HETEROMERIC AND HOMOMERIC INTERACTIONS CORRELATE WITH SIGNALING ACTIVITY AND FUNCTIONAL COOPERATIVITY OF SMAD3 AND SMAD4 DPC4/

Homologs of Drosophila Mad function as downstream mediators of the rec eptors for transforming growth factor beta (TGF-beta)-related factors. Two homologs, the receptor-associated Smad3 and the tumor suppressor Smad4/DPC4, synergize to induce ligand-independent TGF-beta activities and are essential mediators of the natural TGF-beta response, We now show that Smad3 and Smad4 associate in homomeric and heteromeric inter actions, as assessed by yeast two-hybrid and coimmunoprecipitation ana lyses. Heteromeric interactions are mediated through the conserved C-t erminal domains of Smad3 and Smad4. In Smad3, the homomeric interactio n is mediated by the same domain, In contrast, the homomeric associati on of Smad4 requires both the N-terminal domain and the C-terminal dom ain, which by itself does not homomerize. Mutations that have been ass ociated with impaired Mad activity in Drosophila or decreased tumor su ppressor activity of Smad4/DPC4 in pancreas cancer, including a short C-terminal truncation and two point mutations in the conserved C-termi nal domains, impair the ability of Smad3 and Smad4 to undergo homo- an d heteromeric associations, Analyses of the biological activity of Sma d3 and Smad3 and their mutants show that full signaling activity corre lates with their ability to undergo efficient homo- and heteromeric in teractions. Mutations that interfere with these interactions result in decreased signaling activity. Finally, we evaluated the ability of Sm ad3 or Smad4 to induce transcriptional activation in yeast, These resu lts correlate the ability of individual Smads to homomerize with trans criptional activation and additionally with their biological activity in mammalian cells.