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Relationship: 2734

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Antagonism Smoothened leads to Decrease, SMO relocation

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Antagonism Smoothened

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Decrease, SMO relocation

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Antagonism of Smoothened receptor leading to orofacial clefting	adjacent	Moderate	Low	Jacob Reynolds (send email)	Under development: Not open for comment. Do not cite	Under Review

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
human	Homo sapiens	Low	NCBI
mouse	Mus musculus	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	Not Specified

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Embryo	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

The Smoothened (SMO) receptor is Class F G protein coupled receptor involved in signal transduction of the Sonic Hedgehog (SHH) pathway. It includes distinct functional groups including ligand binding pockets, cysteine rich domain (CRD), transmembrane helix (TM), extracellular loop (ECL), intracellular loop (ICL), and a carboxyl-terminal tail (C-term tail) (Arensdorf, Marada et al. 2016). SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016).

In the absence of SHH ligand, the Patched (PTCH) receptor suppresses the activation of SMO. When HH ligand binds to PTCH, suppression on SMO is released and SMO can relocate, accumulate, and signal to intracellular effectors (Denef, Neubüser et al. 2000, Rohatgi and Scott 2007). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009). The exact mechanism through which PTCH and SMO interact is not known.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Pubmed was used as the primary database for evidence collection. Searches are organized by the date and search terms used in the supplementary table. Search results were initially screened through review of the title and abstract for potential for data relating antagonism of SMO and SMO relocation. Each selected publication and its’ data were then examined to determine if support or lack thereof existed for this KER. Papers that did not show any data relating to this KER were discarded. The search is detailed below in Table 1.

Table 1: KER 2734 evidence collection strategy

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

SMO signaling is dependent upon its relocation to a subcellular location. This relocation occurs in the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005). It has been shown that SMO localization to the tip of the primary cilia is essential for the SHH signaling cascade in vertebrates (Corbit, Aanstad et al. 2005, Rohatgi, Milenkovic et al. 2007, Rohatgi, Milenkovic et al. 2009)

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

In vitro
- NIH 3t3 (murine fibroblast) were used to study the effects of three SHH pathway antagonists, SANT 1, SANT2, and cyclopamine on SMO localization using fluorescent microscopy. Cells were treated with increasing concentrations of the antagonists in the presence of SHH ligand. SANT1 and SANT2 both blocked SMO localization in the cilia with IC50 values of 5 and 13nM respectively. Cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10µm (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009).
- A small molecule screen of 10,000 compounds identified six inhibitors of SHH signaling, four of which bind directly to SMO (SANT1-4). Screening was conducted using NIH 3T3 SHH LightII cells cultured in media conditioned from HEK 293 transfected to stably express Shh-N. Cells were dosed with the compound library at 0.714ug/ml and SHH activity was quantified at 30h using Renilla luciferase activity. A fluorescent binding assay using BODIPY-cyclopamine was used to verify binding to SMO for the SANT compounds. Dose response reported as IC50 for the inhibition of SHH signaling was conducting in NIH 3T3 SHH light2, NIH 3T3 SmoA1-Light2, P2 Ptch1-/- (mouse embryonic fibroblasts) (Chen, Taipale et al. 2002).

Compound/Cell	SHH-Light2 (nM)	SmoA1-Light2 (nM)	Ptch1-/- (nM)
SANT-1	20	30	20
SANT-2	30	70	50
SANT-3	100	80	80
SANT-4	200	300	300

