Smoothened (Smo), a distant relative of G protein-coupled receptors, mediates Hedgehog (Hh) signaling during embryonic development and can initiate or transmit ligand-independent pathway activation in tumorigenesis. Although the cellular mechanisms that regulate Smo function remain unclear, the direct inhibition of Smo by cyclopamine, a plant-derived steroidal alkaloid, suggests that endogenous small molecules may be involved. Here we demonstrate that SAG, a chlorobenzothiophene-containing Hh pathway agonist, binds to the Smo heptahelical bundle in a manner that antagonizes cyclopamine action. In addition, we have identified four small molecules that directly inhibit Smo activity but are structurally distinct from cyclopamine. Functional and biochemical studies of these compounds provide evidence for the small molecule modulation of Smo through multiple mechanisms and yield insights into the physiological regulation of Smo activity. The mechanistic differences between the Smo antagonists may be useful in the therapeutic manipulation of Hh signaling. H edgehog (Hh) signaling normally functions to specify embryonic pattern by directing cellular differentiation and proliferation (1), whereas aberrant Hh pathway activation is associated with the formation of tumors such as basal cell carcinoma and medulloblastoma (2-4). Cellular responses to the secreted Hh polypeptide are mediated by two integral membrane proteins, Patched (Ptc) and Smoothened (Smo), which were first identified by genetic screens in Drosophila (5-9). Hh binds to the twelve-pass transmembrane protein Ptc (8, 10, 11), thereby alleviating Ptc-mediated suppression of Smo (12), a distant relative of G protein coupled receptors. Smo activation then triggers a series of intracellular events, culminating in the stabilization of the transcription factor Cubitus interruptus (Ci) and the expression of Ci-dependent genes (13,14). These events are recapitulated during mammalian development and tumorigenesis through multiple protein homologues, including three distinct Hh family members [Sonic (Shh), Indian (Ihh), and Desert (Dhh)], two Ptc proteins (Ptch1 and Ptch2), and three Ci-like transcription factors (Gli1, Gli2, and Gli3; ref. (25), and Ptc is structurally related to the resistance-nodulation-cell division (RND) family of prokaryotic permeases and to the Niemann-Pick C1 (NPC1) protein, both of which are capable of transporting hydrophobic compounds (26, 27). Thus, Ptc might control Smo function by influencing its interactions with cellular small molecules.To study the biochemical basis of Smo activation further, we set out to identify and characterize other small molecules that modulate Smo function. We report here that a family of chlorobenzothiophene molecules identified as Hh pathway agonists (28) act by binding to the Smo heptahelical bundle. We also describe four previously uncharacterized Smo antagonists discovered through small molecule screens for Hh pathway inhibitors. In addition to providing mechanistic insights, such modulators may have ther...

Mutations affecting the transmembrane proteins Patched (Ptc) or Smoothened (Smo) that trigger ligand-independent activity of the Hedgehog (Hh) signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma. Despite extensive genetic studies demonstrating the importance of these receptor components in embryonic patterning and cancer, the mechanism by which Ptc regulates Smo is not understood. Here we report that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, we show that free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; we demonstrate that the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumour suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule.

The amino-terminal signaling domain of the Sonic hedgehog secreted protein (Shh-N), which derives from the Shh precursor through an autoprocessing reaction mediated by the carboxyl-terminal domain, executes multiple functions in embryonic tissue patterning, including induction of ventral and suppression of dorsal cell types in the developing neural tube. An apparent catalytic site within Shh-N is suggested by structural homology to a bacterial carboxypeptidase. We demonstrate here that alteration of residues presumed to be critical for a hydrolytic activity does not cause a loss of inductive activity, thus ruling out catalysis by Shh-N as a requirement for signaling. We favor the alternative, that Shh-N functions primarily as a ligand for the putative receptor Patched (Ptc). This possibility is supported by new evidence for direct binding of Shh-N to Ptc and by a strong correlation between the affinity of Ptc-binding and the signaling potency of Shh-N protein variants carrying alterations of conserved residues in a particular region of the protein surface. These results together suggest that direct Shh-N binding to Ptc is a critical event in transduction of the Shh-N signal.