Smoothened goes molecular: new pieces in the hedgehog signaling puzzle

Abstract

A general aim of studies of signal transduction is to identify mediators of specific signals, order them into pathways, and understand the nature of interactions between individual components and how these interactions alter pathway behavior. Despite years of intensive study and its central importance to animal development and human health, our understanding of the Hedgehog (Hh) signaling pathway remains riddled with gaps, question marks, assumptions, and poorly understood connections. In particular, understanding how interactions between Hh and Patched (Ptc), a 12-pass integral membrane protein, lead to modulation of the function of Smoothened (Smo), a 7-pass integral membrane protein, has defied standard biochemical characterization. Recent structural and biochemical characterizations of Smoothened domains have begun to unlock this riddle, however, and lay the groundwork for improved cancer therapies.

Keywords: Cancer; Cell Signaling; Cholesterol; Development; G Protein-coupled Receptor (GPCR); Hedgehog Signaling Pathway; Patched (Ptc); Smoothened (Smo); Sonic Hedgehog (Shh); Sterol.

FIGURE 1.

Major transmembrane components of Hh signal reception and transduction. Ptc (left) represses Smo (right) through an unknown, indirect mechanism. The interaction of Sonic hedgehog N-terminal domain (ShhN) with Ptc relieves Ptc-mediated repression of Smo. The sterol-sensing domain of Ptc (TM II–TM VI) is colored blue. For Smo, the 8 cysteines mediating 4 disulfide bonds in the Smo ECLs are shown in green; D473H, a Vismodegib resistance mutation, is in blue; W535L, a constitutively activating mutation, is in red; and C-tail sites of serine and threonine phosphorylation (indicated by pS/pT) are in orange.

FIGURE 2.

Structures of the Smo 7TM domain. A, β₂AR in complex with Carazolol (Protein Data Bank (PDB): 2RH1) and Smo in complex with LY2940680 (PDB: 4JKV) colored by GPCR helix number. Key Smo residues are shown in spheres (ECL disulfides are green; D473H is light blue; W535L is orange; and arrows mark D473H and W535L). B, the five Smo 7TM crystal structures with bound ligands shown in spheres. (From left to right, PDB: 4O9R, 4QIN, 4QIM, 4JKV, and 4N4W.)

FIGURE 3.

Structures of class F GPCR CRDs. A, the structure of the mouse Frizzled-8 CRD (Fz8 CRD) shown with the palmitoleic acid moiety (PAM) in red (PDB: 4F0A). The position of Xenopus Wnt8 loop to which PAM is attached is noted by a dashed black line. B, the structure of the zebrafish Smoothened CRD with residues implicated in binding 20(S)-OHC shown in red (PDB: 4C79).

FIGURE 4.

Smoothened-interacting small molecules. A, 7TM-targeting small molecules. B, CRD-targeting small molecules. C, other Smo-targeting small molecules. Activating small molecules are noted by green type.