- Direct binding of cyclopamine to SMO was verified using a photoaffinity form of cyclopamine (PA-cyclopamine). PA-cyclopamine had previously been shown to inhibit SHH signaling in NIH 3T3 Shh-LightII cells with similar IC50 values to cyclopamine (300nm and 150nm respectively) (Taipale, Chen et al. 2000). Binding to SMO was verified using a COS-1 (fibroblast, monkey) line transfected to over express SMO. The location of cyclopamine binding was further investigated using BODIPY- cyclopamine and COS-1 cells modified to lack either a N-terminal, extracellular cysteine-rich domain, or the cytoplasmic C terminal of SMO. The findings support that cyclopamine does not require these domains and instead binds directly to the heptahelical domain (Chen, Taipale et al. 2002).
- To investigate whether SMO localization is regulated by SHH, a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005)
- To determine whether PTCH1 regulates localization of SMO MEFs from PTCH1^-/-mice were used. These showed SHH activity and SMO localization in the primary cilium in the absence of SHH ligand or SAG. Reintroduction of PTCH1 via a retrovirus suppressed SHH activity and prevented SMO accumulation in primary cilia (Rohatgi and Scott 2007)
- A high content assay to detect compounds that block SMO accumulation to the primary cilia in the presence of SHH was used to screen a library of ~5600 compounds. This screen identified 26 hits with DY131 and its analog GSK4716 further investigated as potent hits. These compounds inhibited SHH induced accumulation of SMO::EGFP with IC50s of 0.8um and 2um respectively. DY131 and GSK4716 both inhibited the activation of a Glireporter with IC50s of 2um and 10um respectively (Wang, Arvanites et al. 2012).
In vivo
- Two-week-old mice were dosed with 40mg/kg vismodegib (GDC-0449) via ip injection twice a day for 3 consecutive days. Quantification of immunofluorescence and ciliary length showed that like SMO^fl/+mice, ciliary M71/M72 OR was reduced while cilia lengths were not changed. To determine if SMO regulates ciliary localization an OMP-CRE mouse line was used. It was found that immunofluorescence of M71/M72 was reduced in both SMO^fl/+, SMO^fl/fl, as compared to SMO^+/+ control (Maurya, Bohm et al. 2017).
- Cyclopamine was found to inhibit SHH signaling in White leghorn neural plate explants. Explants were dissected from stage 9-10 embryo chicks and cultured in collagen gels. Tissues were cultured in Shh-N media from COS-1 cells. Cyclopamine was dissolved in ethanol and added to test tissues. Tissues were fixed at 24-29hr and processed for immunofluorescence. 120nm cyclopamine was found to repress SHH induction as determined by Pax7 repression and the blockage of floor plate and motor neuron induction (Incardona, Gaffield et al. 1998).
- - Multiple ciliopathies associated with clefting in humans including Meckel-Gruber syndrome (OMIM 249000) and Ellis-van Creveld syndrome (OMIM 225500)(Brugmann, Cordero et al. 2010)

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

While we know that entry to the cilia is tightly controlled, the exact mechanism of SMO ciliary trafficking is not fully understood. The PC is separated from the plasma membrane by the ciliary pockets and the transition zone which function together to regulate the movement of lipids and proteins in and out of the organelle (Goetz, Ocbina et al. 2009, Rohatgi and Snell 2010). The SHH receptor PTCH contains a ciliary localization sequence in its’ carboxy tail. Localization of PTCH to the PC is essential for inhibition of SMO as deletion of the CLS in PTCH prevents PTCH localization as well as inhibition of SMO (Kim, Hsia et al. 2015) (53). SMO also contains a CLS, but only accumulates in the PC upon ligand binding (Corbit, Aanstad et al. 2005). The entry of SMO into the PC is thought to occur either laterally through the ciliary pockets or internally via recycling endosomes (Milenkovic, Scott et al. 2009). Once inside the PC, SMO can diffuse freely, however it will usually accumulate in specific locations depending upon its’ activation state. Inactive SMO will accumulate more at the base of the PC while active SMO will accumulate in the tip of the PC (Milenkovic, Weiss et al. 2015).

An endogenous ligand for SMO has not been discovered although evidence for one exists and that PTCH controls SMO by controlling its’ availability or accessibility. To support this, it has been shown that PTCH and SMO do not physically interact (Chen and Struhl 1998). PTCH acts catalytically with SMO with one PTCH receptor capable of controlling many (~50) SMO receptors (Taipale, Cooper et al. 2002). Since PTCH includes a sterol sensing domain and shares characteristics of ancient bacterial transporters, a model of PTCH functioning by pumping a sterol-like MSO regulator has been proposed (Mukhopadhyay and Rohatgi 2014). SMO is constitutively active in the absence of PTCH suggesting that the elusive molecule is an agonist (Rohatgi and Scott 2007). Conversely, the discovery that oxysterols bind to the CRD binding domain acting as positive modulators suggest that the molecule could be an agonist with PTCH functioning to sequester away or limit cellular concentration (Corcoran and Scott 2006, Nachtergaele, Mydock et al. 2012)

