Characterizing the Role of the N-terminal MH1 Domain of SMAD6 in BMP Signaling

Endothelial cells (EC), the cells that line blood vessels, can be activated by proangiogenic cues and rearrange their cell-cell junctions to allow for sprouting angiogenesis. Bone morphogenetic protein (BMP) signaling plays a fundamental role in regulating angiogenesis and is pro-angiogenic through ligands BMP2 and BMP6. The BMP pathway is negatively regulated by SMAD6, an intracellular inhibitory SMAD. SMAD6 is anti-angiogenic and modulates EC adherens junction turnover both in vivo and in vitro. SMAD6 is comprised of two MAD homology domains, MH1 and MH2, which correlate to the N-terminal and C-terminal domains of the protein, respectively. The MH2 domain of SMAD6 is responsible for protein interactions important for its function, but the role of the MH1 domain is not understood. Here, I explore the function of the poorly characterized MH1 domain of SMAD6 in vitro by analyzing overexpression effects on BMP signaling outputs. I found that the MH1 domain is necessary for proper SMAD6 cellular localization, as its deletion led to SMAD6 mis-localization in the trans- Golgi network. In the presence of BMP6 ligand, overexpression of either the MH1 or MH2 domain alone did not inhibit BMP signaling nor maintain stable endothelial cell junctions to the degree of full-length SMAD6 overexpression. Collectively, my results demonstrate that the MH1 domain of SMAD6 is important for proper localization of the SMAD6 protein, and localization is important for SMAD6 function. Numerous pathologies are associated with dysfunctional BMP signaling, and thus it is critical to fully understand the regulation of this pathway to inform future treatment. Therefore, understanding how the structure of SMAD6, a key inhibitor of BMP signaling, affects its function has significant therapeutic potential.Bachelor of Scienc