title: An activin mutant with disrupted ALK4 binding blocks signaling via type II receptors.
abstract: Activins control many physiologic and pathophysiologic processes in multiple tissues and, like other TGF-beta superfamily members, signal via type II (ActRII/IIB) and type I (ALK4) receptor serine kinases. ActRII/IIB are promiscuous receptors known to bind at least a dozen TGF-beta superfamily ligands including activins, myostatin, several BMPs, and nodal. Here we utilize a new screening procedure to rapidly identify activin-A mutants with loss of signaling activity. Our goal was to identify activin-A mutants able to bind ActRII but unable to bind ALK4 and which would be, therefore, candidate type II activin receptor antagonists. Using the structure of BMP-2 bound to its type I receptor (ALK3) as a guide, we introduced mutations in the context of the inhibin betaA cDNA and assessed the signaling activity of the resulting mutant proteins. We identified several mutants in the finger (M91E, I105E, M108A) and wrist (activin A/activin C chimera, S60P, I63P) regions of activin-A with reduced signaling activity. Of these the M108A mutant displayed the lowest signaling activity while retaining wild-type-like affinity for ActRII. Unlike wild-type activin-A, the M108A mutant was unable to form a cross-linked complex with ALK4 in the presence of ActRII indicating that its ability to bind ALK4 was disrupted. This data suggested that the M108A mutant might be capable of modulating signaling of activin and related ligands. Indeed, the M108A mutant antagonized activin-A and myostatin, but not TGF-beta, signaling in 293T cells, indicating it may be generally capable of blocking ligands that signal via ActRII/IIB.
authors.affiliation: Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Harrison
authors.forename: Peter C
authors.initials: PC
authors.lastname: Gray
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Fischer
authors.forename: Cindy
authors.initials: C
authors.lastname: Donaldson
authors.forename: Senyon
authors.initials: S
authors.lastname: Choe
authors.forename: Wylie
authors.initials: W
authors.lastname: Vale
mesh_terms: D030301:Activin Receptors, Type II / Q000378:metabolism*; D028341:Activins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D055435:Myostatin; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D000939:Epitopes; D008024:Ligands; C525911:MSTN protein, human; D055435:Myostatin; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D028341:Activins; D005640:Follicle Stimulating Hormone; D030301:Activin Receptors, Type II