Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates

Current Opinion in Cell Biology

Volumn 51, Issue , 2018, Pages 81-88

  Byrne, Eamon FX ^a   Luchetti, Giovanni ^b   Rohatgi, Rajat ^b   Siebold, Christian ^a  

^a UNIVERSITY OF OXFORD   (United Kingdom)

^b STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHOLESTEROL; SMOOTHENED PROTEIN; SONIC HEDGEHOG PROTEIN; DROSOPHILA PROTEIN; LIGAND;

ALLOSTERISM; CELL MEMBRANE; HEDGEHOG SIGNALING; LIGAND BINDING; NONHUMAN; PRIORITY JOURNAL; PROTEIN STRUCTURE; REVIEW; SIGNAL TRANSDUCTION; VERTEBRATE; ANIMAL; BINDING SITE; DROSOPHILA MELANOGASTER; HUMAN; METABOLISM;

ANIMALS; BINDING SITES; DROSOPHILA MELANOGASTER; DROSOPHILA PROTEINS; HEDGEHOG PROTEINS; HUMANS; LIGANDS; SIGNAL TRANSDUCTION; VERTEBRATES;

EID: 85038259669     PISSN: 09550674     EISSN: 18790410     Source Type: Journal    
DOI: 10.1016/j.ceb.2017.10.004     Document Type: Review

References (54)

