The orphan ligand, activin C, signals through activin receptor-like kinase 7
- Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, United States
- Department of Pharmacology and Therapeutics, Centre for Research in Reproduction and Development, McGill University, Canada
- Merck &Co., Inc., United States
Figures
Figure 1 with 1 supplement
Differences in type I receptor utilization by ActA, ActAC, and ActC.
(A) Schematic displaying formation of activin A (ActA), activin AC (ActAC), and activin C (ActC) from dimerization of inhibinβA (blue) and inhibinβC subunits (green). (B) Generalized TGFβ signaling …
Figure 1—figure supplement 1
Luciferase reporter assay displaying activin C (ActC) titration against constant activin A (ActA) (0.62 nM) in untransfected (CAGA)12-luciferase (luc).
Each data point represents the mean ± SD of triplicate experiments measuring relative luminescence units (RLU).
Figure 2 with 2 supplements
ActC binds activin type II receptors with low affinity.
Representative surface plasmon resonance (SPR) sensorgrams of activin A (ActA), activin AC (ActAC), and activin C (ActC) binding to protein A captured ActRIIA-Fc, ActRIIB-Fc, ActRIIB-ALK7-Fc, or …
Figure 2—figure supplement 1
Additional surface plasmon resonance (SPR) sensorgrams of ActA, ActAC, and ActC binding to Alk7 and ActB binding to different receptors.
Figure 2—figure supplement 2
Native gel analysis of type II receptors and ActA, ActAC, and ActC.
Native PAGE analysis of ActRIIA (A) and ActRIIB (B) with activin A (ActA) and activin AC (ActAC). Binary complexes were formed by titrating receptor from 1:1 to 3:1 molar ratio against constant …
Figure 3
ActA and ActAC signal via activin type II receptors.
(A) Schematic representation of activin type II receptor Fc-fusion proteins as decoys. (B and C) HEK293T (CAGA)12-luciferase (luc) cells were transfected with ALK4st and ALK7st and treated with …
Figure 4
Activin C is resistant to inhibition by follistatin but not inhibin A.
(A) HEK293T (CAGA)12-luciferase (luc) cells were transfected with ALK4st and ALK7st and treated with SB-431542 and activin A (ActA), activin AC (ActAC), activin C (ActC), or activin B (ActB) (0.62 …
Figure 5
Type II interface of ActC is distinct from other activins.
(A) Sequence alignment across the activin class displays critical differences at the canonical type II receptor binding site. IAP motif is boxed in black. (B) Structure and schematic representation …
Figure 6 with 1 supplement
ActC activates SMAD2 through ALK7 in differentiated adipocytes.
(A) Representative images of isolated adipose-derived stromal vascular fraction (SVF) or cultured 3T3-L1 cells prior to differentiation (left, Day-0) and following differentiation (right, Day-10). …
Figure 6—figure supplement 1
Supplemental adipocyte-pSMAD2/SMAD2/3 western blots.
Supplemental westerns for representative blots shown in Figure 6. Boxes are drawn to display which data were used for figure generation. Antibodies used: pSMAD2 (cell signaling, 138D4) and SMAD2/3 …
Figure 7 with 3 supplements
Difference between ActA and ActC in type I receptor specificity, type II receptor affinity, and follistatin antagonism.
Gradients of follistatin antagonism (red), activin receptor-like kinase 4 (ALK4)-dependent signaling (blue), activin receptor-like kinase 7 (ALK7)-dependent signaling (green), and activin type II …
Figure 7—figure supplement 1
Sequence alignment of activin and TGFβ ligands.
Sequence comparison of the mature ligand for human activin subclass (activin A [ActA], activin B [ActB], activin C [ActC], activin E [ActE], GDF8 and GDF11), TGFβ1, TGFβ2, and TGFβ3. Numbering …
Figure 7—figure supplement 2
Modeling ActC/ALK7 interactions.
Comparison of the fingertip interface between activin A (ActA)/activin receptor-like kinase 4 (ALK4) (PDB:7OLY) and models of ActA/activin receptor-like kinase 7(ALK7), activin C (ActC)/ALK4, and …
Figure 7—figure supplement 3
Phylogenetic history of ActC.
Phylogenetic analysis of full-length activin C (ActC) across a large variety of species with focus on the type II receptor interface variance. Species with the IQP variant shown in black, with the …
Additional files
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Supplementary file 1
Linear regression analysis.
The activity data corresponding to Figures 1B, D, 4C, 5E were imported into GraphPad prism and fit using a non-linear regression to calculate the EC50 or IC50 with standard error was calculated from triplicate experiments.
- https://cdn.elifesciences.org/articles/78197/elife-78197-supp1-v1.docx
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Supplementary file 2
SPR Kinetic Analysis.
- https://cdn.elifesciences.org/articles/78197/elife-78197-supp2-v1.docx
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Supplementary file 3
qPCR primer sequences.
- https://cdn.elifesciences.org/articles/78197/elife-78197-supp3-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/78197/elife-78197-transrepform1-v1.docx
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Source data 1
Luciferase, SPR and raw image data for all figures.
- https://cdn.elifesciences.org/articles/78197/elife-78197-data1-v1.zip
