The Hedgehog pathway, critical to vertebrate development, is organized in primary cilia. Activation of signaling causes the Hedgehog receptor Ptch1 to exit cilia, allowing a second receptor, Smo, to accumulate in cilia and activate the downstream steps of the pathway. Mechanisms regulating the dynamics of these receptors are unknown, but the ubiquitination of Smo regulates its interaction with the intraflagellar transport system to control ciliary levels. A focused screen of ubiquitin-related genes identified nine required for maintaining low ciliary Smo at the basal state. These included cytoplasmic E3s (Arih2, Mgrn1, and Maea), a ciliary localized E3 (Wwp1), a ciliary localized E2 (Ube2l3), a deubiquitinase (Bap1), and three adaptors (Kctd5, Skp1a, and Skp2). The ciliary E3, Wwp1, binds Ptch1 and localizes to cilia at the basal state. Activation of signaling removes both Ptch1 and Wwp1 from cilia, thus providing an elegant mechanism for Ptch1 to regulate ciliary Smo levels.

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.

Sonic Hedgehog (Shh) is a secreted protein that controls cell fate and mitogenesis in the developing nervous system. Here we show that a constitutively active form of Smoothened (Smo-M2) mimics concentration-dependent actions of Shh in the developing neural tube, including activation of ventral marker genes (HNF3beta, patched, Nkx2.2, netrin-1), suppression of dorsal markers (Pax-3, Gli-3, Ephrin A5) and induction of ventral neurons (dopaminergic, serotonergic) and ventrolateral motor neurons (Islet-1+, Islet-2+, HB9+) and interneurons (Engrailed-1+, CHX10+). Furthermore, Smo-M2's patterning activities were cell autonomous, occurring exclusively in cells expressing Smo-M2. These findings suggest that Smo is a key signaling component in the Hh receptor and that Shh patterns the vertebrate nervous system as a morphogen, rather than through secondary relay signals.

Ischemic stroke occurs as a result of blood supply interruption to the brain causing tissue degeneration, patient disabilities or death. Currently, treatment of ischemic stroke is limited to thrombolytic therapy with a narrow time window of administration. The sonic hedgehog (Shh) signaling pathway has a fundamental role in the central nervous system development, but its impact on neural cell survival and tissue regeneration/repair after ischemic stroke has not been well investigated. Here we report the neuroprotective properties of a small-molecule agonist of the Shh co-receptor Smoothened, purmorphamine (PUR), in the middle cerebral artery occlusion model of ischemic stroke. We found that intravenous administration of PUR at 6 h after injury was neuroprotective and restored neurological deficit after stroke. PUR promoted a transient upregulation of tissue-type plasminogen activator in injured neurons, which was associated with a reduction of apoptotic cell death in the ischemic cortex. We also observed a decrease in blood-brain barrier permeability after PUR treatment. At 14 d postinjury, attenuation of inflammation and reactive astrogliosis was found in PUR-treated animals. PUR increased the number of newly generated neurons in the peri-infarct and infarct area and promoted neovascularization in the ischemic zone. Notably, PUR treatment did not significantly alter the ischemia-induced level of Gli1, a Shh target gene of tumorigenic potential. Thus our study reports a novel pharmacological approach for postischemic treatment using a small-molecule Shh agonist, providing new insights into hedgehog signaling-mediated mechanisms of neuroprotection and regeneration after stroke.

Sonic hedgehog (Shh) and its patched-smoothened receptor complex control a variety of functions in the developing central nervous system, such as neural cell proliferation and differentiation. Recently, Shh signaling components have been found to be expressed at the synaptic level in the postnatal brain, suggesting a potential role in the regulation of synaptic transmission. Using in utero electroporation of constitutively active and negative-phenotype forms of the Shh signal transducer smoothened (Smo), we studied the role of Smo signaling in the development and maturation of GABAergic transmission in the somatosensory cortex. Our results show that enhancing Smo activity during development accelerates the shift from depolarizing to hyperpolarizing GABA in a manner dependent on functional expression of potassium-chloride cotransporter type 2 (KCC2, also known as SLC12A5). On the other hand, blocking Smo activity maintains the GABA response in a depolarizing state in mature cortical neurons, resulting in altered chloride homeostasis and increased seizure susceptibility. This study reveals unexpected functions of Smo signaling in the regulation of chloride homeostasis, through control of KCC2 cell-surface stability, and the timing of the GABA excitatory-to-inhibitory shift in brain maturation.

