Hedgehog 'off' state (Homo sapiens)
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Description
Hedgehog is a secreted morphogen that has evolutionarily conserved roles in body organization by regulating the activity of the Ci/Gli transcription factor family. In Drosophila in the absence of Hh signaling, full-length Ci is partially degraded by the proteasome to generate a truncated repressor form that translocates to the nucleus to represses Hh-responsive genes. Binding of Hh ligand to the Patched (PTC) receptor allows the 7-pass transmembrane protein Smoothened (SMO) to be activated in an unknown manner, disrupting the partial proteolysis of Ci and allowing the full length activator form to accumulate (reviewed in Ingham et al, 2011; Briscoe and Therond, 2013).
While many of the core components of Hh signaling are conserved from flies to humans, the pathways do show points of significant divergence. Notably, the human genome encodes three Ci homologues, GLI1, 2 and 3 that each play slightly different roles in regulating Hh responsive genes. GLI3 is the primary repressor of Hh signaling in vertebrates, and is converted to the truncated GLI3R repressor form in the absence of Hh. GLI2 is a potent activator of transcription in the presence of Hh but contributes only minimally to the repression function. While a minor fraction of GLI2 protein is processed into the repressor form in the absence of Hh, the majority is either fully degraded by the proteasome or sequestered in the full-length form in the cytosol by protein-protein interactions. GLI1 lacks the repression domain and appears to be an obligate transcriptional activator (reviewed in Briscoe and Therond, 2013).
Vertebrate but not fly Hh signaling also depends on the movement of pathway components through the primary cilium. The primary cilium is a non-motile microtubule based structure whose construction and maintenance depends on intraflagellar transport (IFT). Anterograde IFT moves molecules from the ciliary base along the axoneme to the ciliary tip in a manner that requires the microtubule-plus-end directed kinesin KIF3 motor complex and the IFT-B protein complex, while retrograde IFT back to the ciliary base depends on the minus-end directed dynein motor and the IFT-A complex. Genetic screens have identified a number of cilia-related proteins that are required both to maintain Hh in the 'off' state and to transduce the signal when the pathway is activated (reviewed in Hui and Angers, 2011; Goetz and Anderson, 2010). View original pathway at Reactome.
While many of the core components of Hh signaling are conserved from flies to humans, the pathways do show points of significant divergence. Notably, the human genome encodes three Ci homologues, GLI1, 2 and 3 that each play slightly different roles in regulating Hh responsive genes. GLI3 is the primary repressor of Hh signaling in vertebrates, and is converted to the truncated GLI3R repressor form in the absence of Hh. GLI2 is a potent activator of transcription in the presence of Hh but contributes only minimally to the repression function. While a minor fraction of GLI2 protein is processed into the repressor form in the absence of Hh, the majority is either fully degraded by the proteasome or sequestered in the full-length form in the cytosol by protein-protein interactions. GLI1 lacks the repression domain and appears to be an obligate transcriptional activator (reviewed in Briscoe and Therond, 2013).
Vertebrate but not fly Hh signaling also depends on the movement of pathway components through the primary cilium. The primary cilium is a non-motile microtubule based structure whose construction and maintenance depends on intraflagellar transport (IFT). Anterograde IFT moves molecules from the ciliary base along the axoneme to the ciliary tip in a manner that requires the microtubule-plus-end directed kinesin KIF3 motor complex and the IFT-B protein complex, while retrograde IFT back to the ciliary base depends on the minus-end directed dynein motor and the IFT-A complex. Genetic screens have identified a number of cilia-related proteins that are required both to maintain Hh in the 'off' state and to transduce the signal when the pathway is activated (reviewed in Hui and Angers, 2011; Goetz and Anderson, 2010). View original pathway at Reactome.
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Two SCF(beta-TrCP)-dependent degradation sites, Dn and Dc, have been identified in human GLI1. Removal of these sites abrogates the interaction with beta-TrCP, reduces the beta-TrCP-dependent ubiquitination of GLI1 and stabilizes the GLI1 protein levels. As is the case for GLI2 and GLI3, ubiquitination of GLI1 depends on the its prior phosphorylation by PKA, as GLI1 degradation is sensitive to PKA inhibitors and removal of the putative PKA sites abrogates the interaction with beta-TrCP and delays the kinetics of degradation (Huntzicker et al, 2006).
PTCH is a 7 transmembrane protein that is localized to the primary cilium in the absence of Hh ligand (Rohatgi et al, 2007). PTCH regulates SMO in a non-stoichiometric manner and there is little evidence that endogenous PTCH and SMO interact directly (Taipale et al, 2002; reviewed in Huangfu and Anderson, 2006). PTCH has a sterol sensing domain (SSD) and structural similarity to bacterial RND transporters. Mutation in conserved motifs in the RND domain abrogate the ability of PTCH to negatively regulate SMO activity (Taipale et al, 2002). The transmembrane heptahelical domain of SMO has been shown to bind to a number of natural and synthetic molecules, many of which are structurally related to sterols, and this binding can activate or repress SMO activity (Mas et al, 2010; Dwyer et al, 2007; Nachtergaele et al, 2012; Corcoran et al, 2006). Together, these data suggest a speculative model where PTCH regulates SMO activity by controlling the flux of sterol-related SMO agonists and/or antagonists, although this has not been fully substantiated (Khaliullina et al, 2009; reviewed in Rohatgi and Scott, 2007; Briscoe and Therond, 2013).
In the absence of Hh signal, SMO is largely found in intracellular vesicles, with a fraction localized to the plasma membrane (Milenkovic et al, 2009; Huangfu et al, 2006; Corbit et al, 2005; Rohatgi et al, 2007; Wang et al, 2009; Wilson et al, 2009). Like GLI2, 3 and SUFU, however, SMO may traffic through the cilium in the absence of ligand (Wilson et al, 2009; Kim et al, 2009). SMO and PTCH appear to have opposing localizations in both the 'off' and 'on' state, with PTCH exiting and SMO entering the cilium upon Hh pathway activation (Denef et al, 2000; Rohatgi et al, 2007; reviewed in Goetz and Anderson, 2010; Hui and Angers, 2011). Clearance of PTCH from the ciliary membrane in the presence of Hh is promoted by its ubiquitination by the E3 ligase SMURF (Huang et al, 2013; Yue et al, 2014)
Like the Drosophila homologue, vertebrate SMO appears to exists as a constitutive dimer. Dimerization is mediated by the N-terminal Cys-rich domain (CRD) and is required for function (Zhao et al, 2007). The C-terminal tail of SMO has arginine-rich clusters that appear to regulate the conformation of the tails in the dimer, maintaining the SMO dimer in an inactive state. In Drosophila, the inhibitory effect of the arginine-rich region is counteracted upon Hh pathway activation by PKA-mediated phosphorylation of adjacent serine residues. This promotes an open tail conformation that is required for cell surface accumulation and signaling (Zhao et al, 2007; Chen et al, 2010). These consensus PKA motifs are not conserved in the vertebrate SMO C-terminal tail, and a role for PKA-mediated phosphorylation and direct activation of SMO appears not to hold true in mammalian cells (Zhao et al, 2007; Tuson et al, 2011). A similar activating phosphorylation of vertebrate SMO may be CK1 or GRK2-dependent (Chen et al, 2011).