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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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Modulation of Gαs and protein kinase A (PKA) causes Smo accumulation in a proximal region of the primary cilium.(A) Treatment of wild-type MEFs with cholera toxin (CTX) for 24 hours induces Smo (green) translocation to the cilium (red). (B) Quantification of CTX- and FSK-induced Smo ciliary translocation after treatment for the indicated times. Treatment of wild-type MEFs with pertussis toxin (PTX) does not stimulate ciliary translocation of Smo. Error bars indicate +/− SD. (C) Wild-type MEFs were fixed in methanol and stained with antibodies against γ-tubulin (red; label the basal body) and Smo (green). In contrast to the relatively uniform Smo staining on the cilium induced by ShhN, CTX treatment causes Smo accumulation proximal to the basal body. (D) 8xGliBS-luciferase Hh reporter activity in wild-type MEFs with the indicated treatment. CTX and FSK do not activate the Hh pathway despite inducing ciliary localization of Smo. Data are representative of three independent experiments. Hh reporter activity was normalized to β-galactosidase activity produced from a constitutive hsp68-lacZ reporter, as alteration of PKA affected Renilla luciferase activity (data not shown). Error bars indicate +/− SD.
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pone-0005182-g003: Modulation of Gαs and protein kinase A (PKA) causes Smo accumulation in a proximal region of the primary cilium.(A) Treatment of wild-type MEFs with cholera toxin (CTX) for 24 hours induces Smo (green) translocation to the cilium (red). (B) Quantification of CTX- and FSK-induced Smo ciliary translocation after treatment for the indicated times. Treatment of wild-type MEFs with pertussis toxin (PTX) does not stimulate ciliary translocation of Smo. Error bars indicate +/− SD. (C) Wild-type MEFs were fixed in methanol and stained with antibodies against γ-tubulin (red; label the basal body) and Smo (green). In contrast to the relatively uniform Smo staining on the cilium induced by ShhN, CTX treatment causes Smo accumulation proximal to the basal body. (D) 8xGliBS-luciferase Hh reporter activity in wild-type MEFs with the indicated treatment. CTX and FSK do not activate the Hh pathway despite inducing ciliary localization of Smo. Data are representative of three independent experiments. Hh reporter activity was normalized to β-galactosidase activity produced from a constitutive hsp68-lacZ reporter, as alteration of PKA affected Renilla luciferase activity (data not shown). Error bars indicate +/− SD.

Mentions: (A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See Fig. 3C). (B) Quantification of the percentage of Smo-positive (Smo+) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo+ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.


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

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

Modulation of Gαs and protein kinase A (PKA) causes Smo accumulation in a proximal region of the primary cilium.(A) Treatment of wild-type MEFs with cholera toxin (CTX) for 24 hours induces Smo (green) translocation to the cilium (red). (B) Quantification of CTX- and FSK-induced Smo ciliary translocation after treatment for the indicated times. Treatment of wild-type MEFs with pertussis toxin (PTX) does not stimulate ciliary translocation of Smo. Error bars indicate +/− SD. (C) Wild-type MEFs were fixed in methanol and stained with antibodies against γ-tubulin (red; label the basal body) and Smo (green). In contrast to the relatively uniform Smo staining on the cilium induced by ShhN, CTX treatment causes Smo accumulation proximal to the basal body. (D) 8xGliBS-luciferase Hh reporter activity in wild-type MEFs with the indicated treatment. CTX and FSK do not activate the Hh pathway despite inducing ciliary localization of Smo. Data are representative of three independent experiments. Hh reporter activity was normalized to β-galactosidase activity produced from a constitutive hsp68-lacZ reporter, as alteration of PKA affected Renilla luciferase activity (data not shown). Error bars indicate +/− SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664476&req=5

pone-0005182-g003: Modulation of Gαs and protein kinase A (PKA) causes Smo accumulation in a proximal region of the primary cilium.(A) Treatment of wild-type MEFs with cholera toxin (CTX) for 24 hours induces Smo (green) translocation to the cilium (red). (B) Quantification of CTX- and FSK-induced Smo ciliary translocation after treatment for the indicated times. Treatment of wild-type MEFs with pertussis toxin (PTX) does not stimulate ciliary translocation of Smo. Error bars indicate +/− SD. (C) Wild-type MEFs were fixed in methanol and stained with antibodies against γ-tubulin (red; label the basal body) and Smo (green). In contrast to the relatively uniform Smo staining on the cilium induced by ShhN, CTX treatment causes Smo accumulation proximal to the basal body. (D) 8xGliBS-luciferase Hh reporter activity in wild-type MEFs with the indicated treatment. CTX and FSK do not activate the Hh pathway despite inducing ciliary localization of Smo. Data are representative of three independent experiments. Hh reporter activity was normalized to β-galactosidase activity produced from a constitutive hsp68-lacZ reporter, as alteration of PKA affected Renilla luciferase activity (data not shown). Error bars indicate +/− SD.
Mentions: (A) Wild-type mouse embryonic fibroblasts (MEFs) stained with antibodies against endogenous Smo (green), acetylated (AC) tubulin (red), and DAPI (blue). MEFs treated with ShhN-conditioned media (ShhN-CM), purmorphamine, cyclopamine, and jervine display Smo staining along the entire length of the cilium. Note that fixation with paraformaldehyde produces artifactual nuclear staining with the Smo antibody; this nuclear staining is not visible after fixation in methanol (See Fig. 3C). (B) Quantification of the percentage of Smo-positive (Smo+) cilia in MEFs after treatment with ShhN CM and cyclopamine for the indicated times. Cyclopamine induces Smo translocation to a significant number of cilia after 6 hours of treatment, although this is less than that induced by ShhN CM. All quantification of cilia staining in this and subsequent figures represents at least two separate experiments with a minimum of 100 cilia scored per time point and condition. Error bars indicate +/− standard deviation (SD). (C) Quantification of Smo+ cilia in MEFs after treatment with indicated compounds for 24 hours. 20-α-hydroxysterol (20α-OHC), purmorphamine, cyclopamine, and jervine induce Smo translocation to the cilium. Error bars indicate +/− SD.

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