in the literature suggests that there are no differences in the signaling potencies of gdf <dig> and gdf <dig> and that the ligands have identical signaling properties  <cit> .
our results are consistent with this idea, but only at the highest ligand concentrations examined.
interestingly, prior studies also appeared to show that gdf <dig> is more potent or active than gdf <dig>  <cit> , yet many conclusions from these studies maintain the notion that gdf <dig> and gdf <dig> signal and function identically.
the conclusion that gdf <dig> and gdf <dig> are indistinguishable may derive from the fact that subsequent comparisons were performed at concentrations that were above the ec <dig> for both ligands, thus resulting in identical transcriptional outcomes.
in fact, we show that at high concentrations of gdf <dig> and gdf <dig>  smad3-dependent readout is indistinguishable under certain conditions .
however, our data also reveal that gdf <dig> can elicit a significant response under conditions where gdf <dig> appears to have little to no activity.
these results show that the responses generated by gdf <dig> and gdf <dig> are highly dependent on the concentrations of ligand and the available receptors, emphasizing that gdf <dig> and gdf <dig> are not equivalent.
to be clear, these data neither support nor refute claims made by other groups regarding the functional outcomes resulting from gdf <dig> or gdf <dig> signaling.
rather, they demonstrate that the biochemical responses elicited by gdf <dig> and gdf <dig> at equivalent concentrations are significantly different under the conditions we tested.
the physiological relevance, if any, which may result from potency differences, requires additional investigation.
given the nearly identical sequence of their mature ligands, we were initially puzzled by the potency differences between gdf <dig> and gdf <dig>  especially since most of the changes are relatively conserved.
therefore, we turned to x-ray crystallography to determine the structures of gdf <dig> in complex with fs <dig> in addition to gdf <dig> and gdf <dig> in their apo forms.
while this manuscript was in preparation, an additional apo-gdf <dig> structure was resolved  <cit> , thus providing the opportunity to compare multiple structures of each ligand in their fs288-bound and apo states.
as expected, the overall structures of the ligands when bound to fs <dig> are highly similar with minor changes in the fsnd helix of fs <dig>  a region previously shown to be able to accommodate different ligands .
comparison between gdf <dig> and gdf <dig> revealed that differences cluster within and around the type i receptor binding surface.
in particular, differences are observed in residues within or bordering the wrist helix, an area important for type i receptor binding .
to explore if these differences are the basis for increased activity, we used a chimeric approach that introduced gdf11-specific residues into gdf <dig>  apart from the pre-helix loop, the two most prominent differences between the structures of gdf <dig> and gdf <dig> are located at each end of the wrist helix and include gdf <dig> residues q <dig> and g <dig>  in both cases, substitution of the individual gdf <dig> residues h <dig> and a <dig> with the gdf <dig> residues q <dig> and g <dig> resulted in increased activity over wild-type gdf <dig>  moreover, combination of these two mutations  resulted in an additional gain in activity over the single mutations alone, suggesting an additive effect.
in gdf <dig>  these residues appear to stabilize the dimer interface where an additional hydrogen bond is formed between q <dig> and the backbone of the pre-helix loop.
thus, increased dimer stability or stability of the interaction between the wrist and pre-helix loop might be one explanation for the increased ligand activity of gdf <dig>  though we cannot rule out the possibility that these residues directly interact with the receptors.
however, given that activity differences are observed over multiple receptors, it is more likely that potency differences are a result of inherent ligand differences .
furthermore, introducing the gdf11-specific residues in the pre-helix loop , a region known to be important for receptor specificity , also results in increased activity.
additionally, introduction of gdf <dig> fingertip residues, d <dig> and q <dig>  into gdf <dig> also significantly increased ligand activity.
however, our structural data do not provide a molecular explanation for how these variances confer differences in activity.
nevertheless, they do suggest that the potency differences between gdf <dig> and gdf <dig> are a result of a combination of the residue differences within and around the type i binding interface.
resolution of the apo-gdf <dig> and apo-gdf <dig> crystal structures revealed that both ligands exhibit significant conformational flexibility.
interestingly and despite the high sequence identity, nearly identical crystallization conditions resulted in two distinct conformations.
although these conformations may be a result of the crystallization process, it supports the idea that gdf <dig> and gdf <dig> are flexible enough to be trapped in alternate states.
however, an additional apo-gdf <dig> structure revealed the ligand in the classic “open” conformation and contained similar molecular contacts within the wrist region that we observed in the fs288-bound state  <cit> .
furthermore, under similar crystallization conditions, additional tgfβ superfamily growth factors have been solved in their apo states and adopt the classic “open” conformation .
for example, structures of several bmp ligands in the apo state all exhibit the “open” configuration .
the extreme “closed” conformation of gdf <dig> is not entirely unprecedented, as one structure of activin a in complex with actriib showed a similar “closed” configuration  <cit> .
this was initially somewhat controversial; however, multiple activin a structures have since been determined and support the notion that the activin a dimer is flexible  <cit> .
it should be noted that this is not the first example of gdf <dig> captured in an alternate conformation.
recently, the co-crystal structure of gdf <dig> bound to a neutralizing antibody was solved where the gdf <dig> ligand adopts an exaggerated “open” conformation  <cit> , resembling the apo-gdf <dig> structure presented here.
furthermore, similar to activin a and gdf <dig>  ligands of the tgfβ subclass also exhibit significant flexibility .
in fact, the biological activity of different tgfβ isoforms, which also share high overall sequence identity, was shown to correlate with the rigidity of the dimer, specifically the wrist helix  <cit> .
however, we cannot discount that the crystallization process has trapped these ligands in alternate conformations.
additional solution-based biochemical approaches are needed to better understand ligand dynamics.
nevertheless, differences in the wrist region of gdf <dig> could contribute to the increased ligand potency over gdf <dig> by either stabilizing the dimer interface or presentation of the wrist/pre-helix loop to facilitate differential direct interaction with the type i receptors, or a combination thereof.
our data strongly indicate that gdf <dig> is a more potent signaling ligand compared to gdf <dig>  while this observation has not been explicitly realized in the literature, there is evidence suggesting that our results are consistent in that gdf <dig> is a more potent ligand than gdf <dig>  <cit> .
