Selective translocation of intracellular Smoothened to the primary cilium in response to Hedgehog pathway modulation

Abstract

Smoothened (Smo), a 7-pass transmembrane protein, is essential for transduction of a Hedgehog (Hh) signal across the cell membrane. Smo is also the principle therapeutic target for several candidate drugs in the treatment of Hh-related diseases. Mammalian Smo translocates to the primary cilium in response to Sonic hedgehog (Shh) ligand-mediated signaling. A mechanistic understanding of Smo translocation and its interactions with drug candidates is pivotal to our understanding of Hh signaling and the design, development and application of successful drugs. We established a system in which Smo was dual-labeled with GFP and a 12-aa tag whose recognition by an enzymatic process enables the posttranslational labeling of Smo in the cell membrane within the living cell. These tools enable the simultaneous visualization of all cellular Smo, and more specifically, the cell membrane restricted subpopulation. Using this system, we demonstrate that cyclopamine, a widely used Hh antagonist, induces a cilial translocation of Smo similar to that reported for Shh ligand and several Hh agonists. In contrast, other antagonists abrogate the Shh-induced, cilial translocation of Smo. We present evidence that the majority of cilial-localized Smo originates from an intracellular source and may traffic to the primary cilium through an intraflagellar transport (IFT) pathway.