The activity of SMO is controlled by ligand binding (Kobilka 2007). Two separate binding pockets, one in the groove of the extracellular CRD and the other in the helices of the TMD have been identified (Nachtergaele, Mydock et al. 2012, Rana, Carroll et al. 2013, Wang, Wu et al. 2013, Byrne, Sircar et al. 2016, Huang, Zheng et al. 2018). These two binding pockets have been shown to interact in an allosteric manner (Nachtergaele, Mydock et al. 2012). The binding pocket in the helices of the TMD binds several SMO agonists including SAG as well as antagonists Vismodegib and Sonidegib. The CRD binding pocket binds cholesterol and its’ oxidized derivates (Byrne, Luchetti et al. 2018). The antagonist cyclopamine binds to the TMD binding pocket and inhibits SHH signal transduction. However, in mSMO carrying the mutations D477G/E552K that disable the TMD binding pocket, cyclopamine binds to the CRD pocket and activates the pathway (Huang, Nedelcu et al. 2016). To date several oxysterols including 20(S)-hydroxylcholesterol, 22(S)-hydroxylcholesterol, 7-keto-25-hydroxylcholesterol and 7-keto-27-hydroxylcholesterol have been identified as activators of SMO (Dwyer, Sever et al. 2007, Nachtergaele, Mydock et al. 2012, Myers, Sever et al. 2013). A binding site for 24(S),25-epoxycholesterol has been identified in the TMD pocket using cryo-EM of SMO in complex with 24(S),25-epoxycholesterol (Qi, Liu et al. 2019).

While it is well understood that cyclopamine is an antagonist of SMO, contradictory in vivo data was found regarding whether cyclopamine blocks SMO relocation to the primary cilia. Rohatgi et al used NIH 3T3s cell and found that cyclopamine did not inhibit the accumulation of SMO in the cilia even when dosed at 5-10um (>10 fold above kd). All three antagonists inhibited SHH pathway transduction and target gene expression (Rohatgi, Milenkovic et al. 2009). Corbit et al used a renal epithelial MDCK (Madin-Darby canine kidney) line was engineered to express Myc-tagged SMO. Following culture for 1hr in SHH conditioned media SMO presence in the primary cilium is upregulated while cells cultured in the presence of cyclopamine see a downregulation of SMO in the primary cilia (Corbit, Aanstad et al. 2005). Further work is required to determine if SMO antagonism via cyclopamine results in decrease in SMO relocation.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

The data presented in support of this KER includes both in vitro and in vivo studies. The in vivo work identifies multiple antagonists of SMO and validates that they directly bind to SMO. These studies also offer data to show that antagonism of SMO causes a down regulation in SMO relocation the primary cilia. Dose dependent SMO localization is seen in the studies performed by Rohtagi et al 2009 and Chen et al 2002.The response time of SMO antagonism and subsequent time for a decrease in SMO relocation and activation has not been reported. No dose dependent in vivo data for antagonism of SMO and relocation to the cilia was found and all in vivo evidence is conducted under steady state exposure. Dose response data for disruption of SHH using the antagonists exists and is well charactered however quantification of ciliary relocation is lacking. Further studies are needed to expand our quantitative understanding of this linkage.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

No studies identified

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Relocation of SMO to the PC typically occurs within ~20 minutes of agonist stimulation (Arensdorf, Marada et al. 2016). No data was found on how fast antagonism of SMO will stop its’ relocation to the primary cilia.

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

None identified

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

The relationship between antagonism of SMO and a decrease in SMO relocation and activation has been shown repeatedly in mice models as detailed in the empirical evidence section. The relationship is biologically plausible in human, but to date no specific experiments have addressed this question. The SHH pathway is well understood to be fundamental to proper embryonic development and that aberrant SHH signaling during embryonic development can cause birth defects indculding orofacial clefts (OFCs). For this reason, this KER is applicable to the embryonic stage with a high level of confidence.

References

List of the literature that was cited for this KER description. More help

Alexandre, C., A. Jacinto and P. W. Ingham (1996). "Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins." Genes Dev 10(16): 2003-2013.