however, a recent study utilizing a cell line with modified and potentially functionally inactivated type ii and type i receptors showed that gdf <dig> and gdf <dig> are nearly indistinguishable in terms of their potential for type ii and type i heterodimerization  <cit> .
in this same study and using another assay, there was little difference in the potentiation of downstream smad2/ <dig> responsive elements between gdf <dig> and gdf <dig>  <cit> .
interestingly, we observed that differences in smad2/ <dig> responsiveness between gdf <dig> and gdf <dig> were less pronounced in the hepg <dig> cell line, the same cell line utilized in  <cit> .
we observed more robust differences between gdf <dig> and gdf <dig> in other cell lines and in vivo.
therefore, a potential explanation for these differences may be due to the receptor profile of a particular cell type or tissue in addition to other confounding factors such as co-receptors.
given that gdf <dig> may not utilize the type i receptors as effectively as gdf <dig>  it is tempting to speculate that both potency and receptor utilization differentiate the biological actions of gdf <dig> and gdf <dig>
CONCLUSIONS
in conclusion, we present evidence supporting the notion that gdf <dig> and gdf <dig>  despite their high sequence identity, are not functionally equivalent.
our data show that gdf <dig> is a more potent activator of smad2/ <dig> in vitro and in vivo.
gdf <dig> has been considered a somewhat unique ligand of the tgfβ family due to dual utilization of the type i receptors alk <dig> and alk <dig>  our data suggest that gdf <dig> possesses these same attributes but, due to differences in amino acid composition, utilizes these receptors more effectively to initiate signal transduction.
apart from additional regulatory alterations between gdf <dig> and gdf <dig>  these ligands may have evolved differences in relative potency through selective pressure in order to provide an evolutionary advantage.

 the transforming growth factor β superfamily of secreted proteins comprises more than structurally related, yet functionally distinct proteins that play critical roles in embryological tissue development and patterning, wound healing, and adult tissue maintenance. the tgfβ superfamily can be divided into three subclasses: tgfβs, bone morphogenetic proteins, and activins/inhibins. growth/differentiation factors and are two closely related members of the activin/inhibin subclass that share ~90% sequence identity in their mature, c-terminal signaling domains and ~52% identity in their n-terminal prodomains. gdf and gdf bind to similar receptors and extracellular antagonists, leading to the assumption that mature gdf and gdf ligands are functionally indistinguishable. while it is clear that gdf and gdf share many commonalities, including overlapping or redundant roles in certain biological processes, there is accumulating evidence that the two ligands may be functionally distinct. gdf is expressed postnatally by skeletal and cardiac muscle and therein negatively regulates skeletal muscle mass by suppressing both the number and size of individual muscle fibers. in contrast, gdf appears to act more broadly, regulating anterior/posterior patterning and development of multiple organs/tissues. many tissues express gdf postnatally, including the spleen, pancreas, kidney, and skeletal muscle. however, determination of gdf11’s exact role in the adult has remained elusive due to the embryonic lethality of gdf11 -/- mice. in stark contrast, gdf8 -/- mice survive into adulthood and have a profound hypermuscular phenotype, which can be recapitulated in wild-type mice using natural occurring antagonists of gdf such as follistatin, follistatin-like, and growth/differentiation factor-associated serum protein. interestingly, gdf8 -/- /gdf11 -/- mice have exaggerated homeotic axial transformations compared to gdf11 -/- mice, suggesting that gdf and gdf have redundant functions in skeletal patterning. however, muscle-specific knockout of gdf does not result in significant increases in muscle mass and circulating gdf does not overcome the hypermuscular phenotype found in gdf8 -/- mice, suggesting that gdf and gdf do not serve redundant roles in regulating skeletal muscle mass. thus, while it is clear that loss of one ligand compared to the other yields drastically different phenotypes, it has been argued that these differences relate primarily to differential localization of ligand expression and do not reflect differences in ligand signaling. similar to other tgfβ ligands, gdf and gdf are disulfide-linked dimers that are initially synthesized as precursors, which are cleaved by furin-like proteases to separate the n-terminal prodomain from the c-terminal mature domain. unlike most tgfβ ligands, mature gdf and gdf remain tightly bound to their prodomains, holding them in a latent state. ligand activation requires additional cleavage of the prodomain by bmp1/tolloid metalloproteinases. the ligand dimer elicits signal transduction by symmetrically binding two type ii and two type i transmembrane serine/threonine kinase receptors. ligand-induced receptor clustering leads to phosphorylation of smad and smad transcription factors by the type i receptor. subsequent accumulation of smad2/ in the nucleus results in activation or repression of gdf and gdf responsive genes. similar to other ligands in the activin/inhibin subclass, gdf and gdf predominantly signal through the type ii receptors, activin receptor kinase iia and actriib and the type i receptors, activin-like receptor kinase and alk. there is also evidence that gdf can signal using the type i receptor alk. furthermore, signaling by both gdf and gdf is controlled by extracellular protein antagonists, including fs, fstl, gasp and gasp.fig. gdf is a more potent ligand than gdf a overview of the well-established canonical activin a, activin b, gdf gdf and tgfβ receptor utilization and downstream smad pathway. b, c, d potency differences between gdf and gdf luciferase reporter gene assay promoter) following titration of gdf and gdf ligands in hek and hepg cells. luciferase activity was assessed 18–24 h post ligand treatment. the black arrows in indicate the ligand concentrations utilized in panels e and f. in d, mouse gonadotrope cells were treated with increasing doses of gdf or gdf. follicle-stimulating hormone release was measured 24 h later, as previously described. refer to additional file 1: table s for a corresponding analysis of the activation curves. e short exposure to gdf results in a significantly enhanced smad3-dependent response compared to gdf the experimental design is such that the ligand was added to hek cells stably transfected with the promoter driving the luciferase gene for the indicated time followed by replacement of media without ligand. activity was measured 24 h after initial treatment. cells were treated with gdf or gdf at a ligand concentration of pm and pm. f time-dependent differences in the smad activation by gdf and gdf similar experimental design as in e, but instead cells were lysed and assessed for luciferase activity at the indicated time of ligand treatment. cells were treated with gdf or gdf at a ligand concentration of pm and pm. data information: in b, c and e, f, data are presented as fold activation above background. each concentration was performed in triplicate and shown as the mean ± standard error of the mean of three independent experiments. in d, data are presented as fold fsh release above background. each