Hedgehog (Hh) signaling is essential for normal growth, patterning, and homeostasis of many tissues in diverse organisms, and is misregulated in a variety of diseases including cancer. Cytoplasmic Hedgehog signaling is activated by multisite phosphorylation of the seven-pass transmembrane protein Smoothened (Smo) in its cytoplasmic C-terminus. Aside from a short membrane-proximal stretch, the sequence of the C-terminus is highly divergent in different phyla, and the evidence suggests that the precise mechanism of Smo activation and transduction of the signal to downstream effectors also differs. To clarify the conserved role of G-protein-coupled receptor kinases (GRKs) in Smo regulation, we mapped four clusters of phosphorylation sites in the membrane-proximal C-terminus of Drosophila Smo that are phosphorylated by Gprk2, one of the two fly GRKs. Phosphorylation at these sites enhances Smo dimerization and increases but is not essential for Smo activity. Three of these clusters overlap with regulatory phosphorylation sites in mouse Smo and are highly conserved throughout the bilaterian lineages, suggesting that they serve a common function. Consistent with this, we find that a C-terminally truncated form of Drosophila Smo consisting of just the highly conserved core, including Gprk2 regulatory sites, can recruit the downstream effector Costal-2 and activate target gene expression, in a Gprk2-dependent manner. These results indicate that GRK phosphorylation in the membrane proximal C-terminus is an evolutionarily ancient mechanism of Smo regulation, and point to a higher degree of similarity in the regulation and signaling mechanisms of bilaterian Smo proteins than has previously been recognized.

The smoothened receptor (Smo) plays a key role in Hedgehog (Hh) signaling pathway and it has been regarded as an efficacious therapeutic target for basal cell carcinoma (BCC) and medulloblastoma (MB). Nevertheless, the resistance mutation and active mutants of Smo have put forward the requirement of finding more effective inhibitors. Herein, we performed metadynamics simulations on Smo bound with vismodegib (Smo-Vismod) and with cholesterol (Smo-CLR), respectively, to explore the inhibition mechanism of vismodegib. The simulation results indicated that vismodegib-induced shifts of TM5, TM6, and TM7, which permitted the extracellular extension of TM6 and extracellular loop3 (ECL3) to enter the extracellular cysteine-rich domain (CRD) groove. Therefore, an open CRD groove that has not been noticed previously was observed in Smo-Vismod complex. As a consequence, the occupied CRD groove prevents the binding of cholesterol. In addition, the HD and ECLs play crucial roles in the interaction of CRD and TMD. These results reveal that TM5, TM6, and TM7 play important roles in allosteric inhibition the activation of Smo and disrupting cholesterol binding by vismodegib binding. Our results are expected to contribute to understanding the allosteric inhibition mechanism of Smo by vismodegib. Moreover, the detailed conformational changes contribute to the development of novel Smo inhibitors against resistance mutation and active mutants of Smo.

To determine the influence of the sonic hedgehog (shh) pathway and its receptor smoothened (smo), on the survival and functionality of dopaminergic (DA) neurons.

One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases.

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

Dynamic changes in membrane protein composition of the primary cilium are central to development and homeostasis, but we know little about mechanisms regulating membrane protein flux. Stimulation of the sonic hedgehog (Shh) pathway in vertebrates results in accumulation and activation of the effector Smoothened within cilia and concomitant disappearance of a negative regulator, the orphan G protein-coupled receptor (GPCR), Gpr161. Here, we describe a two-step process determining removal of Gpr161 from cilia. The first step involves β-arrestin recruitment by the signaling competent receptor, which is facilitated by the GPCR kinase Grk2. An essential factor here is the ciliary trafficking and activation of Smoothened, which by increasing Gpr161-β-arrestin binding promotes Gpr161 removal, both during resting conditions and upon Shh pathway activation. The second step involves clathrin-mediated endocytosis, which functions outside of the ciliary compartment in coordinating Gpr161 removal. Mechanisms determining dynamic compartmentalization of Gpr161 in cilia define a new paradigm for down-regulation of GPCRs during developmental signaling from a specialized subcellular compartment.