1. Ingham, P.W., Nakano, Y., Seger, C., Mechanisms and functions of Hedgehog signalling across the metazoa. Nat Rev Genet 12 (2011), 393–406.
2. Briscoe, J., Therond, P.P., The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol 14 (2013), 416–429.
3. Wu, F., Zhang, Y., Sun, B., McMahon, A.P., Wang, Y., Hedgehog signaling: from basic biology to cancer therapy. Cell Chem Biol 24 (2017), 252–280.
4. Beachy, P.A., Hymowitz, S.G., Lazarus, R.A., Leahy, D.J., Siebold, C., Interactions between Hedgehog proteins and their binding partners come into view. Genes Dev 24 (2010), 2001–2012.
5. Allen, B.L., Song, J.Y., Izzi, L., Althaus, I.W., Kang, J.S., Charron, F., Krauss, R.S., McMahon, A.P., Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function. Dev Cell 20 (2011), 775–787.
6. Izzi, L., Levesque, M., Morin, S., Laniel, D., Wilkes, B.C., Mille, F., Krauss, R.S., McMahon, A.P., Allen, B.L., Charron, F., Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev Cell 20 (2011), 788–801.
7. Taipale, J., Cooper, M.K., Maiti, T., Beachy, P.A., Patched acts catalytically to suppress the activity of Smoothened. Nature 418 (2002), 892–897.
8. Denef, N., Neubuser, D., Perez, L., Cohen, S.M., Hedgehog induces opposite changes in turnover and subcellular localization of patched and Smoothened. Cell 102 (2000), 521–531.
9. Ingham, P.W., Nystedt, S., Nakano, Y., Brown, W., Stark, D., van den Heuvel, M., Taylor, A.M., Patched represses the Hedgehog signalling pathway by promoting modification of the Smoothened protein. Curr Biol 10 (2000), 1315–1318.
10. Rubin, L.L., de Sauvage, F.J., Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 5 (2006), 1026–1033.
11. Bazan, J.F., de Sauvage, F.J., Structural ties between cholesterol transport and morphogen signaling. Cell 138 (2009), 1055–1056.
12. Wang, C., Wu, H., Katritch, V., Han, G.W., Huang, X.P., Liu, W., Siu, F.Y., Roth, B.L., Cherezov, V., Stevens, R.C., Structure of the human Smoothened receptor bound to an antitumour agent. Nature 497 (2013), 338–343.
13. Nachtergaele, S., Whalen, D.M., Mydock, L.K., Zhao, Z., Malinauskas, T., Krishnan, K., Ingham, P.W., Covey, D.F., Siebold, C., Rohatgi, R., Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. Elife, 2, 2013, e01340.
14. Rana, R., Carroll, C.E., Lee, H.J., Bao, J., Marada, S., Grace, C.R., Guibao, C.D., Ogden, S.K., Zheng, J.J., Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling. Nat Commun, 4, 2013, 2965.
15. Sharpe, H.J., Wang, W., Hannoush, R.N., de Sauvage, F.J., Regulation of the oncoprotein Smoothened by small molecules. Nat Chem Biol 11 (2015), 246–255.
16. Weierstall, U., James, D., Wang, C., White, T.A., Wang, D., Liu, W., Spence, J.C., Bruce Doak, R., Nelson, G., Fromme, P., et al. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat Commun, 5, 2014, 3309.
17. Byrne, E.F., Sircar, R., Miller, P.S., Hedger, G., Luchetti, G., Nachtergaele, S., Tully, M.D., Mydock-McGrane, L., Covey, D.F., Rambo, R.P., et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535 (2016), 517–522 This study describes multi-domain structures of SMO, which represent the first high-resolution views of a G protein coupled receptor with a large extracellular domain. These structures, with accompanying functional analyses, demonstrated the interaction of cholesterol with the SMO extracellular domain, revealed how structural communication between the extracellular and transmembrane domains can regulate signalling, and provided a structural explanation for SMO mutations that cause resistance to a clinically-used anti-cancer drug.
18. Zhang, X., Zhao, F., Wu, Y., Yang, J., Han, G.W., Zhao, S., Ishchenko, A., Ye, L., Lin, X., Ding, K., et al. Crystal structure of a multi-domain human Smoothened receptor in complex with a super stabilizing ligand. Nat Commun, 8, 2017, 15383 The authors presented the crystal structure of a multi-domain human SMO protein, bound and stabilized by the designed ligand TC114. By combining structural analysis with molecular dynamics simulations and deuterium exchange experiments, they identified key structural elements in SMO — the TMD helix VI and the ECL3 loop — that are important for communication between the extracellular and transmembrane domains.
19. Wang, C., Wu, H., Evron, T., Vardy, E., Han, G.W., Huang, X.P., Hufeisen, S.J., Mangano, T.J., Urban, D.J., Katritch, V., et al. Structural basis for Smoothened receptor modulation and chemoresistance to anticancer drugs. Nat Commun, 5, 2014, 4355.