concentration was performed in triplicate and shown as the mean ± sem of one experiment; data shown are representative of independent experiments. in e, f, curves were compared using two-way anova with bonferroni correction. ligand sources: for b, e, f, gift from acceleron pharma; for c, d, purchased from r&d systems; cat. no. 788-g8-cf and cat. no. 1958-gd-010-cf) there have been conflicting reports comparing the biological functions of gdf and gdf with some question as to whether the two ligands are functionally equivalent. specifically, gdf was identified as a circulating factor that declines with age, and exogenous delivery of the mature ligand reversed many pathologies associated with aging, including reducing age-related cardiac hypertrophy, improving skeletal muscle performance and repair, improving olfaction, and increasing neurogenesis. conversely, other reports have challenged these claims, arguing that gdf and gdf are functionally and biologically equivalent. however, it is difficult to compare the results, given that many of these studies did not utilize the same experimental design, sources of recombinant proteins, or strategies for detection of gdf and gdf proteins. therefore, the extent of functional overlap between mature gdf and gdf without the prodomain remains to be determined. furthermore, a rigorous side-by-side biochemical and biological comparison of mature gdf and gdf has yet to be performed to determine if distinct features exist between these growth factors. in this study, using a variety of in vitro and in vivo systems, we show that gdf is significantly more potent than gdf and that the enhanced activity is due to differences in type i receptor utilization. in addition, we present three new x-ray crystal structures of apo-gdf apo-gdf and gdf in complex with follistatin. these structures, including an additional recently solved apo-gdf crystal structure, reveal that differences between gdf and gdf cluster in the type i receptor binding epitope. intriguingly, our structural analysis has revealed unique and alternate conformations of both gdf and gdf suggesting that both ligands are inherently flexible. gdf8/gdf chimeras, in which particular gdf residues are substituted into the gdf sequence, show that increased potency can be conferred to gdf taken together, our results demonstrate that, despite the high sequence identity between mature gdf and gdf the ligands indeed possess different signaling properties.  gdf is a more potent ligand than gdf8 recent reports have attributed unique in vivo properties to gdf relative to gdf. therefore, we first aimed to determine if signaling differences exist between these two growth factors using hek and hepg cells stably or transiently transfected, respectively, with the smad3-responsive luciferase reporter. side-by-side titration of mammalian-derived ligands revealed that gdf was more potent than gdf in both cell lines. remarkably, we determined that the half-maximal effective concentration values for gdf in hek and hepg cells were nm and nm, respectively, compared to nm and nm for gdf respectively. interestingly, the maximal smad response achieved by gdf was ~ fourfold higher compared to gdf in hepg cells, suggesting that there may be differences in ligand potency and/or in the repertoire of receptors and their relative utilization. furthermore, we found that gdf more potently stimulated the release of follicle-stimulating hormone than gdf in murine lβt pituitary gonadotrope cells. finally, gdf activated smad with ~ a sevenfold lower ec compared to gdf in a cells transfected with the luciferase reporter. these results are consistent with previous reports, which likewise indicated that gdf is a more potent activator of smad3-dependent signaling than gdf. note that ligands from different sources were utilized and were directly compared using the smad3-responsive luciferase reporter assay. for clarity, we have indicated the ligand sources utilized for each study in the figure legends. given that gdf stimulated greater smad activation in a concentration-dependent fashion compared to gdf we wanted to determine if this difference was maintained when we performed a pulse-chase exposure to each ligand. to examine this, we performed two separate experiments. in the first, we treated hek cells with gdf or gdf at three different concentrations for the indicated times, after which the culture medium was removed and replaced with ligand-free culture medium for the duration of the experiment. at 24 h, the cells were lysed and assayed for smad3-dependent luciferase activity. gdf elicited a stronger response compared to gdf in both a concentration- and a time-dependent manner. in fact, at lower concentrations and exposure times, treatment with gdf but not gdf resulted in productive signaling. a second experiment was performed where the cells were treated for the indicated time and immediately assayed for activity. interestingly, at the lower concentration, we observed a significant difference in smad activation compared to gdf at most time points. these differences were less pronounced or non-existent at higher concentrations. together, these results indicate that the cellular response to mature gdf and gdf can be significantly different depending on the concentration and duration of exposure. modulation of gdf and gdf activity by known extracellular antagonists like many tgfβ ligands, extracellular antagonists modulate gdf and gdf signaling. here, we determined if there were differences in the effectiveness of the known antagonists fs fstl gasp and gasp to inhibit gdf or gdf using our hek luciferase assay. fs and gasp similarly inhibited gdf or gdf. however, fstl and gasp more potently inhibited the actions of gdf relative to gdf. curiously, inhibition by fstl for gdf revealed a more negative hill slope compared to gdf suggesting that, while they are both potently inhibited, the binding interactions between the ligands and antagonists may not be identical.fig. inhibition of gdf and gdf by known antagonists. a, b, c, d luciferase reporter assays showing the inhibitory activity following titration of fs, fstl, gasp, and gasp against a constant concentration of gdf or gdf in hek cells. refer to table  for a corresponding analysis of the inhibition curves. data information: in a–d, data are presented as fraction activation. each concentration was performed in triplicate and shown as the mean ± sem of two to three independent experiments. data from independent experiments were combined and fit to non-linear regression with a variable slope. ligand source: gift from acceleron pharma a ci confidence interval b sem standard error of the mean structure of gdf bound to fs288 the complex of the gdf dimer bound to two molecules of fs was resolved using x-ray crystallography to  Å. this is the first structure of gdf bound to a known antagonist. similar to previous ligand:follistatin structures, two molecules of fs bind symmetrically to wrap around the gdf dimer occluding both type ii and type i receptor binding sites. as expected, follistatin domains and d overlap with the type ii binding epitope, whereas the follistatin n-terminal domain occupies the type i binding slot. the overall structure of gdf11:fs is highly similar to that of the gdf8:fs complex  =   Å). nonetheless, the structure of gdf11:fs reveals minor changes in the positioning of residues in the helix of nd. for example, f of fs is pushed inward to accommodate the larger side chain of m in gdf versus v in gdf. this is consistent with