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Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium.

Wilson CW, Chen MH, Chuang PT - PLoS ONE (2009)

Bottom Line: Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation.Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium.We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

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ShhN ligand and SANT-1 act dominantly to PKA-induced Smo translocation.(A) Wild-type MEFs treated with indicated compounds for 24 hours stained with antibodies against acetylated (AC) tubulin (red), Smo (green) and DAPI (blue). ShhN causes Smo distribution along the length of the cilium when PKA is activated, whereas SANT-1 inhibits any type of Smo trafficking on the cilium. (B) Quantification of Smo+ cilia in wild-type MEFs after treatment for 24 hours. Nearly all cilia are Smo+ when treated with ShhN and CTX or FSK. Error bars indicate +/− SD. (C) Quantification of Smo+ cilia in wild-type MEFs after 24 hours. SANT-1 inhibits CTX- and FSK-mediated stimulation of Smo translocation to the proximal region of the primary cilium. Error bars indicate +/− SD. (D) 8xGliBS-luciferase reporter activity in wild-type MEFs with indicated treatments for 48 hours. CTX and FSK, but not PTX inhibit ShhN stimulation of the reporter. Normalization was performed as in Figure 3. Error bars indicate +/− SD.
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pone-0005182-g004: ShhN ligand and SANT-1 act dominantly to PKA-induced Smo translocation.(A) Wild-type MEFs treated with indicated compounds for 24 hours stained with antibodies against acetylated (AC) tubulin (red), Smo (green) and DAPI (blue). ShhN causes Smo distribution along the length of the cilium when PKA is activated, whereas SANT-1 inhibits any type of Smo trafficking on the cilium. (B) Quantification of Smo+ cilia in wild-type MEFs after treatment for 24 hours. Nearly all cilia are Smo+ when treated with ShhN and CTX or FSK. Error bars indicate +/− SD. (C) Quantification of Smo+ cilia in wild-type MEFs after 24 hours. SANT-1 inhibits CTX- and FSK-mediated stimulation of Smo translocation to the proximal region of the primary cilium. Error bars indicate +/− SD. (D) 8xGliBS-luciferase reporter activity in wild-type MEFs with indicated treatments for 48 hours. CTX and FSK, but not PTX inhibit ShhN stimulation of the reporter. Normalization was performed as in Figure 3. Error bars indicate +/− SD.

Mentions: We examined the relationship of PKA-mediated Smo translocation to the proximal cilium with ShhN-mediated pathway activation and SANT-1 inhibition. Exposure of MEFs to both ShhN and CTX or FSK resulted in restoration of Smo staining along the entire length of the primary cilium (Fig. 4A, 4B). In contrast, SANT-1 inhibited PKA stimulation of Smo trafficking to the proximal cilium (Fig. 4A, 4C). Two conclusions may be drawn from these results. First, the Smo conformation adopted when bound to SANT-1 is refractory to both cyclopamine- and PKA-stimulated cilium trafficking, suggesting that SANT-1 may act upstream to sequester Smo from or inhibit its interaction with β-arrestins or IFT particles [25]. Second, addition of ShhN overrides accumulation of Smo in the proximal region of the primary cilium. Restricted Smo localization to this region could be a prerequisite for pathway activation, or a means to inhibit Smo trafficking or coupling to downstream components. Consistent with previous reports [34]–[37], we observed that activation of the Hh reporter was blocked by CTX- and FSK-mediated PKA stimulation, but PTX treatment produced only a modest reduction (Fig 4D). We speculate that if PKA positively regulates Smo while promoting Gli repressor production, any potential positive effect on Hh signaling due to partial Smo translocation induced by CTX or FSK may be offset by negative regulation of Gli factors by PKA [38]. Additional studies using endogenous levels of PKA-refractory forms of the Gli proteins will be necessary to rigorously resolve this issue.


Smoothened adopts multiple active and inactive conformations capable of trafficking to the primary cilium.

