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Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling.

ABSTRACT: Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that the Smo CRD may bind its own endogenous ligand. Here we present the NMR solution structure of the Drosophila Smo CRD, and describe interactions between the glucocorticoid budesonide (Bud) and the Smo CRDs from both Drosophila and human. Our results highlight a function of the Smo CRD, demonstrating its role in binding to small-molecule modulators.

SUBMITTER: Rana R

PROVIDER: S-EPMC3890372 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling.

Rana Rajashree R Carroll Candace E CE Lee Ho-Jin HJ Bao Ju J Marada Suresh S Grace Christy R R CR Guibao Cristina D CD Ogden Stacey K SK Zheng Jie J JJ

Nature communications 20130101

Smoothened (Smo) is a member of the Frizzled (FzD) class of G-protein-coupled receptors (GPCRs), and functions as the key transducer in the Hedgehog (Hh) signalling pathway. Smo has an extracellular cysteine-rich domain (CRD), indispensable for its function and downstream Hh signalling. Despite its essential role, the functional contribution of the CRD to Smo signalling has not been clearly elucidated. However, given that the FzD CRD binds to the endogenous Wnt ligand, it has been proposed that ...[more]

PMID: 24351982

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An essential role for Grk2 in Hedgehog signalling downstream of Smoothened.

Project description:The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.

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Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain.

Project description:Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.

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E3 ligase Herc4 regulates Hedgehog signalling through promoting Smoothened degradation.

Project description:Hedgehog (Hh) signalling plays conserved roles in controlling embryonic development; its dysregulation causes many diseases including cancers. The G protein-coupled receptor Smoothened (Smo) is the key signal transducer of the Hh pathway, whose posttranslational regulation has been shown to be critical for its accumulation and activation. Ubiquitination has been reported an essential posttranslational regulation of Smo. Here, we identify a novel E3 ligase of Smo, Herc4, which binds to Smo, and regulates Hh signalling by controlling Smo ubiquitination and degradation. Interestingly, our data suggest that Herc4-mediated Smo degradation is regulated by Hh in PKA-primed phosphorylation-dependent and independent manners.

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The cation-π interaction in cysteine-rich domain of Smoothened is critical for its cholesterylation and function.

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Structural Basis of Smoothened Activation in Hedgehog Signaling.

Project description:The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.

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G protein Galphai functions immediately downstream of Smoothened in Hedgehog signalling.

Project description:The hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer. Hh pathway activity is absolutely dependent on signalling by the seven-transmembrane protein smoothened (Smo), which is regulated by the Hh receptor patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci). In the absence of Hh, this complex regulates the cleavage of full-length Ci to a truncated repressor protein, Ci75, in a process that is dependent on the proteasome and priming phosphorylations by Protein kinase A (PKA). Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G-protein-coupled receptors (GPCR), a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role that G proteins may have in Hh signal transduction is unclear and quite controversial, which has led to widespread speculation that Smo signals through a variety of novel G-protein-independent mechanisms. Here we present in vitro and in vivo evidence in Drosophila that Smo activates a G protein to modulate intracellular cyclic AMP levels in response to Hh. Our results demonstrate that Smo functions as a canonical GPCR, which signals through Galphai to regulate Hh pathway activation.

| S-EPMC2744466 | biostudies-literature

Structural insights into human Kif7, a kinesin involved in Hedgehog signalling.

Project description:Kif7, a member of the kinesin 4 superfamily, is implicated in a variety of diseases including Joubert, hydrolethalus and acrocallosal syndromes. It is also involved in primary cilium formation and the Hedgehog signalling pathway and may play a role in cancer. Its activity is crucial for embryonic development. Kif7 and Kif27, a closely related kinesin in the same subfamily, are orthologues of the Drosophila melanogaster kinesin-like protein Costal-2 (Cos2). In vertebrates, they work together to fulfil the role of the single Cos2 gene in Drosophila. Here, the high-resolution structure of the human Kif7 motor domain is reported and is compared with that of conventional kinesin, the founding member of the kinesin superfamily. These data are a first step towards structural characterization of a kinesin-4 family member and of this interesting molecular motor of medical significance.

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Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling.

Project description:Oxysterols bind the seven-transmembrane protein Smo (Smo) and potently activate vertebrate Hedgehog (Hh) signaling, a pathway essential in embryonic development, adult stem cell maintenance and cancer. It is unknown, however, whether oxysterols are important for normal vertebrate Hh signaling and whether antagonizing oxysterols can inhibit the Hh pathway. We developed azasterols that block Hh signaling by binding the oxysterol-binding site of Smo. We show that the binding site for oxysterols and azasterols maps to the extracellular, cysteine-rich domain of Smo and is completely separable from the site bound by other small-molecule modulators, located within the heptahelical bundle of Smo. Smo mutants in which oxysterol binding is abolished no longer respond to oxysterols and cannot be maximally activated by the Hh ligand. Our results show that oxysterol binding to vertebrate Smo is required for normal Hh signaling and that targeting the oxysterol-binding site is an effective strategy to inhibit Smo.

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Interaction of smoothened with integrin-linked kinase in primary cilia mediates Hedgehog signalling.

Project description:Here we report that ILK localizes in the mouse primary cilium, a sensory organelle required for signalling by the Hedgehog (Hh) pathway. Genetic or pharmacological inhibition of ILK blocks ciliary accumulation of the Hh pathway effector smoothened (Smo) and suppresses the induction of Gli transcription factor mRNAs by SHh. Conditional deletion of ILK or Smo also inhibits SHh-driven activation of Gli2 in the embryonic mouse cerebellum. ILK regulation of Hh signalling probably requires the physical interaction of ILK and Smo in the cilium, and we also show selective cilia-associated interaction of ILK with ?-arrestin, a known mediator of Smo-dependent signalling.

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Role of lipid metabolism in smoothened derepression in hedgehog signaling.

Project description:The binding of Hedgehog (Hh) to its receptor Patched causes derepression of Smoothened (Smo), resulting in the activation of the Hh pathway. Here, we show that Smo activation is dependent on the levels of the phospholipid phosphatidylinositol-4 phosphate (PI4P). Loss of STT4 kinase, which is required for the generation of PI4P, exhibits hh loss-of-function phenotypes, whereas loss of Sac1 phosphatase, which is required for the degradation of PI4P, results in hh gain-of-function phenotypes in multiple settings during Drosophila development. Furthermore, loss of Ptc function, which results in the activation of Hh pathway, also causes an increase in PI4P levels. Sac1 functions downstream of STT4 and Ptc in the regulation of Smo membrane localization and Hh pathway activation. Taken together, our results suggest a model in which Ptc directly or indirectly functions to suppress the accumulation of PI4P. Binding of Hh to Ptc derepresses the levels of PI4P, which, in turn, promotes Smo activation.

| S-EPMC2945252 | biostudies-literature

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