other ligand:fs structures, showing that the nd helix is quite plastic in its “molding” to accommodate ligand differences at the type i interface. as observed in previous ligand:fs structures, the nd from one molecule interacts with d from the other follistatin in a head-to-tail fashion. similar to gdf8:fs, the electrostatic surface potential of gdf11:fs depicts a large, continuous electropositive surface on one side of the complex. this is formed by the combination of the heparin binding motif of the two fs molecules with positively charged residues of gdf. as such, the gdf11:fs complex has increased affinity for heparin as compared to fs alone, and slightly stronger affinity than gdf8:fs.fig. the structure of the gdf11:fs complex. a comparison of the gdf11:fs and gdf8:fs 3hh2; ) structures. the inset shows the differences between the interaction of the fsnd helix and the ligand type i receptor epitope. recombinant human gdf was obtained from acceleron pharma for resolution of this crystal structure. b isoelectric surface representation of the gdf11:fs and gdf8:fs structures. surfaces are colored blue and red on a scale of – to kbt/ec using the apbs plugin for pymol. c heparin affinity analysis of fs alone, gdf11:fs and gdf8:fs complexes. gdf11:fs complex has higher affinity for heparin than fs alone indicated by elution at a higher ionic strength. purified proteins and complexes were applied to a heparin column and eluted with a linear sodium chloride gradient. the approximate nacl concentration for protein elution is shown at the peak maxima. ligand source: gift from acceleron pharma       r merge   r pim  mn b  cc1/2 c   r work, r free  clashscoree avalues in parentheses are for highest resolution shell bmn is defined as < merged < ih >/sd  > ≈ signal/noise ccc1/ for highest resolution shell d r free calculated from 5% of initial total number of reflections edetermined by molprobity gdf and gdf structural differences: follistatin-bound versus apo states initial investigation of the sequence alignment between gdf and gdf mature domains did not provide a clear explanation for why gdf is more potent than gdf. in other words, there did not appear to be major amino acid differences between the two ligands that would indicate significant differences in how they would interact with binding partners or receptors. however, when the differences between gdf and gdf were plotted on the structure, we saw that the majority are found near the type i receptor interface. therefore, to determine if there were any unique structural consequences of these amino acid differences, we made a more thorough examination of the x-ray crystal structures of mature gdf and gdf in the follistatin-bound and apo states.fig. sequence alignment and structural comparison of gdf and gdf in their fs288-bound state. a sequence alignment of mature gdf and gdf secondary structure for each ligand in the fs288-bound form is shown above and below the gdf and gdf sequences, respectively, where arrows represent β-sheet and cylinders represent α-helix. cysteines are highlighted in yellow, and residues that are different between gdf and gdf are highlighted in blue. solid black lines joining two cysteines indicate intramolecular disulfide bonds. the dotted black line indicates the cysteine responsible for the intermolecular disulfide bond. b distribution of the amino acid differences between gdf and gdf gdf is shown as ribbon, and non-identical amino acids are shown as orange spheres. symmetrical type i and type ii interfaces are depicted with blue and yellow lines. insets represent zoomed-in views depicting the molecular differences between fs288-bound gdf and fs288-bound gdf in the vicinity of the type i receptor binding interface. the arrow points to the hydrogen bond in the fs288-bound gdf between q and carbonyl oxygen of y superposition of the mature ligands from their respective fs complex structures did not reveal any major structural differences in the type ii interface of the ligand. however, examination of the type i interface revealed two unique interactions in the wrist of gdf compared to gdf. the wrist region is known to be critically important for dictating ligand type i receptor affinity and specificity. first, superposition of the ligands showed that y55a of gdf was shifted by ~  Å inward toward the n-terminal side of the wrist helix on the adjacent monomer. this was facilitated by the lack of a side chain in g100b of gdf. in gdf the corresponding residue is a100b where the methyl side chain sterically occludes y55a. this difference allows y55a to more intimately interact with the opposing chain and facilitates additional hydrophobic interactions with m79b and the aliphatic side chain of k54a. the second unique feature of gdf is the formation of an additional hydrogen bond between the backbone of the pre-helix loop and q62a located on the c-terminal side of the wrist helix. in gdf the corresponding residue is h62a and does not interact with the pre-helix loop in the fs288-bound gdf crystal structure. in fact, h62a does not interact with the pre-helix loop in additional crystal structures of gdf bound to either fstl or a neutralizing antibody. thus, amino acid differences between gdf and gdf impose unique intramolecular contacts in gdf that are not present in gdf given that these changes occur at the type i interface, it is intriguing to speculate that they could contribute to differences in ligand activity. however, we cannot exclude the possibility that these newly identified interactions are a result of binding to fs to explore this possibility, we examined the x-ray crystal structures of apo-gdf and apo-gdf.fig. structural comparison of gdf and gdf in fs288-bound and apo-conformations reveal ligand flexibility. cartoon diagrams of the bound and apo states of gdf and gdf. each monomer is colored independently to highlight the differences in conformation. superposition of each conformation using monomerb is shown on the left. protein data bank ids for bound ligands and apo ligands. resolution of apo-gdf was determined from recombinant human gdf produced and purified as previously described. resolution of fs288-bound and apo-gdf was determined from recombinant human gdf produced and purified by acceleron pharma we resolved the structure of apo-gdf to  Å and the apo-gdf structure to  Å using x-ray crystallography, thus allowing a direct comparison to the recently published apo-gdf structure. unexpectedly, resolution of the apo-gdf structure revealed a unique conformation such that the fingertips of each monomer are positioned proximally in a “closed” conformation compared to the classical distal positioning or “open” conformation observed for many ligands, including structures of fs-bound gdf. while the structure of apo-gdf adopted an unexpected “closed” conformation, a similar conformation has been observed for activin a when in complex with actriib. in both cases, the wrist region is disordered and not resolved in the crystal structure. our new apo-gdf structure, solved under similar crystallization conditions as apo-gdf adopted an exaggerated “open” conformation such that the fingers extend past the horizontal plane. this conformation shares a similar exaggerated “open” conformation to the crystal structure of gdf bound to a neutralizing antibody. however, the wrist region in our apo-gdf structure is also disordered and not resolved in the crystal structure, similar to apo-gdf. the previously published apo-gdf crystal structure adopts the classic “open” conformation