Smoothened oligomerization/higher order clustering in lipid rafts is essential for high Hedgehog activity transduction.

The Hedgehog (Hh) signaling pathway plays evolutionarily conserved roles in controlling embryonic development and tissue homeostasis, and its dysregulation has been implicated in many human diseases including congenital disorder and cancer. The Hh pathway has a unique signal reception system that includes two membrane proteins, the receptor Patched (Ptc) and the transducer Smoothened (Smo). In the Hh signaling cascade, Smo plays a critical role in controlling transduction of Hh gradient signal from the outside into the inside of cells. Although the Smo downstream signal transduction has been intensively studied, the mechanism by which Smo on the plasma membrane is regulated has not been fully understood. As a specific membrane structure of metazoan cells, lipid rafts act as a platform to regulate signal transduction by forming a nanoscale cluster through protein-protein or protein-lipid interactions. However, it remains largely unknown whether lipid rafts are also involved in the regulation of Hh signal transduction. Here, we show that Smo extracellular domain (N terminus) and transmembrane domains form oligomers/higher order clusters in response to Hh signal. Furthermore, we identify that lipid rafts on the plasma membrane are essential for high level activity of Smo during the Hh signal transduction. Finally, our observation suggests that oligomerization/higher order clustering of Smo C-terminal cytoplasmic tail (C-tail) is essential for the transduction of high level Hh signal. Collectively, our data support that in response to Hh gradient signals, Smo transduces high level Hh signal by forming oligomers/higher order clusters in the lipid rafts of cell plasma membrane.