The sonic hedgehog (Shh) signaling pathway plays a fundamental role in the central nervous system (CNS) development, but its effects on neural cell survival and brain repair after subarachnoid hemorrhage (SAH) has not been well-investigated. The present study was undertaken to evaluate the influence of an agonist of the Shh co-receptor Smoothened (Smo), purmorphamine (PUR), on early brain injury (EBI) as well as the underlying mechanisms after SAH. PUR was administered via an intraperitoneal injection with a dose of 0.5, 1, and 5 mg/kg at 2, 6, 24, and 46 h after SAH in rat model. The results showed that PUR treatment significantly ameliorated brain edema, improved neurobehavioral function, and attenuated neuronal cell death in the prefrontal cortex (PFC), associated with a decrease in Bax/Bcl-2 ratio and suppression of caspase-3 activation at 48 h after SAH. PUR also promoted phospho-ERK levels. Additionally, PUR treatment markedly decreased MDA concentration accompanied with the elevation in the expression of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 in PFC. Notably, PUR treatment significantly reversed the changes of Shh pathway mediators containing Patched, Gli1, and Shh by SAH insult, and the neuroprotection of PUR on SAH was blocked by Smo antagonist cyclopamine. These results indicated that PUR exerts neuroprotection against SAH-evoked injury in rats, mediated in part by anti-apoptotic and anti-oxidant mechanism, up-regulating phospho-ERK levels, mediating Shh signaling molecules in the PFC.

Smoothened (smo) is a segment polarity gene required for correct patterning of every segment in Drosophila. The earliest defect in smo mutant embryos is loss of expression of the Hedgehog-responsive gene wingless between 1 and 2 hr after gastrulation. Since smo mutant embryos cannot respond to exogenous Hedgehog (Hh) but can respond to exogenous Wingless, the smo product functions in Hh signaling. Smo acts downstream of or in parallel to Patched, an antagonist of the Hh signal. The smo gene encodes an integral membrane protein with characteristics of G protein-coupled receptors and shows homology to the Drosophila Frizzled protein. Based on its predicted physical characteristics and on its position in the Hh signaling pathway, we suggest that smo encodes a receptor for the Hh signal.

In the Drosophila wing, Hedgehog is made by cells of the posterior compartment and acts as a morphogen to pattern cells of the anterior compartment. High Hedgehog levels instruct L3/4 intervein fate, whereas lower levels instruct L3 vein fate. Transcriptional responses to Hedgehog are mediated by the balance between repressor and activator forms of Cubitus interruptus, CiR and CiA. Hedgehog regulates this balance through its receptor, Patched, which acts through Smoothened and thence a regulatory complex that includes Fused, Costal, Suppressor of Fused and Cubitus interruptus. It is not known how the Hedgehog signal is relayed from Smoothened to the regulatory complex nor how responses to different levels of Hedgehog are implemented. We have used chimeric and deleted forms of Smoothened to explore the signaling functions of Smoothened. A Frizzled/Smoothened chimera containing the Smo cytoplasmic tail (FFS) can induce the full spectrum of Hedgehog responses but is regulated by Wingless rather than Hedgehog. Smoothened whose cytoplasmic tail is replaced with that of Frizzled (SSF) mimics fused mutants, interfering with high Hedgehog responses but with no effect on low Hedgehog responses. The cytoplasmic tail of Smoothened with no transmembrane or extracellular domains (SmoC) interferes with high Hedgehog responses and allows endogenous Smoothened to constitutively initiate low responses. SmoC mimics costal mutants. Genetic interactions suggest that SSF interferes with high signaling by titrating out Smoothened, whereas SmoC drives constitutive low signaling by titrating out Costal. These data suggest that low and high signaling (1) are qualitatively different, (2) are mediated by distinct configurations of the regulatory complex and (3) are initiated by distinct activities of Smoothened. We present a model where low signaling is initiated when a Costal inhibitory site on the Smoothened cytoplasmic tail shifts the regulatory complex to its low state. High signaling is initiated when cooperating Smoothened cytoplasmic tails activate Costal and Fused, driving the regulatory complex to its high state. Thus, two activities of Smoothened translate different levels of Hedgehog into distinct intracellular responses.

It has been widely demonstrated that the hedgehog pathway is strongly associated with basal cell carcinoma of the skin (NBCCS). To assess potential DNA alterations related to keratocystic odontogenic tumors (KCOTs), we sequenced smoothened (SMO) genes in 12 sporadic KCOTs.

The oncoprotein Smoothened (SMO), a Frizzled-class-G-protein-coupled receptor, is the central transducer of hedgehog (Hh) signaling. While canonical SMO signaling is best understood in the context of cilia, evidence suggests that SMO has other functions in cancer biology that are unrelated to canonical Hh signaling. Herein, we provided evidence that elevated levels of human SMO show a strong correlation with elevated levels of insulin-like growth factor 1 receptor (IGF1R) and reduced survival in diffuse large B-cell lymphoma (DLBCL). As an integral component of raft microdomains, SMO plays a fundamental role in maintaining the levels of IGF1R in lymphoma and breast cancer cells as well IGF1R-associated activation of protein kinase B (AKT). Silencing of SMO increases lysosomal degradation and favors a localization of IGF1R to late endosomal compartments instead of early endosomal compartments from which much of the receptor would normally recycle. In addition, loss of SMO interferes with the lipid raft localization and retention of the remaining IGF1R and AKT, thereby disrupting the primary signaling context for IGF1R/AKT. This activity of SMO is independent of its canonical signaling and represents a novel and clinically relevant contribution to signaling by the highly oncogenic IGF1R/AKT signaling axis.