with an ordered wrist helix that includes the additional interchain hydrogen bond between q62a and the pre-helix loop observed in the fs288-bound gdf crystal structure. taken together, crystallization of both gdf and gdf in their apo states reveals intrinsic structural flexibility in these ligands. gdf signals more effectively through type i receptors than gdf8 to better understand the potency differences between gdf and gdf we compared their receptor binding affinities and utilization. given that the structural differences between gdf and gdf are positioned at the type i receptor interface, we hypothesized that potency differences are likely through differences in type i interactions. in order to rule out potential differences in type ii receptor binding, we first compared the inhibitory potential of the soluble receptors, actriib extracellular domain and fc-actriib-ecd using our hek luciferase assay. we found no difference in the ic value for actriib-ecd between gdf and gdf, whereas fc-actriib-ecd was ~ threefold more potent in inhibiting gdf than gdf. while the calculated hill slope for the actriib-ecd was similar for gdf and gdf we observed a more negative hill slope for the inhibition of gdf by the fc-actriib-ecd compared to gdf similar to our earlier results showing a more negative hill slope when gdf was antagonized by fs fstl and gasp.fig. enhanced type i receptor utilization by gdf compared to gdf a, b, luciferase reporter assays showing the inhibitory activity following titration of actriib-ecd, and fc-actriib-ecd against a constant concentration of gdf or gdf in hek cells. refer to table  for a corresponding analysis of the inhibition curves. c differences in type i receptor utilization by gdf following transient transfection of a single type i receptor or a combination of type i receptors. rib l cells were co-transfected with  ng of the individual receptor dna alone or various receptor dna combinations and  ng promoter plasmid driving the luciferase gene. purified recombinant ligands were added 12 h post transfection. cells were then lysed and assessed for luciferase activity 8 h post ligand treatment. d treatment of rib l cells with dmso or the type i receptor small molecule inhibitor sb following transfection of empty vector or alk s270t. rib l cells were co-transfected with  ng of the individual receptor dna and  ng promoter plasmid driving the luciferase gene. purified recombinant ligands were added 12 h post transfection. cells were then lysed and assessed for luciferase activity 8 h post ligand treatment. data information: in a, b, data are presented as fraction activation. each concentration was performed in triplicate and shown as the mean ± sem of two independent experiments. data from independent experiments were combined and fit to non-linear regression with a variable slope. in c, d, data are presented as fold activation defined as the total activation from each ligand compared to cells only transfected with the reporter construct. each bar is the mean ± sem. a representative experiment is shown of at least two independent experiments in which each concentration was performed in duplicate or triplicate. only comparisons between gdf and gdf were made. *p ≤  and **p ≤ . ligand sources: gdf and gdf gift from acceleron pharma; activin a, activin b, and tgfβ produced and purified as described in “methods” a ci confidence interval b sem standard error of the mean c ecd extracellular domain next, we determined if gdf and gdf signal through similar type i receptors. numerous studies have shown that gdf and gdf utilize the type i receptors alk and alk. in addition, gdf can signal through alk, although it is unclear if gdf also utilizes this receptor. to our knowledge, a study directly comparing signaling by both ligands via the three receptors has not been reported. therefore, we assessed the relative luciferase reporter response to gdf or gdf following transient expression of individual type i receptors in rib l cells. this cell line lacks alk expression and expresses low levels of alk. the level of alk expression in these cells has not been reported. as expected, activin a robustly signaled in cells transfected with alk, activin b signaled through both alk and alk, and tgfβ specifically signaled through alk. consistent with previous reports, gdf and gdf readily signaled through alk and alk. additionally, we confirmed that gdf signaled through alk. strikingly, gdf induced greater reporter responses than gdf via alk alk or alk. we should note that activin a, activin b, gdf and to a lesser extent gdf stimulated some reporter activity in the absence of exogenous receptors, suggesting low levels of endogenous alk and alk expression by these cells. consistent with this idea, activin b, gdf and gdf induction of reporter activity was completely blocked by addition of the alk4/5/ small molecule inhibitor sb. importantly, signaling could be rescued by transfection of an sb431542-resistant form of human alk, confirming that all three ligands can signal via alk with relative potencies of activin b > gdf11 > gdf collectively, our results indicate that gdf can generate a greater smad3-dependent signal compared to gdf through all three type i receptors. given that type i receptor expression is not ubiquitous in all cells, we next wanted to determine if differences in alk4/5/ combinations would further distinguish or normalize response differences between gdf and gdf different receptor combinations modestly enhanced signaling relative to individual receptors. interestingly, when alk and alk were co-transfected, there was no statistically significant difference in response to gdf and gdf, in contrast to when either receptor was expressed alone. co-transfection of alk and alk resulted in a more robust response to gdf over gdf taken together, these results further suggest that ligand sensitivity can be driven, in part, by the receptor expressed on a particular cell type, thereby providing a possible avenue by which cells may discriminate between highly similar ligands, such as gdf and gdf structural studies describing the interaction between the type i receptors and the activin/inhibin subclass are lacking, predominantly due to the inability to generate recombinant receptors whose folding and biological activity can be confirmed unequivocally. while this barrier remains unresolved for alk and alk we can reliably study the interactions between ligands and the alk5-ecd. we tested binding of gdf and gdf to recombinant alk5-ecd in the absence or presence of actriib-ecd using native-page and surface plasmon resonance. neither mature gdf nor gdf alone were readily resolved using native-page. however, addition of actriib-ecd to either ligand resulted in a mobility shift compared to actriib-ecd alone, indicative of complex formation between the ligand and type ii receptor. mixing the ligands with alk5-ecd alone did not result in a detectable alk5-ecd-ligand complex. however, when we co-incubated either ligand with a constant amount of actriib-ecd followed by titration with alk5-ecd, we observed a noticeable shift in the migration pattern for gdf especially apparent at excess molar ratios of alk5-ecd. under similar conditions, little to no change was observed with gdf. although high concentrations were required, these results suggest that a low-affinity ternary complex composed of gdf11:actriib:alk was more readily formed than the corresponding complex with gdf fig. binding of gdf to the type i receptor alk a basic native-page of