Region between α-helices 3 and 4 of the Mad homology 2 domain of Smad4: Functional roles in oligomer formation and transcriptional activation

Abstract

Background: Smad4 has a unique region of 35 amino acids between α- helices 3 and 4 (termed H3/4 loop) of the Mad homology (MH) 2 domain. In order to elucidate the functional importance of the H3/4 loop, we prepared chimeric constructs of Smad4 containing the region corresponding to the α- helix 3, H3/4 loop and α-helix 4 of different Smads, including a chimera containing that of Smad2 (Smad4-HL2). Results: Smad4-HL2 constitutively induced the transcriptional activation of p3TP-Lux, a TGF-β-responsive reporter construct. However, co-transfection of Smad2 with Smad4-HL2 did not induce a further increase in the activation of p3TP-Lux. Smad4-HL2 did not induce the activation of pAR3-Lux, which contains FAST1-binding sites and is activated by a complex composed of FAST1, Smad2 and Smad4. Smad4-HL2 formed a homo-oligomer more efficiently than wild-type Smad4 in mammalian cells. Moreover, Smad4-HL2 bound to DNA containing the Smad-binding sites with a greater affinity than the wild-type Smad4. Conclusion: Smad4-HL2 spontaneously forms a homo-oligomer, which may bind to DNA with relatively high affinity and induce transcriptional activation of p3TP-Lux. The H3/4 loop of Smad4 may thus play a role in precluding the spontaneous oligomer formation of Smad4.