The Hedgehog pathway is critical for animal development and has been implicated in multiple human malignancies. Despite great interest in targeting the pathway pharmacologically, many of the principles underlying the signal transduction cascade remain poorly understood. Hedgehog ligands are recognized by a unique receptor system that features the transporter-like protein Patched and the G protein-coupled receptor (GPCR)-like Smoothened (SMO). The biochemical interaction between these transmembrane proteins is the subject of intensive efforts. Recent structural and functional studies have provided great insight into the small-molecule regulation of SMO through identification of two distinct ligand-binding sites. In this Perspective, we review these recent findings and relate them to potential mechanisms for the endogenous regulation of SMO.

The seven transmembrane protein Smoothened is required for Hedgehog signaling during embryonic development and adult tissue homeostasis. Inappropriate activation of the Hedgehog signalling pathway leads to cancers such as basal cell carcinoma and medulloblastoma, and Smoothened inhibitors are now available clinically to treat these diseases. However, resistance to these inhibitors rapidly develops thereby limiting their efficacy. The determination of Smoothened crystal structures enables structure-based discovery of new ligands with new chemotypes that will be critical to combat resistance. In this study, we docked 3.2 million available, lead-like molecules against Smoothened, looking for those with high physical complementarity to its structure; this represents the first such campaign against the class Frizzled G-protein coupled receptor family. Twenty-one high-ranking compounds were selected for experimental testing, and four, representing three different chemotypes, were identified to antagonize Smoothened with IC50 values better than 50 μM. A screen for analogs revealed another six molecules, with IC50 values in the low micromolar range. Importantly, one of the most active of the new antagonists continued to be efficacious at the D473H mutant of Smoothened, which confers clinical resistance to the antagonist vismodegib in cancer treatment.

Transduction of Hedgehog signals across the plasma membrane is facilitated by the class F G-protein-coupled-receptor (GPCR) Smoothened (SMO). Recent studies suggest that SMO is modulated via interactions of its transmembrane (TM) domain with cholesterol. We apply molecular dynamics simulations of SMO embedded in cholesterol containing lipid bilayers, revealing a direct interaction of cholesterol with the TM domain at regions distinct from those observed in class A GPCRs. In particular the extracellular tips of helices TM2 and TM3 form a well-defined cholesterol interaction site. Potential of mean force calculations yield a free energy landscape for cholesterol binding. Alongside analysis of equilibrium cholesterol occupancy, this reveals the existence of a dynamic "greasy patch" interaction with the TM domain of SMO, which may be compared with previously identified lipid interaction sites on other membrane proteins. These predictions provide molecular-level insights into cholesterol interactions with a class F GPCR, suggesting potential druggable sites.

Cyclopamine is a natural alkaloid that is known to act as an agonist when it binds to the Cysteine Rich Domain (CRD) of the Smoothened receptor and as an antagonist when it binds to the Transmembrane Domain (TMD). To study the effect of cyclopamine binding to each binding site experimentally, mutations in the other site are required. Hence, simulations are critical for understanding the WT activity due to binding at different sites. Additionally, there is a possibility that cyclopamine could bind to both sites simultaneously especially at high concentration, the implications of which remain unknown. We performed three independent sets of simulations to observe the receptor activation with cyclopamine bound to each site independently (CRD, TMD) and bound to both sites simultaneously. Using multi-milliseconds long aggregate MD simulations combined with Markov state models and machine learning, we explored the dynamic behavior of cyclopamine's interactions with different domains of WT SMO. A higher population of the active state at equilibrium, a lower activation free energy barrier of ~ 2 kcal/mol, and expansion of the hydrophobic tunnel to facilitate cholesterol transport agrees with the cyclopamine's agonistic behavior when bound to the CRD of SMO. A higher population of the inactive state at equilibrium, a higher free energy barrier of ~ 4 kcal/mol and restricted the hydrophobic tunnel to impede cholesterol transport showed cyclopamine's antagonistic behavior when bound to TMD. With cyclopamine bound to both sites, there was a slightly larger inactive population at equilibrium and an increased free energy barrier (~ 3.5 kcal/mol). The tunnel was slightly larger than when solely bound to TMD, and showed a balance between agonism and antagonism with respect to residue movements exhibiting an overall weak antagonistic effect.