Arensdorf, A. M., S. Marada and S. K. Ogden (2016). "Smoothened Regulation: A Tale of Two Signals." Trends Pharmacol Sci 37(1): 62-72.

Brugmann, S. A., D. R. Cordero and J. A. Helms (2010). "Craniofacial ciliopathies: A new classification for craniofacial disorders." Am J Med Genet A 152A(12): 2995-3006.

Byrne, E. F. X., G. Luchetti, R. Rohatgi and C. Siebold (2018). "Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates." Current Opinion in Cell Biology 51: 81-88.

Byrne, E. F. X., R. Sircar, P. S. Miller, G. Hedger, G. Luchetti, S. Nachtergaele, M. D. Tully, L. Mydock-McGrane, D. F. Covey, R. P. Rambo, M. S. P. Sansom, S. Newstead, R. Rohatgi and C. Siebold (2016). "Structural basis of Smoothened regulation by its extracellular domains." Nature 535(7613): 517-522.

Chen, J. K., J. Taipale, M. K. Cooper and P. A. Beachy (2002). "Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened." Genes Dev 16(21): 2743-2748.

Chen, J. K., J. Taipale, K. E. Young, T. Maiti and P. A. Beachy (2002). "Small molecule modulation of Smoothened activity." Proc Natl Acad Sci U S A 99(22): 14071-14076.

Chen, Y. and G. Struhl (1998). "In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex." Development 125(24): 4943-4948.

Corbit, K. C., P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier and J. F. Reiter (2005). "Vertebrate Smoothened functions at the primary cilium." Nature 437(7061): 1018-1021.

Corcoran, R. B. and M. P. Scott (2006). "Oxysterols stimulate Sonic hedgehog signal transduction and proliferation of medulloblastoma cells." Proc Natl Acad Sci U S A 103(22): 8408-8413.

Denef, N., D. Neubüser, L. Perez and S. M. Cohen (2000). "Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened." Cell 102(4): 521-531.

Dwyer, J. R., N. Sever, M. Carlson, S. F. Nelson, P. A. Beachy and F. Parhami (2007). "Oxysterols are novel activators of the hedgehog signaling pathway in pluripotent mesenchymal cells." J Biol Chem 282(12): 8959-8968.

Goetz, S. C., P. J. Ocbina and K. V. Anderson (2009). "The primary cilium as a Hedgehog signal transduction machine." Methods Cell Biol 94: 199-222.

Heyne, G. W., C. G. Melberg, P. Doroodchi, K. F. Parins, H. W. Kietzman, J. L. Everson, L. J. Ansen-Wilson and R. J. Lipinski (2015). "Definition of critical periods for Hedgehog pathway antagonist-induced holoprosencephaly, cleft lip, and cleft palate." PLoS One 10(3): e0120517.

Huang, P., D. Nedelcu, M. Watanabe, C. Jao, Y. Kim, J. Liu and A. Salic (2016). "Cellular Cholesterol Directly Activates Smoothened in Hedgehog Signaling." Cell 166(5): 1176-1187.e1114.

Huang, P., S. Zheng, B. M. Wierbowski, Y. Kim, D. Nedelcu, L. Aravena, J. Liu, A. C. Kruse and A. Salic (2018). "Structural Basis of Smoothened Activation in Hedgehog Signaling." Cell 174(2): 312-324.e316.

Huangfu, D. and K. V. Anderson (2005). "Cilia and Hedgehog responsiveness in the mouse." Proc Natl Acad Sci U S A 102(32): 11325-11330.

Incardona, J. P., W. Gaffield, R. P. Kapur and H. Roelink (1998). "The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction." Development 125(18): 3553-3562.

Kim, J., E. Y. Hsia, A. Brigui, A. Plessis, P. A. Beachy and X. Zheng (2015). "The role of ciliary trafficking in Hedgehog receptor signaling." Sci Signal 8(379): ra55.

Kobilka, B. K. (2007). "G protein coupled receptor structure and activation." Biochimica et Biophysica Acta (BBA) - Biomembranes 1768(4): 794-807.

Maurya, D. K., S. Bohm and M. Alenius (2017). "Hedgehog signaling regulates ciliary localization of mouse odorant receptors." Proc Natl Acad Sci U S A 114(44): E9386-e9394.

