Structure-function analysis of the EGF-CFC family member Cripto identifies residues essential for nodal signalling.
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 (MZoep) 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 MZoep 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.
Minchiotti
G
G
International Institute of Genetics and Biophysics, CNR, Via G. Marconi 12, 80125 Naples, Italy. minchiot@iigb.na.cnr.it
Manco
G
G
Parisi
S
S
Lago
C T
CT
Rosa
F
F
Persico
M G
MG
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D003545:Cysteine; D004627:Embryonic Induction; D004815:Epidermal Growth Factor; D005816:Genetic Complementation Test; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*
D018398:Homeodomain Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D029961:Zebrafish Proteins; C110484:tdgf1 protein, zebrafish; D004815:Epidermal Growth Factor; D003545:Cysteine