20. Chen, J.K., Taipale, J., Cooper, M.K., Beachy, P.A., Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16 (2002), 2743–2748.
21. Chen, J.K., Taipale, J., Young, K.E., Maiti, T., Beachy, P.A., Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A 99 (2002), 14071–14076.
22. Frank-Kamenetsky, M., Zhang, X.M., Bottega, S., Guicherit, O., Wichterle, H., Dudek, H., Bumcrot, D., Wang, F.Y., Jones, S., Shulok, J., et al. Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists. J Biol, 1, 2002, 10.
23. Robarge, K.D., Brunton, S.A., Castanedo, G.M., Cui, Y., Dina, M.S., Goldsmith, R., Gould, S.E., Guichert, O., Gunzner, J.L., Halladay, J., et al. GDC-0449 — a potent inhibitor of the hedgehog pathway. Bioorg Med Chem Lett 19 (2009), 5576–5581.
24. Pan, S., Wu, X., Jiang, J., Gao, W., Wan, Y., Cheng, D., Han, D., Liu, J., Englund, N.P., Wang, Y., et al. Discovery of NVP-LDE225, a potent and selective Smoothened antagonist. ACS Med Chem Lett 1 (2010), 130–134.
25. Khaliullina, H., Bilgin, M., Sampaio, J.L., Shevchenko, A., Eaton, S., Endocannabinoids are conserved inhibitors of the Hedgehog pathway. Proc Natl Acad Sci U S A 112 (2015), 3415–3420.
26. Arensdorf, A.M., Dillard, M.E., Menke, J.M., Frank, M.W., Rock, C.O., Ogden, S.K., Sonic Hedgehog activates phospholipase A2 to enhance Smoothened ciliary translocation. Cell Rep 19 (2017), 2074–2087.
27. Wang, Y., Davidow, L., Arvanites, A.C., Blanchard, J., Lam, K., Xu, K., Oza, V., Yoo, J.W., Ng, J.M., Curran, T., et al. Glucocorticoid compounds modify Smoothened localization and hedgehog pathway activity. Chem Biol 19 (2012), 972–982.
28. Nachtergaele, S., Mydock, L.K., Krishnan, K., Rammohan, J., Schlesinger, P.H., Covey, D.F., Rohatgi, R., Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8 (2012), 211–220.
29. Myers, B.R., Sever, N., Chong, Y.C., Kim, J., Belani, J.D., Rychnovsky, S., Bazan, J.F., Beachy, P.A., Hedgehog pathway modulation by multiple lipid binding sites on the Smoothened effector of signal response. Dev Cell 26 (2013), 346–357.
30. Nedelcu, D., Liu, J., Xu, Y., Jao, C., Salic, A., Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling. Nat Chem Biol 9 (2013), 557–564.
31. Huang, P., Nedelcu, D., Watanabe, M., Jao, C., Kim, Y., Liu, J., Salic, A., Cellular cholesterol directly activates Smoothened in hedgehog signaling. Cell 166 (2016), 1176–1187.e1114.
32. Luchetti, G., Sircar, R., Kong, J.H., Nachtergaele, S., Sagner, A., Byrne, E.F., Covey, D.F., Siebold, C., Rohatgi, R., Cholesterol activates the G-protein coupled receptor Smoothened to promote morphogenetic signaling. Elife, 2016, 5 Both studies from Huang et al. and Luchetti et al. showed that cholesterol is sufficient to activate Hh signalling in fibroblasts and neural precursor cells in the absence of native Hh ligands. Mutations in the SMO cysteine-rich domain (CRD) that prevent cholesterol binding also prevent signalling by native Hh ligands, showing that the CRD-cholesterol interaction is important for endogenous signaling. Using mutagenesis, both studies provided evidence that the endogenous CRD ligand is likely to be cholesterol, not oxysterols as previously proposed. Huang et al. also presented high-resolution crystal structures of the isolated Xenopus SMO CRD in complex with an activating oxysterol and with cyclopamine, a SMO inhibitor that binds to both the CRD and TMD sites.
33. Xiao, X., Tang, J.J., Peng, C., Wang, Y., Fu, L., Qiu, Z.P., Xiong, Y., Yang, L.F., Cui, H.W., He, X.L., et al. Cholesterol modification of Smoothened is required for Hedgehog signaling. Mol Cell, 66, 2017 154–162.e110 The authors presented the unexpected observation that cholesterol can form a covalent ester bond between its 3′-hydroxyl and Asp95 in the sterol-binding groove of the SMO CRD and that PTC1 can inhibit the formation of this SMO-cholesterol adduct. An Asp95N mutation impairs embryonic development in a mouse, pheno-copying a null allele of SMO and highlighting the importance of the CRD sterol-binding groove in the context of an intact animal.
34. Yavari, A., Nagaraj, R., Owusu-Ansah, E., Folick, A., Ngo, K., Hillman, T., Call, G., Rohatgi, R., Scott, M.P., Banerjee, U., Role of lipid metabolism in Smoothened derepression in hedgehog signaling. Dev Cell 19 (2010), 54–65.
35. Jiang, K., Liu, Y., Fan, J., Zhang, J., Li, X.A., Evers, B.M., Zhu, H., Jia, J., PI(4)p promotes phosphorylation and conformational change of Smoothened through interaction with its C-terminal tail. PLoS Biol, 14, 2016, e1002375.
