Abstract
Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Gαs, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.
Highlights
The Hedgehog (Hh) signal transduction cascade is critical for many aspects of embryonic development, and aberrant regulation of the pathway results in a wide variety of congenital defects and cancers [1]
When exposed to conditioned media (CM) collected from cells expressing the N-terminal signaling fragment of Sonic hedgehog (ShhN), wild-type mouse embryonic fibroblasts (MEFs) accumulated Smo in primary cilia, and nearly 100% of cilia were positive for Smo (Smo+) after 6 hours of treatment (Fig. 1A, 1B)
In agreement with previously published results [7], a reduced number of cilia were Smo+ after brief (1 hour) treatment with cyclopamine, a teratogen derived from the Veratrum genus of plants and a welldefined Smo antagonist known to bind to the heptahelical bundle of Smo (Fig. 1A, 1B) [18]
Summary
The Hedgehog (Hh) signal transduction cascade is critical for many aspects of embryonic development, and aberrant regulation of the pathway results in a wide variety of congenital defects and cancers [1]. When exposed to conditioned media (CM) collected from cells expressing the N-terminal signaling fragment of Sonic hedgehog (ShhN), wild-type mouse embryonic fibroblasts (MEFs) accumulated Smo in primary cilia, and nearly 100% of cilia were positive for Smo (Smo+) after 6 hours of treatment (Fig. 1A, 1B). This unexpected result prompted us to screen a number of previously reported modulators of Smo activity for their ability to induce or prevent Smo translocation to the primary cilium.
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