Roles of activin/follistatin in neural induction

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The aim of this project is to elucidate the roles of activin and follistatin in neural induction in embryogenesis. In this period, we have obtained the following findings.(1) Identification and characterization of a PDZ protein.We have identified a mouse PDZ protein that interacts with the activin type IIA receptor (ActRIIA), which we named ARIP1 (activin receptor interacting protein 1 ). By using yeast two-hybrid screening, we isolated a cDNA clone of ARIP1 from a mouse brain cDNA library. ARIPI had one guanylate kinase domain (Guk) in the NH2-terminal region, followed by two WW domains and five PDZ domains (PDZ1-5). ARIP1 interacted with ActRIIA through PDZ5. The COOH-terminal residues of ActRIIA (E-S-S-L) agree with a PDZ-binding consensus motif, and ARIP1 recognized the consensus sequence. ARIP1 interacts specifically with ActRIIA among the receptors for TGF-β family. Interestingly, ARIP1 also interacted with Smad3, which is an activin/TGF-β intracellular signaling molecule. The mR … More NA of ARIP1 was more abundant in the brain that in other tissues. Overexpressin of ARIP1 controls activin-induced and Smad3-induced transcription in activin-responsive cell lines. These findings suggest that ARIP1 has a significant role in assembling activin signaling molecules at specific subcellular sites and in regulating signal transduction in neuronal cells.(2) Characterization of a novel follistatin-like protein as an activin-binding protein.We have identified a novel follistatin-like protein in humans and mice, which was found to differ from known follistatin gene products. Follistatin has three follistatin domains that are presumed to be growth factor binding motifs. Both the human and mouse follistatin-like proteins possessed only two follistatin domains. Northen blotting revealed that mRNAs for the novel follistatin-like proteins were abundantly expressed in placenta, adrenal gland, lung, heart, pituitary, testis and ovary, tissues where activins are also abundantly expressed. The recombinant proteins were found to have an activin-bindig activity as revealed by coimmunoprecipitation analysis. Furthermore, the recombinant protein inhibited activin-induced transcriptional responses in activin-responsive cells I n a dose-dependent manner. Ligand blotting using ィイD1125ィエD1I-activin and GST-pull down analysis revealed that the second follistatin domain in the C-terminal region on the molecule was responsible for the activin-binding activity. In summary, we have identified a novel follistatin possessing activin-binding activity. Our finding implies that cellular signaling of activin is tightly regulated by multiple members of the follistatin family. Less