gdf and gdf ligand, receptor, and ligand:receptor complexes. to test for ligand-receptor complex formation, receptors were first diluted into 20 mm hepes ph  followed by addition of the ligand. the ligand:receptor mixtures were held at room temperature for 15 min before being loaded and run in the gel. amount loaded for each protein: ligand, alk5-ecd, ligand + alk5-ecd, actriib-ecd  = 3 μg each; ligand  =   μg; ligand + actriib-ecd  =   μg ligand and 3 μg actriib-ecd; for alk5-ecd titration ligand =   μg, actriib-ecd = 3 μg, and or 8 μg alk5-ecd. proteins were visualized by colloidal coomassie stain. b surface plasmon resonance sensorgrams of gdf and gdf binding to fc-alk5-ecd. fc-alk5-ecd was captured by protein a that was amine coupled to a cm chip. serial dilutions of each ligand alone or in the presence of a twofold molar excess of actriib-ecd were allowed to associate for 120 s at a 70 μl/min flow rate followed by a 280 s dissociation at 37 °c. sensorgrams were double referenced using an average of two nm ligand injections. data information: ligand sources: gdf and gdf gift from acceleron pharma; activin a, activin b, and tgfβ produced and purified as described in “methods” to clarify these results, we attempted to detect direct binding of either ligand to alk5-ecd, fc-alk5-ecd, and fc-actriib using spr. in this experiment, the receptor was immobilized on the spr biosensor chip with standard amine coupling or captured using protein a. as expected, both ligands readily bound the fc-actriib-ecd. qualitatively, gdf interacted with both alk5-ecd and fc-alk5-ecd in a dose-dependent manner and exhibited a fast association and rapid dissociation. in contrast, gdf did not bind to alk5-ecd or fc-alk5-ecd in a dose-dependent fashion. given that alk binding to tgfβ is significantly enhanced through a cooperative interaction with tβrii, we speculated that actriib might improve gdf or gdf binding to alk5-ecd. interestingly, co-injection of actriib-ecd with gdf did not appear to alter the association or dissociation with alk5-ecd compared to gdf alone. however, we did observe a detectable improvement in binding of gdf to alk5-ecd in the presence of actriib-ecd. these results suggest that gdf binding to alk is likely more favorable compared to gdf however, it is clear that additional experiments will be necessary to fully resolve the interactions between these ligands and their cognate type i receptors. substitution of gdf11-like residues into gdf enhances ligand activity to determine which residues are important for conferring enhanced activity to gdf we generated a number of gdf8/gdf chimera constructs. site-directed mutagenesis was performed on the full-length human gdf to introduce gdf11-like substitutions. we compared the activity of the chimeras to wild-type gdf by transient co-transfection of the constructs with tolloid-like and furin using hek luciferase cells. we focused our efforts on the regions showing the most divergence between gdf and gdf specifically the type i interface consisting of the pre-helix loop and wrist. interestingly, transient expression of a number of chimeric mutants revealed a significant gain in activity. the increased activity was observed at nearly every concentration tested, with differences between the chimeric mutants and wt gdf more pronounced at lower concentrations. thus, incorporation of gdf11-like residues into gdf can increase ligand potency. consistent with our overall hypothesis, we found that full substitution of all different residues between mature gdf and gdf resulted in a very potent ligand compared to wt gdf particularly at lower concentrations. remarkably, a significant gain in activity was observed in the chimeras that incorporated the gdf residues, q and g, which were shown in the structure to form additional, stabilizing interactions at the ligand dimer interface.fig. generation of gdf8/gdf chimeric ligand confers potency to gdf a luciferase reporter gene assay promoter) following transient co-transfection of hek cells with gdf8/gdf chimera constructs, furin, and tll. all constructs contain human wild-type gdf prodomain followed by human wild-type or mutated mature gdf 18–24 h post transfection, the culture media were replaced with serum-free media and allowed to incubate for an additional 24 h, at which point the cells were lysed and measured for luciferase activity. wt = gdf8; point mutations are indicated by number. for chimera number, each non-identical residue between gdf and gdf was mutated to generate the equivalent wt gdf ligand. a surface representation of gdf dimer is shown. each monomer is colored independently. non-identical residues are shown in orange. b, c luciferase reporter gene assay promoter) following exogenous addition of purified empty vector, wt, or gdf8/gdf chimeric latent protein complexes. the latent complexes were activated by treatment with acid, or the hek cells were transiently transfected with tll prior to protein addition. aa acid activated, not aa no acid activation, not aa + gasp no acid activation but complexes added to cells in the presence of nm gasp aa + gasp acid activation in the presence of nm gasp ev empty vector transfected, ev + gasp empty vector transfected, but complexes added in the presence of nm gasp tll transfected with tll tll2 + gasp cells transfected with tll complexes added in the presence of nm gasp data information: in a, b, c, data are presented as a ratio of the fold activation where each was normalized to the response of empty vector control. each concentration was performed in duplicate or triplicate, and each bar is shown as the mean ± sem from two to three independent experiments. a p ≤ , b p ≤ , c p ≤ , and ns = not significant. ligand sources: produced and purified as described in “methods” to rule out that activity differences were attributed to differences in the relative amounts of protein expression, we produced and purified wt and selected mutant latent complexes. to determine their specific activity relative to wt gdf, we used two methods known to activate the latent complex: acid activation and activation with a member of the tld family, such as tolloid-like. overall, our results from treatment with purified protein were consistent with the results from transient transfection. as expected, little to no luciferase activity was detected in cells treated with the latent complexes unless they were acid activated or applied to cells transfected with tll. in addition, we performed the experiments in the presence of gasp a specific antagonist of gdf and gdf, to confirm that the signal was specific to wt gdf or gdf8/gdf chimeras. both activation methods revealed similar findings with the exception of the acid-activated empty vector sample. the acid-activated ev sample showed a significant response that could not be attenuated with gasp, indicating possible contamination by additional ligands or ph-dependent artifacts. despite this possibility, most of the signal with gdf or gdf8/gdf chimeras was attenuated by gasp suggesting that the majority of the signal was specific to the ligands added. moreover, the higher background signal was not observed in the tll experiment. together, these data support the conclusions that mature gdf is more active than gdf and that gdf activity can be enhanced through substitution of specific residues from gdf gdf is a more potent inducer than gdf of phosphorylated smad2/ in mouse primary skeletal myoblasts in vitro and the mouse myocardium in vivo since we have shown that gdf is more potent than gdf in multiple cell lines, we next sought to determine if similar differences in potency could be seen in primary cells. consistent with a previous report, western analysis of cultured myoblasts treated with mature gdf or gdf at a range of concentrations showed robust, dose-dependent induction of phosphorylated smad2/ compared to untreated cultures. further, gdf treatment elicited a significantly greater induction of psmad2/ compared to equivalent concentrations of gdf.fig. exogenous treatment of gdf potently activates smad2/ in cultured primary skeletal muscle myoblasts. a, b western blot and quantification showing phosphorylated smad2/, total smad2/, and gapdh of cultured primary skeletal muscle myoblasts following treatment with gdf or gdf cells were treated with a range of ligand concentrations for 1 h, lysed, and probed for the indicated proteins. 