Signaling by NODAL is essential for patterning of the axes of the embryo and formation of mesoderm and endoderm (reviewed in Schier 2009, Shen 2007). The NODAL proprotein is secreted and cleaved extracellularly to yield mature NODAL. Mature NODAL homodimerizes and can also form heterodimers with LEFTY1, LEFTY2, or CERBERUS, which negatively regulate NODAL signaling. NODAL also forms heterodimers with GDF1, which increases NODAL activity. NODAL dimers bind the NODAL receptor comprising a type I Activin receptor (ACVR1B or ACVR1C), a type II Activin receptor (ACVR2A or ACVR2B), and an EGF-CFC coreceptor (CRIPTO or CRYPTIC). After binding NODAL, the type II activin receptor phosphorylates the type I activin receptor which then phosphorylates SMAD2 and SMAD3 (R-SMADs). Phosphorylated SMAD2 and SMAD3 form hetero-oligomeric complexes with SMAD4 (CO-SMAD) and transit from the cytosol to the nucleus. Within the nucleus the SMAD complexes interact with transcription factors such as FOXH1 to activate transcription of target genes.
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Either FURIN or PACE4 endoproteases cleave the 321 amino acid NODAL proprotein to yield the 110 amino acid NODAL mature protein. In cultured mouse cells the CRIPTO coreceptor at the plasma membrane recruits both NODAL proprotein and FURIN or PACE4 endoprotease.
NODAL binds a receptor comprising a type I activin receptor (ACVR1B or ACVR1C), a type II activin receptor (ACVR2 or ACVR2B), and a EGF-CFC coreceptor (CRIPTO or CRYPTIC). Though NODAL is able to signal via the ACVR1C (ALK7) receptor (Reissman et al. 2001), experiments in mouse indicate NODAL signaling via ALK7 is dispensable during embryogenesis (Jornvall et al. 2004).
As inferred from the response of the activin receptor to activin, the type II component of the NODAL receptor phosphorylates the type I component in response to NODAL binding. Experiments with human proteins in frog oocytes show NODAL can signal via the CRIPTO:ACVR1B(ALK4):ACVR2 complex (Yeo and Whitman 2001).
LEFTY1 and LEFTY2 are able to inhibit NODAL signaling by binding the EGF-CFC coreceptor (CRIPTO or CRYPTIC) and thereby preventing the coreceptor from interacting with other components of the NODAL receptor.
As inferred from mouse (Chen and Shen 2004) both LEFTY1 and LEFTY2 can bind NODAL and inhibit NODAL signaling. The stoichiometry of the resulting LEFTY:NODAL complex is unknown.
As inferred from Xenopus (Piccolo 1999) and as observed in human cells (Aykul et al. 2015) CERBERUS binds NODAL and inhibits NODAL signaling. CERBERUS and NODAL are believed to bind as dimer to form a tetrameric complex (Aykul et al. 2015)
NODAL receptors signal by phosphorylating SMAD2 and SMAD3 (Bondestam et al. 2001, Kumar et al. 2001, DaCosta Byfield et al. 2004). As in TGF-beta signaling, Smad anchor for receptor activation (SARA) may bind and present SMAD2 and SMAD3 for phosphorylation but this has not yet been demonstrated in NODAL signaling.
As inferred from the response of the activin receptor to activin, the type II component of the NODAL receptor phosphorylates the type I component in response to NODAL binding. As inferred from mouse and frog (Xenopus) NODAL can signal via the ACVR1C (ALK7) type I activin receptor (Reissman et al. 2001) though this may be dispensable for development in mouse (Jornvall et al. 2004).
SMAD2 and SMAD3 do not bind DNA efficiently. They must interact with DNA-binding proteins to activate transcription. FOXH1 interacts with phospho-SMAD2 and phospho-SMAD3 complexed with CO-SMAD (SMAD4) at promoters containing the Activin Response Element (Zhou et al. 1998, Yanagisawa et al. 2000, inferred from Xenopus in Chen et al. 1996, Chen et al. 1997, Yeo et al. 1999). Follicle-stimulating hormone beta subunit (FSHB) and the Lim1 homeobox gene (LXH1) are examples of genes regulated by Activin.
FOXO3 (FOXO3A) interacts with phospho-SMAD2 and phospho-SMAD3 complexed with CO-SMAD (SMAD4) at a promoter containing the FoxO3a-binding Element (Fu and Peng 20110).
The phosphorylated C-terminal tail of R-SMAD induces a conformational change in the MH2 domain (Qin et al. 2001, Chacko et al. 2004), which now acquires high affinity towards Co-SMAD i.e. SMAD4 (common mediator of signal transduction in TGF-beta/BMP signaling). The R-SMAD:Co-SMAD complex (Nakao et al. 1997) most likely is a trimer of two R-SMADs with one Co-SMAD (Kawabata et al. 1998). It is important to note that the Co-SMAD itself cannot be phosphorylated as it lacks the C-terminal serine motif.
ZFYVE16 (endofin) promotes SMAD heterotrimer formation. ZFYVE16 can bind TGFBR1 and facilitate SMAD2 phosphorylation, and it can also bind SMAD4, but the exact mechanism of ZFYVE16 (endofin) action in the context of TGF-beta receptor signaling is not known (Chen et al. 2007).
The phosphorylated R-SMAD:CO-SMAD complex rapidly translocates to the nucleus (Xu et al. 2000, Kurisaki et al. 2001, Xiao et al. 2003) where it binds directly to DNA and interacts with a plethora of transcription co-factors. Regulation of target gene expression can be either positive or negative. A classic example of a target gene of the pathway are the genes encoding for I-SMADs. Thus, TGF-beta/SMAD signaling induces the expression of the negative regulators of the pathway (negative feedback loop).
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ZFYVE16 (endofin) promotes SMAD heterotrimer formation. ZFYVE16 can bind TGFBR1 and facilitate SMAD2 phosphorylation, and it can also bind SMAD4, but the exact mechanism of ZFYVE16 (endofin) action in the context of TGF-beta receptor signaling is not known (Chen et al. 2007).