Structure-function analysis of the EGF-CFC family member Cripto identifies residues essential for nodal signalling

• Published in: Development (Cambridge) Vol. 128; no. 22; pp. 4501 - 4510
• Main Authors: Minchiotti, G, Manco, G, Parisi, S, Lago, C T, Rosa, F, Persico, M G
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Limited 15.11.2001
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Computer Simulation; cripto gene; Cripto protein; Cysteine; Danio rerio; Embryonic Induction; Epidermal Growth Factor; Genetic Complementation Test; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Neoplasm Proteins - administration & dosage; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; oep gene; one-eyed pinhead gene; Point Mutation; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins - administration & dosage; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Transcription Factors - genetics; Transcription Factors - metabolism; Zebrafish - embryology; Zebrafish Proteins - administration & dosage; Zebrafish Proteins - genetics; Zebrafish Proteins - metabolism
• ISSN: 0950-1991; 1477-9129
• DOI: 10.1242/dev.128.22.4501

cripto is the founding member of the family of EGF-CFC genes, a class of extracellular factors essential for early vertebrate development. In this study we show that injection of Cripto recombinant protein in mid to late zebrafish Maternal-Zygotic one-eyed pinhead (MZ oep ) blastulae was able to fully rescue the mutant phenotype, thus providing the first direct evidence that Cripto activity can be added extracellularly to recover oep -encoded function in zebrafish early embryos. Moreover, 15 point mutations and two deletion mutants were generated to assess in vivo their functional relevance by comparing the ability of cripto wild-type and mutant RNAs to rescue the zebrafish MZ oep mutant. From this study we concluded that the EGF-CFC domain is sufficient for Cripto biological activity and identified ten point mutations with a functional defective phenotype, two of which, located in the EGF-like domain, correspond to loss-of-function mutations. Finally, we have developed a three-dimensional structural model of Cripto protein and used it as a guide to predict amino acid residues potentially implicated in protein-protein interaction.