Milenkovic, L., M. P. Scott and R. Rohatgi (2009). "Lateral transport of Smoothened from the plasma membrane to the membrane of the cilium." J Cell Biol 187(3): 365-374.

Milenkovic, L., L. E. Weiss, J. Yoon, T. L. Roth, Y. S. Su, S. J. Sahl, M. P. Scott and W. E. Moerner (2015). "Single-molecule imaging of Hedgehog pathway protein Smoothened in primary cilia reveals binding events regulated by Patched1." Proc Natl Acad Sci U S A 112(27): 8320-8325.

Millington, G., K. H. Elliott, Y. T. Chang, C. F. Chang, A. Dlugosz and S. A. Brugmann (2017). "Cilia-dependent GLI processing in neural crest cells is required for tongue development." Dev Biol 424(2): 124-137.

Mukhopadhyay, S. and R. Rohatgi (2014). "G-protein-coupled receptors, Hedgehog signaling and primary cilia." Semin Cell Dev Biol 33: 63-72.

Myers, B. R., L. Neahring, Y. Zhang, K. J. Roberts and P. A. Beachy (2017). "Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium." Proc Natl Acad Sci U S A 114(52): E11141-E11150.

Myers, Benjamin R., N. Sever, Yong C. Chong, J. Kim, Jitendra D. Belani, S. Rychnovsky, J. F. Bazan and Philip A. Beachy (2013). "Hedgehog Pathway Modulation by Multiple Lipid Binding Sites on the Smoothened Effector of Signal Response." Developmental Cell 26(4): 346-357.

Nachtergaele, S., L. K. Mydock, K. Krishnan, J. Rammohan, P. H. Schlesinger, D. F. Covey and R. Rohatgi (2012). "Oxysterols are allosteric activators of the oncoprotein Smoothened." Nat Chem Biol 8(2): 211-220.

Qi, X., H. Liu, B. Thompson, J. McDonald, C. Zhang and X. Li (2019). "Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi." Nature 571(7764): 279-283.

Rana, R., C. E. Carroll, H.-J. Lee, J. Bao, S. Marada, C. R. R. Grace, C. D. Guibao, S. K. Ogden and J. J. Zheng (2013). "Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling." Nature Communications 4(1): 2965.

Rohatgi, R., L. Milenkovic, R. B. Corcoran and M. P. Scott (2009). "Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process." Proc Natl Acad Sci U S A 106(9): 3196-3201.

Rohatgi, R., L. Milenkovic and M. P. Scott (2007). "Patched1 regulates hedgehog signaling at the primary cilium." Science 317(5836): 372-376.

Rohatgi, R. and M. P. Scott (2007). "Patching the gaps in Hedgehog signalling." Nat Cell Biol 9(9): 1005-1009.

Rohatgi, R. and W. J. Snell (2010). "The ciliary membrane." Curr Opin Cell Biol 22(4): 541-546.

Taipale, J., J. K. Chen, M. K. Cooper, B. Wang, R. K. Mann, L. Milenkovic, M. P. Scott and P. A. Beachy (2000). "Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine." Nature 406(6799): 1005-1009.

Taipale, J., M. K. Cooper, T. Maiti and P. A. Beachy (2002). "Patched acts catalytically to suppress the activity of Smoothened." Nature 418(6900): 892-896.

Von Ohlen, T. and J. E. Hooper (1997). "Hedgehog signaling regulates transcription through Gli/Ci binding sites in the wingless enhancer." Mech Dev 68(1-2): 149-156.

Wang, C., H. Wu, V. Katritch, G. W. Han, X. P. Huang, W. Liu, F. Y. Siu, B. L. Roth, V. Cherezov and R. C. Stevens (2013). "Structure of the human smoothened receptor bound to an antitumour agent." Nature 497(7449): 338-343.

Wang, Y., A. C. Arvanites, L. Davidow, J. Blanchard, K. Lam, J. W. Yoo, S. Coy, L. L. Rubin and A. P. McMahon (2012). "Selective identification of hedgehog pathway antagonists by direct analysis of smoothened ciliary translocation." ACS Chem Biol 7(6): 1040-1048.