20 μg total protein loaded. primary skeletal myoblasts were obtained from three different animals. data information: in b, data are presented as a ratio of psmad2/ to total smad2/ to obtain this ratio, psmad2/ or smad2/ was first normalized to gapdh. the ratio of this quotient was then graphed. data are presented as the mean ± sem. *p ≤  and **p ≤ . ligand sources: peprotech cat. no. 120- and cat. no. 120-11 to determine if similar potency differences between gdf and gdf may also occur in vivo, we next examined smad phosphorylation in the mouse myocardium following intravenous injection of various doses of gdf and gdf. psmad levels were significantly increased in the myocardium 1 h after injection of  mg/kg of gdf or gdf. at equivalent doses, gdf stimulated significantly more psmad than gdf. in fact, nearly eightfold more gdf than gdf was required to achieve similar levels of psmad. these data suggest that, depending on dose, intravenous gdf or gdf can result in a significantly different smad2/3-dependent response in the myocardium.fig. intravenous injection of gdf but not gdf elicits robust phosphorylation of smad2/ in the mouse myocardium. a, b, c, d representative western blots showing phosphorylated smad, total smad2/, and gapdh and corresponding quantification in the mouse myocardium 1 h post tail-vein injection of saline, gdf or gdf a, c show a direct comparison in the level of psmad in the myocardium following injection of  mg/kg gdf or gdf. b, d show that nearly eightfold more gdf than gdf is required to achieve a similar psmad response. data information: in c, d, data are presented as a ratio of psmad2/total smad2/ to obtain this ratio, psmad or smad2/ was first normalized to gapdh. the ratio of this quotient was then graphed. data are presented as the mean ± sem. *p ≤  and ***p ≤ . ligand sources: peprotech cat. no. 120- and cat. no. 120-11 discussion previous studies indicate that gdf and gdf have both divergent and overlapping roles in developmental processes and tissue maintenance in adult animals. while the role of gdf during development is well defined, its role in the adult remains poorly understood. however, recent evidence suggests that gdf is an important circulating factor that declines with age and exogenous treatment with gdf can reverse age-related effects including reducing cardiac hypertrophy, improving skeletal muscle regeneration, and promoting neurogenesis. however, other studies have contradicted certain aspects of these claims, fueling significant controversy on the functional similarities and differences between gdf and gdf. controversy notwithstanding, in this study, we investigated the signaling activity and structural features of the mature gdf and gdf ligands. at the outset of our studies, we made the unexpected observation that gdf is a more potent signaling ligand than gdf this observation was consistently and reproducibly observed across multiple laboratories, using different ligand preparations, in multiple cell lines and cultured primary cells, and recapitulated in vivo in the mouse myocardium. the current dogma in the literature suggests that there are no differences in the signaling potencies of gdf and gdf and that the ligands have identical signaling properties. our results are consistent with this idea, but only at the highest ligand concentrations examined. interestingly, prior studies also appeared to show that gdf is more potent or active than gdf, yet many conclusions from these studies maintain the notion that gdf and gdf signal and function identically. the conclusion that gdf and gdf are indistinguishable may derive from the fact that subsequent comparisons were performed at concentrations that were above the ec for both ligands, thus resulting in identical transcriptional outcomes. in fact, we show that at high concentrations of gdf and gdf smad3-dependent readout is indistinguishable under certain conditions. however, our data also reveal that gdf can elicit a significant response under conditions where gdf appears to have little to no activity. these results show that the responses generated by gdf and gdf are highly dependent on the concentrations of ligand and the available receptors, emphasizing that gdf and gdf are not equivalent. to be clear, these data neither support nor refute claims made by other groups regarding the functional outcomes resulting from gdf or gdf signaling. rather, they demonstrate that the biochemical responses elicited by gdf and gdf at equivalent concentrations are significantly different under the conditions we tested. the physiological relevance, if any, which may result from potency differences, requires additional investigation. given the nearly identical sequence of their mature ligands, we were initially puzzled by the potency differences between gdf and gdf especially since most of the changes are relatively conserved. therefore, we turned to x-ray crystallography to determine the structures of gdf in complex with fs in addition to gdf and gdf in their apo forms. while this manuscript was in preparation, an additional apo-gdf structure was resolved, thus providing the opportunity to compare multiple structures of each ligand in their fs288-bound and apo states. as expected, the overall structures of the ligands when bound to fs are highly similar with minor changes in the fsnd helix of fs a region previously shown to be able to accommodate different ligands. comparison between gdf and gdf revealed that differences cluster within and around the type i receptor binding surface. in particular, differences are observed in residues within or bordering the wrist helix, an area important for type i receptor binding. to explore if these differences are the basis for increased activity, we used a chimeric approach that introduced gdf11-specific residues into gdf apart from the pre-helix loop, the two most prominent differences between the structures of gdf and gdf are located at each end of the wrist helix and include gdf residues q and g in both cases, substitution of the individual gdf residues h and a with the gdf residues q and g resulted in increased activity over wild-type gdf moreover, combination of these two mutations resulted in an additional gain in activity over the single mutations alone, suggesting an additive effect. in gdf these residues appear to stabilize the dimer interface where an additional hydrogen bond is formed between q and the backbone of the pre-helix loop. thus, increased dimer stability or stability of the interaction between the wrist and pre-helix loop might be one explanation for the increased ligand activity of gdf though we cannot rule out the possibility that these