title: Biochemical and Cellular Analysis Reveals Ligand Binding Specificities, a Molecular Basis for Ligand Recognition, and Membrane Association-dependent Activities of Cripto-1 and Cryptic.
abstract: Transforming growth factor β (TGF-β) pathways are key determinants of cell fate in animals. Their basic mechanism of action is simple. However, to produce cell-specific responses, TGF-β pathways are heavily regulated by secondary factors, such as membrane-associated EGF-CFC family proteins. Cellular activities of EGF-CFC proteins have been described, but their molecular functions, including how the mammalian homologs Cripto-1 and Cryptic recognize and regulate TGF-β family ligands, are less clear. Here we use purified human Cripto-1 and mouse Cryptic produced in mammalian cells to show that these two EGF-CFC homologs have distinct, highly specific ligand binding activities. Cripto-1 interacts with BMP-4 in addition to its known partner Nodal, whereas Cryptic interacts only with Activin B. These interactions depend on the integrity of the protein, as truncated or deglycosylated Cripto-1 lacked BMP-4 binding activity. Significantly, Cripto-1 and Cryptic blocked binding of their cognate ligands to type I and type II TGF-β receptors, indicating that Cripto-1 and Cryptic contact ligands at their receptor interaction surfaces and, thus, that they could inhibit their ligands. Indeed, soluble Cripto-1 and Cryptic inhibited ligand signaling in various cell-based assays, including SMAD-mediated luciferase reporter gene expression, and differentiation of a multipotent stem cell line. But in agreement with previous work, the membrane bound form of Cripto-1 potentiated signaling, revealing a critical role of membrane association for its established cellular activity. Thus, our studies provide new insights into the mechanism of ligand recognition by this enigmatic family of membrane-anchored TGF-β family signaling regulators and link membrane association with their signal potentiating activities.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Senem
authors.initials: S
authors.lastname: Aykul
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Anthony
authors.initials: A
authors.lastname: Parenti
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Kit Yee
authors.initials: KY
authors.lastname: Chu
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Jake
authors.initials: J
authors.lastname: Reske
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Monique
authors.initials: M
authors.lastname: Floer
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319.
authors.forename: Amy
authors.initials: A
authors.lastname: Ralston
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319 emh@msu.edu.
authors.forename: Erik
authors.initials: E
authors.lastname: Martinez-Hackert
mesh_terms: D000595:Amino Acid Sequence; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism*; D058851:GPI-Linked Proteins / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D008024:Ligands; D009363:Neoplasm Proteins / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000378:metabolism*
chemical_list: C491268:CFC1 protein, human; D058851:GPI-Linked Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D009363:Neoplasm Proteins; D018125:Receptors, Transforming Growth Factor beta; C070636:TDGF1 protein, human; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II
keywords: activin A receptor, type I (ACVR1); bone morphogenetic protein (BMP); protein-protein interaction; signal transduction; surface plasmon resonance (SPR); transforming growth factor β (TGF-β)