Wilson CW, Chen MH, Chuang PT - PLoS ONE (2009)

ShhN ligand and SANT-1 act dominantly to PKA-induced Smo translocation.(A) Wild-type MEFs treated with indicated compounds for 24 hours stained with antibodies against acetylated (AC) tubulin (red), Smo (green) and DAPI (blue). ShhN causes Smo distribution along the length of the cilium when PKA is activated, whereas SANT-1 inhibits any type of Smo trafficking on the cilium. (B) Quantification of Smo+ cilia in wild-type MEFs after treatment for 24 hours. Nearly all cilia are Smo+ when treated with ShhN and CTX or FSK. Error bars indicate +/− SD. (C) Quantification of Smo+ cilia in wild-type MEFs after 24 hours. SANT-1 inhibits CTX- and FSK-mediated stimulation of Smo translocation to the proximal region of the primary cilium. Error bars indicate +/− SD. (D) 8xGliBS-luciferase reporter activity in wild-type MEFs with indicated treatments for 48 hours. CTX and FSK, but not PTX inhibit ShhN stimulation of the reporter. Normalization was performed as in Figure 3. Error bars indicate +/− SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664476&req=5

pone-0005182-g004: ShhN ligand and SANT-1 act dominantly to PKA-induced Smo translocation.(A) Wild-type MEFs treated with indicated compounds for 24 hours stained with antibodies against acetylated (AC) tubulin (red), Smo (green) and DAPI (blue). ShhN causes Smo distribution along the length of the cilium when PKA is activated, whereas SANT-1 inhibits any type of Smo trafficking on the cilium. (B) Quantification of Smo+ cilia in wild-type MEFs after treatment for 24 hours. Nearly all cilia are Smo+ when treated with ShhN and CTX or FSK. Error bars indicate +/− SD. (C) Quantification of Smo+ cilia in wild-type MEFs after 24 hours. SANT-1 inhibits CTX- and FSK-mediated stimulation of Smo translocation to the proximal region of the primary cilium. Error bars indicate +/− SD. (D) 8xGliBS-luciferase reporter activity in wild-type MEFs with indicated treatments for 48 hours. CTX and FSK, but not PTX inhibit ShhN stimulation of the reporter. Normalization was performed as in Figure 3. Error bars indicate +/− SD.
Mentions: We examined the relationship of PKA-mediated Smo translocation to the proximal cilium with ShhN-mediated pathway activation and SANT-1 inhibition. Exposure of MEFs to both ShhN and CTX or FSK resulted in restoration of Smo staining along the entire length of the primary cilium (Fig. 4A, 4B). In contrast, SANT-1 inhibited PKA stimulation of Smo trafficking to the proximal cilium (Fig. 4A, 4C). Two conclusions may be drawn from these results. First, the Smo conformation adopted when bound to SANT-1 is refractory to both cyclopamine- and PKA-stimulated cilium trafficking, suggesting that SANT-1 may act upstream to sequester Smo from or inhibit its interaction with β-arrestins or IFT particles [25]. Second, addition of ShhN overrides accumulation of Smo in the proximal region of the primary cilium. Restricted Smo localization to this region could be a prerequisite for pathway activation, or a means to inhibit Smo trafficking or coupling to downstream components. Consistent with previous reports [34]–[37], we observed that activation of the Hh reporter was blocked by CTX- and FSK-mediated PKA stimulation, but PTX treatment produced only a modest reduction (Fig 4D). We speculate that if PKA positively regulates Smo while promoting Gli repressor production, any potential positive effect on Hh signaling due to partial Smo translocation induced by CTX or FSK may be offset by negative regulation of Gli factors by PKA [38]. Additional studies using endogenous levels of PKA-refractory forms of the Gli proteins will be necessary to rigorously resolve this issue.

Bottom Line: Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation.Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium.We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Activation of Hedgehog (Hh) signaling requires the transmembrane protein Smoothened (Smo), a member of the G-protein coupled receptor superfamily. In mammals, Smo translocates to the primary cilium upon binding of Hh ligands to their receptor, Patched (Ptch1), but it is unclear if ciliary trafficking of Smo is sufficient for pathway activation. Here, we demonstrate that cyclopamine and jervine, two structurally related inhibitors of Smo, force ciliary translocation of Smo. Treatment with SANT-1, an unrelated Smo antagonist, abrogates cyclopamine- and jervine-mediated Smo translocation. Further, activation of protein kinase A, either directly or through activation of Galphas, causes Smo to translocate to a proximal region of the primary cilium. We propose that Smo adopts multiple inactive and active conformations, which influence its localization and trafficking on the primary cilium.

Show MeSH
Related in: MedlinePlus