Abstract
The inhibitory Smads (I-Smads), i.e. Smad6 and Smad7, are negative regulators of transforming growth factor-β (TGF-β) family signaling. I-Smads inhibit TGF-β family signaling principally through physical interaction with type I receptors (activin receptor-like kinases), so as to compete with receptor-regulated Smads (R-Smads) for activation. However, how I-Smads interact with type I receptors is not well understood. In the present study, we found that Smad7 has two modes of interaction with type I receptors. One is through a three-finger-like structure in the MH2 domain, consisting of residues 331-361, 379-387, and the L3 loop. The other is through a basic groove in the MH2 domain (Mochizuki, T., Miyazaki, H., Hara, T., Furuya, T., Imamura, T., Watabe, T., and Miyazono, K. (2004) J. Biol. Chem. 279, 31568-31574). We also found that Smad6 principally utilizes a basic groove in the MH2 domain for interaction with type I receptors. Smad7 thus has an additional mode of interaction with TGF-β family type I receptors not possessed by Smad6, which may play roles in mediating the inhibitory effects unique to Smad7.
Highlights
MH2 Domains Are Responsible for the Differential Inhibitory Effects of inhibitory Smads (I-Smads) on Signaling from ALK-2—Smad7 efficiently inhibits bone morphogenetic proteins (BMPs) signaling from both ALK-2 and ALK-3
Smad7C inhibited ALK-2 signaling as effectively as wild-type Smad7, whereas Smad6C had less inhibitory effect on ALK-2 signaling, similar to the full-length Smad6. Consistent with these results, Smad7C interacted with ALK-2 more stably than Smad6C, whereas the N domains of both I-Smads failed to interact with ALK-2 and ALK-3
Replacement of the L3 loop of S7-S4L3s by that of Smad7 resulted in recovery of inhibition (Fig. 2D) as well as interaction with ALK-2 and ALK-3. These findings indicate that the L3 loops of I-Smads are required for their inhibitory effects on ALK-2 and ALK-3 through physical interaction
Summary
We constructed mutants of Smad6 and 7 in which the regions spanning the L3 loop to the C terminus had been swapped (Fig. 2, A and B, left) and determined their inhibitory effects on ALK-2 and -3 signaling. The L3 loops of I-Smads are required for but not determinative of the differential effects of Smad6 and Smad7 on ALK-2 signaling, suggesting that other region(s) in I-Smads play crucial roles in determining the specificity of interaction with BMP type I receptors.
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