Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling

Abstract

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants.DOI: http://dx.doi.org/10.7554/eLife.01340.001

A Functional and Putative Physiological Role of Calcitriol in Patched1/Smoothened Interaction

“…In addition, the synergistic inhibition of Hh signaling by calcitriol and ITZ indicated that calcitriol and ITZ bind to different Smo sites. To validate the hypothesis that calcitriol binds to Smo outside the 7TM or CRD, we tested if calcitriol or ITZ might compete for direct Smo binding with BODIPY-labeled CP in HEK293S cells (20,24), because the binding sites of CP have been already mapped to the 7TM domains of Smo and to some extend to the CRD (18,20). Indeed, the known 7TM binders, CP (Fig.…”

“…Calcitriol Efficiently Inhibits CRD-deleted Smo-The CRD of Smo has been described as an exclusive binding site for oxysterols that are discussed to be physiological regulators of Hh signaling activity (18,19 (Fig. 3, a and b (Fig.…”

“…These facts suggest that calcitriol might be an attractive candidate for the development of therapies against Hhassociated tumors. Thus we tested if the antitumoral effects of calcitriol could be enhanced by a combination with the potent Smo-inhibitor ITZ (40) or with CP that binds Smo almost exclusively at the 7TM (18).…”

“…cyclopamine (CP) (20), vismodegib (21)) and activators (Smoothened agonist; SAG (22,23)). Although no endogenous molecule has been identified that regulates Smo activity upon binding to the 7TM, endogenous oxidized derivatives of cholesterol (oxysterols) (15,24,25) such as 20(S)-hydroxycholesterol (20(S)-OHC) can bind and activate Smo via its CRD (18,19).…”

“…Smo, the signaling partner of Ptch, consists of a 7-transmembrane domain (7TM), an intracellular C-terminal tail and an extracellular N-terminal region containing a cysteine-rich domain (CRD) (18,19). The 7TM harbors a binding site for Smo inhibitors (e.g.…”