Here we report the discovery of oncogenic mutations in the Hedgehog and mitogen-activated protein kinase (MAPK) pathways in over 80% of ameloblastomas, locally destructive odontogenic tumors of the jaw, by genomic analysis of archival material. Mutations in SMO (encoding Smoothened, SMO) are common in ameloblastomas of the maxilla, whereas BRAF mutations are predominant in tumors of the mandible. We show that a frequently occurring SMO alteration encoding p.Leu412Phe is an activating mutation and that its effect on Hedgehog-pathway activity can be inhibited by arsenic trioxide (ATO), an anti-leukemia drug approved by the US Food and Drug Administration (FDA) that is currently in clinical trials for its Hedgehog-inhibitory activity. In a similar manner, ameloblastoma cells harboring an activating BRAF mutation encoding p.Val600Glu are sensitive to the BRAF inhibitor vemurafenib. Our findings establish a new paradigm for the diagnostic classification and treatment of ameloblastomas.

Summary Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo), by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

Highlights d Patched structure reveals a hydrophobic conduit with sterollike contents d Patched mediates Hedgehog-reversible reduction of inner leaflet cholesterol activity d Hydrophobic conduit is essential for cholesterol effect and Smoothened suppression d Inner leaflet cholesterol likely mediates Hedgehog-Patched regulation of Smoothened

A coordinated sequence of cell growth, proliferation, and death is critical for tissue maintenance and homeostasis. Elements that influence cell physiology include growth factors, nutrients, and stress signals. An intracellular signaling network integrates multiple and occasionally conflicting signals to coordinate the response. FKBP12-rapamycin-associated protein (FRAP) plays an important role in this network and ensures that cell growth occurs under optimal conditions, in part by regulating a p70 S6 kinase (p70S6K) phosphatase (1). Rapamycin is a specific modulator of FRAP, and treatment of cells with rapamycin results in a rapid dephosphorylation of p70S6K by means of the formation of an FKBP12-rapamycin-FRAP ternary complex (2). p70S6K activity has been shown to be sensitive to a wide variety of inputs that include serum, nutrients, wortmanin (a phosphatidylinositol 3-kinase inhibitor), and osmotic stress (3). Mitogens and growth factors regulate p70S6K primarily by means of the upstream kinases such as phosphatidylinositol 3-kinase, phosphoinositide-dependent kinase, and Akt. This kinase-dependent phosphorylation is in equilibrium with unidentified ''subordinate'' phosphatases such that when cells are deprived of serum these subordinate phosphatases dephosphorylate and deactivate p70S6K. In addition to kinase-directed regulation, p70S6K is regulated by a ''dominant,'' FRAPregulated phosphatase that is constitutively associated with p70S6K (4). Activation of this PP2A-type phosphatase results in rapid dephosphorylation of p70S6K even in the presence of mitogens, suggesting that this mechanism is dominant over kinase-directed signaling. Rapamycin dephosphorylates p70S6K by activating the p70S6K phosphatase in a FRAP-dependent manner (4).A rapamycin-resistant allele of p70S6K generated by truncating its N and C termini (NTCT-p70S6K) has proved to be a valuable reagent in segregating the p70S6K regulatory signals (5). NTCT-p70S6K is sensitive to kinase-directed signals initiated by mitogens and growth factors, but it is insensitive to signals that pass through the FRAP-regulated phosphatase. NTCT-p70S6K is unable to interact with this phosphatase. Accordingly, NTCT-p70S6K is sensitive to wortmanin (phosphatidylinositol 3-kinase inhibitor) but insensitive to rapamycin. Signals emanating from amino acid deprivation (6) and hyperosmolarity (7) do not affect NTCT-p70S6K activity, indicating that they are mediated by FRAP and not by the growth factor-activated kinases. Despite the compelling nature of experiments conducted with NTCT-p70S6K, one cannot rule out the possibility that NTCT-p70S6K is also insensitive to as-yetunknown FRAP-independent signals impinging on p70S6K.This correlative use of NTCT-p70S6K has more recently been extended to investigations of mitochondrial function. Mitochondrial poisons that deplete the mitochondrial proton-gradient and lower the concentration of intracellular ATP result in deactivation of p70S6K (8, 9). An NTCT-like allele of p70S6K is resistant to mitochondrial poisons, suggesti...

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3–5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results—combined with signalling studies and molecular dynamics simulations—delineate the structural basis for PTCH1 -SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na to regulate Smoothened in our assays, raising the possibility that a Na gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

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TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.