residues directly interact with the receptors. however, given that activity differences are observed over multiple receptors, it is more likely that potency differences are a result of inherent ligand differences. furthermore, introducing the gdf11-specific residues in the pre-helix loop, a region known to be important for receptor specificity, also results in increased activity. additionally, introduction of gdf fingertip residues, d and q into gdf also significantly increased ligand activity. however, our structural data do not provide a molecular explanation for how these variances confer differences in activity. nevertheless, they do suggest that the potency differences between gdf and gdf are a result of a combination of the residue differences within and around the type i binding interface. resolution of the apo-gdf and apo-gdf crystal structures revealed that both ligands exhibit significant conformational flexibility. interestingly and despite the high sequence identity, nearly identical crystallization conditions resulted in two distinct conformations. although these conformations may be a result of the crystallization process, it supports the idea that gdf and gdf are flexible enough to be trapped in alternate states. however, an additional apo-gdf structure revealed the ligand in the classic “open” conformation and contained similar molecular contacts within the wrist region that we observed in the fs288-bound state. furthermore, under similar crystallization conditions, additional tgfβ superfamily growth factors have been solved in their apo states and adopt the classic “open” conformation. for example, structures of several bmp ligands in the apo state all exhibit the “open” configuration. the extreme “closed” conformation of gdf is not entirely unprecedented, as one structure of activin a in complex with actriib showed a similar “closed” configuration. this was initially somewhat controversial; however, multiple activin a structures have since been determined and support the notion that the activin a dimer is flexible. it should be noted that this is not the first example of gdf captured in an alternate conformation. recently, the co-crystal structure of gdf bound to a neutralizing antibody was solved where the gdf ligand adopts an exaggerated “open” conformation, resembling the apo-gdf structure presented here. furthermore, similar to activin a and gdf ligands of the tgfβ subclass also exhibit significant flexibility. in fact, the biological activity of different tgfβ isoforms, which also share high overall sequence identity, was shown to correlate with the rigidity of the dimer, specifically the wrist helix. however, we cannot discount that the crystallization process has trapped these ligands in alternate conformations. additional solution-based biochemical approaches are needed to better understand ligand dynamics. nevertheless, differences in the wrist region of gdf could contribute to the increased ligand potency over gdf by either stabilizing the dimer interface or presentation of the wrist/pre-helix loop to facilitate differential direct interaction with the type i receptors, or a combination thereof. our data strongly indicate that gdf is a more potent signaling ligand compared to gdf while this observation has not been explicitly realized in the literature, there is evidence suggesting that our results are consistent in that gdf is a more potent ligand than gdf. however, a recent study utilizing a cell line with modified and potentially functionally inactivated type ii and type i receptors showed that gdf and gdf are nearly indistinguishable in terms of their potential for type ii and type i heterodimerization. in this same study and using another assay, there was little difference in the potentiation of downstream smad2/ responsive elements between gdf and gdf. interestingly, we observed that differences in smad2/ responsiveness between gdf and gdf were less pronounced in the hepg cell line, the same cell line utilized in. we observed more robust differences between gdf and gdf in other cell lines and in vivo. therefore, a potential explanation for these differences may be due to the receptor profile of a particular cell type or tissue in addition to other confounding factors such as co-receptors. given that gdf may not utilize the type i receptors as effectively as gdf it is tempting to speculate that both potency and receptor utilization differentiate the biological actions of gdf and gdf  in conclusion, we present evidence supporting the notion that gdf and gdf despite their high sequence identity, are not functionally equivalent. our data show that gdf is a more potent activator of smad2/ in vitro and in vivo. gdf has been considered a somewhat unique ligand of the tgfβ family due to dual utilization of the type i receptors alk and alk our data suggest that gdf possesses these same attributes but, due to differences in amino acid composition, utilizes these receptors more effectively to initiate signal transduction. apart from additional regulatory alterations between gdf and gdf these ligands may have evolved differences in relative potency through selective pressure in order to provide an evolutionary advantage. 
here we show that, despite their high similarity, gdf is a more potent activator of smad2/ and signals more effectively through the type i activin-like receptor kinase receptors alk4/5/ than gdf resolution of the gdf11:fs complex, apo-gdf and apo-gdf crystal structures reveals unique properties of both ligands, specifically in the type i receptor binding site.
 growth/differentiation factor and gdf are two highly similar members of the transforming growth factor β family. while gdf has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of gdf and whether gdf has beneficial effects on age-related dysfunction. to address whether gdf and gdf are functionally identical, we compared their signaling and structural properties.  here we show that, despite their high similarity, gdf is a more potent activator of smad2/ and signals more effectively through the type i activin-like receptor kinase receptors alk4/5/ than gdf resolution of the gdf11:fs complex, apo-gdf and apo-gdf crystal structures reveals unique properties of both ligands, specifically in the type i receptor binding site. lastly, substitution of gdf residues into gdf confers enhanced activity to gdf  these studies identify distinctive structural features of gdf that enhance its potency, relative to gdf8; however, the biological consequences of these differences remain to be determined. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ligandsmyostatinreceptorstructuretransforming growth factor β http://dx.doi.org/ /100000049national institute on agingr56ag048917r01ag048917r01ag047131r01ag040019r03ag049657wagers amy j. lee richard t. paul f. glenncenter grantwagers amy j. http://dx.doi.org/ /100000069national institute of arthritis and musculoskeletal and skin diseasesr41ar06880401martinez-hackert erik http://dx.doi.org/ /100000057national institute of general medical sciencesr01gm58670r01gm114640hinck andrew p. thompson thomas b. http://dx.doi.org/ /100000054national cancer instituter01ca172886hinck andrew p. http://dx.doi.org/ /501100000024canadian institutes of health researchmop-133394bernard daniel j. http://dx.doi.org/ /501100000925national health and medical research council1078907harrison craig a. http://dx.doi.org/ /100005202muscular dystrophy association240087thompson thomas b. issue-copyright-statement© the author 2017 