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Key Event Title
Decrease, Smoothend relocation and activation
|Level of Biological Organization|
Key Event Components
|protein localization to cilium||smoothened||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP||Point of Contact||Author Status||OECD Status|
|Anatagonsim SMO leads to OFC||KeyEvent||Arthur Author (send email)||Under development: Not open for comment. Do not cite|
Key Event Description
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). This relocation then leads to signaling to effectors resulting 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.
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).
How It Is Measured or Detected
- Fluorescent proteins can be used tag SMO, cilia and the plasma membrane to determine if SMO has relocated to the cilia (Filipova, Diaz Garcia et al. 2020).
- Fluorescent binding assay can be used to verify if a compound binds to SMO (Chen, Taipale et al. 2002).
- Cell lines can be engineered to express Myc-tagged SMO. This gives a user friendly readout of SMO activation. (Corbit, Aanstad et al. 2005).
Domain of Applicability
- Sex- SMO and cilia are present in both male and females and differences in gene expression has not been demonstrated.
- Life stages- The Hedgehog pathway is a major pathway in embryonic development.
- Taxonomic-SMO relocation to the tip of primary cilia occurs in vertebrates Huangfu and Anderson 2005)
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.
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.
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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.
Filipova, A., D. Diaz Garcia, J. Dvorak, S. Filip, M. Jelicova and Z. Sinkorova (2020). "Simple Detection of Primary Cilia by Immunofluorescence." J Vis Exp(159).
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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.
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.
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.
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.
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.