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Event: 2027

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Antagonism, Smoothened receptor

Short name

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Antagonism Smoothened

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Biological Context

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Level of Biological Organization
Molecular

Cell term

The location/biological environment in which the event takes place.The biological context describes the location/biological environment in which the event takes place. For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable. For individual/population events, the organ context is not applicable. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Cell term
mesenchymal cell

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
regulation of receptor activity	smoothened	decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help

AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
Anatagonsim SMO leads to OFC	MolecularInitiatingEvent	Arthur Author (send email)	Under development: Not open for comment. Do not cite

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 KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Term	Scientific Term	Evidence	Link
Vertebrates	Vertebrates		NCBI

Life Stages

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

Life stage	Evidence
Embryo	High
All life stages	High

Sex Applicability

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

Term	Evidence
Unspecific

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. 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 occurs in the plasma membrane for flies (Denef, Neubüser et al. 2000) and the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005).

In the absence of Hedgehog (HH) ligand, the Patched (PTCH) receptor suppresses the activation of SMO. When HH ligand binds to PTCH, suppression on SMO is released and SMO is able to relocate, accumulate, and signal to intracellular effectors (Denef, Neubüser et al. 2000). This signaling to effectors results in the activation of the GLI transcription factors and the subsequent induction of HH target gene expression(Alexandre, Jacinto et al. 1996, Von Ohlen and Hooper 1997). The exact mechanism through which PTCH and SMO interact is not known.

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).

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Verification of binding and affinity for SMO can be measured using fluorescence binding assays and photoaffinity labeling respectively (Chen, Taipale et al. 2002). qRT-PCR can be used to determine the expression level of SMO (Lou, Li et al. 2020).

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

Sex- SMO is present in both male and females and differences in activation or antagonism between sex have not been demonstrated.
Life stages- The Hedgehog pathway is a major pathway in embryonic development. Aberrant activation of HH signalling is known to cause cancer (Dahmane, Lee et al. 1997, Kimura, Stephen et al. 2005). For these reasons all stages of life are of relevance.
Taxonomic- SMO is conserved in both vertebrates and invertebrates. SMO signaling is dependent upon its relocation to a subcellular location. This occurs in the plasma membrane for flies (Denef, Neubüser et al. 2000) and the primary cilium (PC) in vertebrates (Huangfu and Anderson 2005).

References

List of the literature that was cited for this KE 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.

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.

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.

Dahmane, N., J. Lee, P. Robins, P. Heller and A. Ruiz i Altaba (1997). "Activation of the transcription factor Gli1 and the Sonic hedgehog signalling pathway in skin tumours." Nature 389(6653): 876-881.

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.

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.

Kimura, H., D. Stephen, A. Joyner and T. Curran (2005). "Gli1 is important for medulloblastoma formation in Ptc1+/- mice." Oncogene 24(25): 4026-4036.

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

Lou, H., H. Li, A. R. Huehn, N. I. Tarasova, B. Saleh, S. K. Anderson and M. Dean (2020). "Genetic and Epigenetic Regulation of the Smoothened Gene (SMO) in Cancer Cells." Cancers (Basel) 12(8).

Meiss, F., H. Andrlová and R. Zeiser (2018). "Vismodegib." Recent Results Cancer Res 211: 125-139.

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

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." Nature Chemical Biology 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. and M. P. Scott (2007). "Patching the gaps in Hedgehog signalling." Nat Cell Biol 9(9): 1005-1009.

Sharpe, H. J., W. Wang, R. N. Hannoush and F. J. de Sauvage (2015). "Regulation of the oncoprotein Smoothened by small molecules." Nat Chem Biol 11(4): 246-255.

Sinha, S. and J. K. Chen (2006). "Purmorphamine activates the Hedgehog pathway by targeting Smoothened." Nat Chem Biol 2(1): 29-30.

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, T. Evron, E. Vardy, G. W. Han, X. P. Huang, S. J. Hufeisen, T. J. Mangano, D. J. Urban, V. Katritch, V. Cherezov, M. G. Caron, B. L. Roth and R. C. Stevens (2014). "Structural basis for Smoothened receptor modulation and chemoresistance to anticancer drugs." Nat Commun 5: 4355.

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.