Structural basis for potency differences between GDF8 and GDF11.

BACKGROUND: Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor β (TGFβ) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties. RESULTS: Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8. CONCLUSIONS: These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined

Murine FSH Production Depends on the Activin Type II Receptors ACVR2A and ACVR2B.

Activins are selective regulators of FSH production by pituitary gonadotrope cells. In a gonadotrope-like cell line, LβT2, activins stimulate FSH via the activin type IIA receptor (ACVR2A) and/or bone morphogenetic protein type II receptor (BMPR2). Consistent with these observations, FSH is greatly reduced, though still present, in global Acvr2a knockout mice. In contrast, FSH production is unaltered in gonadotrope-specific Bmpr2 knockout mice. In light of these results, we questioned whether an additional type II receptor might mediate the actions of activins or related TGF-β ligands in gonadotropes. We focused on the activin type IIB receptor (ACVR2B), even though it does not mediate activin actions in LβT2 cells. Using a Cre-lox strategy, we ablated Acvr2a and/or Acvr2b in murine gonadotropes. The resulting conditional knockout (cKO) animals were compared with littermate controls. Acvr2a cKO (cKO-A) females were subfertile (~70% reduced litter size), cKO-A males were hypogonadal, and both sexes showed marked decreases in serum FSH levels compared with controls. Acvr2b cKO (cKO-B) females were subfertile (~20% reduced litter size), cKO-B males had a moderate decrease in testicular weight, but only males showed a significant decrease in serum FSH levels relative to controls. Simultaneous deletion of both Acvr2a and Acvr2b in gonadotropes led to profound hypogonadism and FSH deficiency in both sexes; females were acyclic and sterile. Collectively, these data demonstrate that ACVR2A and ACVR2B are the critical type II receptors through which activins or related TGF-β ligands induce FSH production in mice in vivo

Additional file 1: Table S1. of Structural basis for potency differences between GDF8 and GDF11

Analysis of GDF8 and GDF11 activity in HEK293, HepG2 and LβT2 cells [57, 94–96]. (DOC 30 kb

Additional file 2: Figure S1. of Structural basis for potency differences between GDF8 and GDF11

Potency of recombinant GDF8 and GDF11 from different sources. Luciferase reporter gene assay ((CAGA)12 promoter) following titration of GDF8 (blue) and GDF11 (orange) ligands in HEK293 cells. Luciferase activity was assessed 18–24 h post ligand treatment. The calculated EC50 value for each ligand source using non-linear regression with variable slope is shown in the table below the graph. Data information: Data are presented as percent GDF11 activation after background subtraction (0 nM ligand concentration). Each point is the mean ± SEM of three to four independent experiments. Ligand sources are indicated in the graph. (TIF 750 kb

Additional file 4: Figure S3. of Structural basis for potency differences between GDF8 and GDF11

Sequence alignment of human BMP2, GDF8, and GDF11. Gray bars above and below the sequence depict gross topology of the ligands. Residues that interact with the type I receptor (blue) and type II receptor (yellow) are shown on BMP2 based on the BMP2:ALK3:ActRIIA co-crystal structure (Protein Data Bank (PDB): 2GOO; [97]). The non-identical residues between GDF8 and GDF11 are highlighted in gray. (TIF 566 kb

Additional file 2: Figure S1. of Structural basis for potency differences between GDF8 and GDF11

Potency of recombinant GDF8 and GDF11 from different sources. Luciferase reporter gene assay ((CAGA)12 promoter) following titration of GDF8 (blue) and GDF11 (orange) ligands in HEK293 cells. Luciferase activity was assessed 18–24 h post ligand treatment. The calculated EC50 value for each ligand source using non-linear regression with variable slope is shown in the table below the graph. Data information: Data are presented as percent GDF11 activation after background subtraction (0 nM ligand concentration). Each point is the mean ± SEM of three to four independent experiments. Ligand sources are indicated in the graph. (TIF 750 kb

Additional file 6: Figure S5. of Structural basis for potency differences between GDF8 and GDF11

Purification and quantification of GDF8/GDF11 chimeric ligands. A, B Representation of purified protein from selected GDF8/GDF11 chimeras under non-reduced (A) and reduced (B) conditions (4–15% gradient gel). Chimeras or empty vector control were produced transiently using HEK293T cells and purified using size exclusion chromatography. The resultant peak containing the prodomain:mature ligand complex was pooled and concentrated. For empty vector control, corresponding fractions from a similar retention volume were pooled. The lane labeled “pro domain + mature” serves as a control for the molecular weight of purified wt GDF8 prodomain and wt GDF8 mature ligand. Note the expected changes in mass of the mature ligand (blue arrows) under non-reducing (dimer) and reducing (monomer) conditions while the prodomain mass is relatively unaffected (gray arrow). Protein is visualized by colloidal Coomassie stain. To ensure that comparable amounts of each GDF8/GDF11 chimeric protein were being administered in the cell-based assays, we first normalized protein concentrations based on the amount of dimer present in a non-reduced SDS-PAGE gel stained with colloidal Coomassie. The samples were then normalized and reexamined by SDS-PAGE gel under non-reducing and reducing gel. The subsequent bands were quantified (bottom, below gel) under non-reduced (A) and reduced (B) gels using ImageJ showing that the protein concentrations were indeed normalized. 500 ng of recombinant GDF8 prodomain and purified GDF8 mature were loaded for reference. (TIF 3694 kb

Additional file 5: Figure S4. of Structural basis for potency differences between GDF8 and GDF11

Binding of GDF11 to the type I receptor ALK5. A, B, C Steady state analysis for SPR traces shown in Fig. 7b and calculated values. The maximum response at each concentration is plotted to a steady state binding equation using Biacore T200 Evaluation Software version 1.0 (Biacore). Sensorgrams were double referenced using an average of two 0 nM ligand injections. Ligand sources: GDF8 and GDF11, gift from Acceleron Pharma; Activin A, Activin B, and TGFβ3, produced and purified as described in “Methods.” D, E, F Ligand binding to Fc-ActRIIB-ECD (A), Fc-ALK5-ECD (B), and ALK5-ECD (C) amine coupled to a CM5 biosensor chip. Ligands were at 500 nM. TβRII, the type II receptor, was required for TGFβ3 binding to Fc-ALK5-ECD and ALK5-ECD. The receptor concentration was at 1 μM for this experiment. Experiments were performed using 40 μL/min flow rate at 37 °C. (TIF 1166 kb