Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-β (TGF-β) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-β and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-β signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-β and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-β signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-β type I receptor (TβR-I) more efficiently, and were more potent in repressing TGF-β signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-β receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-β signaling.

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Bone morphogenetic proteins (BMPs) are pleiotropic growth and differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily. Signals of the TGF-β-like ligands are propagated to the nucleus through specific interaction of transmembrane serine/threonine kinase receptors and Smad proteins. GCCGnCGC has been suggested as a consensus binding sequence for DrosophilaMad regulated by a BMP-like ligand, Decapentaplegic. Smad1 is one of the mammalian Smads activated by BMPs. Here we show that Smad1 binds to this motif upon BMP stimulation in the presence of the common Smad, Smad4. The binding affinity is likely to be relatively low, because Smad1 binds to three copies of the motif weakly, but more repeats of the motif significantly enhance the binding. Heterologous reporter genes (GCCG-Lux) with multiple repeats of the motif respond to BMP stimulation but not to TGF-β or activin. Mutational analyses reveal several bases critical for the responsiveness. A natural BMP-responsive reporter, pTlx-Lux, is activated by BMP receptors in P19 cells but not in mink lung cells. In contrast, GCCG-Lux responds to BMP stimulation in both cells, suggesting that it is a universal reporter that directly detects Smad phosphorylation by BMP receptors.

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Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-beta superfamily. Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.