alpha-Helix 2 in the amino-terminal mad homology 1 domain is responsible for specific DNA binding of Smad3

Smads, signal transducers of the transforming growth factor-beta (TGF-beta) superfamily proteins, directly bind to DNA and regulate transcription of t arget genes. Smad3 binds to CAGA box, whereas Smad1 and Smad5 preferentiall y bind to GC-rich sequences. The beta -hairpin loop in the amino-terminal M ad homology 1 (MH1) domain is the direct DNA-binding site of Smad3; however , the amino acid sequences of the beta -hairpin loop of Smad3 and Smad1/5 a re identical, suggesting that other regions may be responsible for the diff erential DNA binding of Smad3 and Smad1/5. To identify regions other than t he beta -hairpin loop responsible for specific DNA binding of Smad3, we gen erated chimeras containing various regions of Smad3 and Smad1. Luciferase a ssays using a TGF-beta -responsive reporter (CAGA)(9)-MLP-Luc and gel-mobil ity shift assays using 3xCAGA as a probe revealed that alpha -helix 2 (H2) in the amino-terminal part of the MH1 domain plays an important role in spe cific DNA binding and transcriptional activation of Smad3. Luciferase assay s using natural TGF-beta -responsive reporters also revealed the functional importance of H2 in the Smad3 AMI domain in direct DNA binding. Smad3 thus binds to DNA directly through the beta -hairpin loop, and H2 supports spec ific DNA binding of Smad3.