36. Dijkgraaf, G.J., Alicke, B., Weinmann, L., Januario, T., West, K., Modrusan, Z., Burdick, D., Goldsmith, R., Robarge, K., Sutherlin, D., et al. Small molecule inhibition of GDC-0449 refractory Smoothened mutants and downstream mechanisms of drug resistance. Cancer Res 71 (2011), 435–444.
37. Yauch, R.L., Dijkgraaf, G.J., Alicke, B., Januario, T., Ahn, C.P., Holcomb, T., Pujara, K., Stinson, J., Callahan, C.A., Tang, T., et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326 (2009), 572–574.
38. Kim, J., Tang, J.Y., Gong, R., Kim, J., Lee, J.J., Clemons, K.V., Chong, C.R., Chang, K.S., Fereshteh, M., Gardner, D., et al. Itraconazole, a commonly used antifungal that inhibits Hedgehog pathway activity and cancer growth. Cancer Cell 17 (2010), 388–399.
39. Cooper, M.K., Porter, J.A., Young, K.E., Beachy, P.A., Teratogen-mediated inhibition of target tissue response to Shh signaling. Science 280 (1998), 1603–1607.
40. Cooper, M.K., Wassif, C.A., Krakowiak, P.A., Taipale, J., Gong, R., Kelley, R.I., Porter, F.D., Beachy, P.A., A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat Genet 33 (2003), 508–513.
41. Incardona, J.P., Gaffield, W., Kapur, R.P., Roelink, H., The teratogenic Veratrum alkaloid cyclopamine inhibits Sonic hedgehog signal transduction. Development 125 (1998), 3553–3562.
42. Incardona, J.P., Gaffield, W., Lange, Y., Cooney, A., Pentchev, P.G., Liu, S., Watson, J.A., Kapur, R.P., Roelink, H., Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesterol transport. Dev Biol 224 (2000), 440–452.
43. Kelley, R.L., Roessler, E., Hennekam, R.C., Feldman, G.L., Kosaki, K., Jones, M.C., Palumbos, J.C., Muenke, M., Holoprosencephaly in RSH/Smith-Lemli-Opitz syndrome: does abnormal cholesterol metabolism affect the function of Sonic Hedgehog?. Am J Med Genet 66 (1996), 478–484.
44. Lanoue, L., Dehart, D.B., Hinsdale, M.E., Maeda, N., Tint, G.S., Sulik, K.K., Limb, genital, CNS, and facial malformations result from gene/environment-induced cholesterol deficiency: further evidence for a link to sonic hedgehog. Am J Med Genet 73 (1997), 24–31.
45. Sever, N., Mann, R.K., Xu, L., Snell, W.J., Hernandez-Lara, C.I., Porter, N.A., Beachy, P.A., Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols. Proc Natl Acad Sci U S A 113 (2016), 5904–5909.
46. Blassberg, R., Macrae, J.I., Briscoe, J., Jacob, J., Reduced cholesterol levels impair Smoothened activation in Smith-Lemli-Opitz syndrome. Hum Mol Genet, 2015 By studying an in vitro model for the autosomal-recessive disorder Smith–Lemli–Opitz syndrome (SLOS), the authors showed that a deficit in cholesterol, rather than the accumulation of an inhibitor precursor sterol, is likely to impair SMO activation and its localization to the primary cilium in this disease. This study suggested that endogenous cholesterol levels or distribution may regulate SMO activation in cells.
47. Cunningham, D., DeBarber, A.E., Bir, N., Binkley, L., Merkens, L.S., Steiner, R.D., Herman, G.E., Analysis of hedgehog signaling in cerebellar granule cell precursors in a conditional Nsdhl allele demonstrates an essential role for cholesterol in postnatal CNS development. Hum Mol Genet 24 (2015), 2808–2825.
48. Gimpl, G., Interaction of G protein coupled receptors and cholesterol. Chem Phys Lipids 199 (2016), 61–73.
49. Carstea, E.D., Morris, J.A., Coleman, K.G., Loftus, S.K., Zhang, D., Cummings, C., Gu, J., Rosenfeld, M.A., Pavan, W.J., Krizman, D.B., et al. Niemann–Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277 (1997), 228–231.
50. Bidet, M., Joubert, O., Lacombe, B., Ciantar, M., Nehme, R., Mollat, P., Bretillon, L., Faure, H., Bittman, R., Ruat, M., et al. The hedgehog receptor patched is involved in cholesterol transport. PLoS ONE, 6, 2011, e23834.
51. Das, A., Brown, M.S., Anderson, D.D., Goldstein, J.L., Radhakrishnan, A., Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis. Elife, 2014, 3.
52. Mukhopadhyay, S., Wen, X., Ratti, N., Loktev, A., Rangell, L., Scales, S.J., Jackson, P.K., The ciliary G-protein-coupled receptor Gpr161 negatively regulates the Sonic hedgehog pathway via cAMP signaling. Cell 152 (2013), 210–223.
53. DeCamp, D.L., Thompson, T.M., de Sauvage, F.J., Lerner, M.R., Smoothened activates Galphai-mediated signaling in frog melanophores. J Biol Chem 275 (2000), 26322–26327.
54. Riobo, N.A., Saucy, B., Dilizio, C., Manning, D.R., Activation of heterotrimeric G proteins by Smoothened. Proc Natl Acad Sci U S A 103 (2006), 12607–12612.