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

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

Functional divergence in the role of N-linked glycosylation in smoothened signaling

The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice

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

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

Identification of Smo N-linked glycosylation sites.

<p><b>A</b>. A multiple sequence alignment of Smo proteins from different phyla are shown. Consensus sequences for N-linked glycosylation are highlighted in gray and the Asn acceptor residues are bold. Sites conserved across vertebrate proteins are indicated as N1-N4. The predicted <i>D</i>. <i>melanogaster</i> sites are not tightly conserved with vertebrates. <b>B</b>. Drosophila Smo is N-glycosylated. Cell lysates prepared from Cl8 cells expressing Hh with wild type or NQ5 dSmo proteins were treated with the indicated deglycosylating enzymes. Wild type Smo demonstrated ER (arrow) and post-ER (arrowhead) glycosylation species. NQ5 migrated similarly to the fully deglycosylated species under all conditions (arrow). <b>C</b>. Mouse Smo is N-glycosylated. Cellular lysates from <i>Smo-/-</i> cells stably expressing mSmoWT or mSmoNQ4 were treated with deglycosylating enzymes and subjected to SDS-PAGE and western blot. mSmoWT resolves as two distinct forms (lane 2). The arrow marks the ER form and the arrowhead indicates the post-ER form. mSmoNQ4 migrates in SDS-PAGE similarly to the PNGase-treated wild type protein (lanes 4–5). <b>C’</b>. mSmoNQ4 is O-glycosylated. Lysates were prepared from NIH3T3 cells expressing mSmoNQ4 and subjected to lambda phosphatase, PNGase and O-glycosidase/neuraminidase treatments. The upper band collapsed upon O-glycosidase/neuraminidase treatment. <b>D</b>. Expression of individual N to Q dSmo mutants. The indicated N to Q dSmo mutants were expressed in Cl8 cells and cell lysates were analyzed by SDS-PAGE and western blot against the Myc tag. Kin serves as loading control. <b>E</b>. Extracellular mSmo consensus sites are N-glycosylated. Mutation of individual extracellular mSmo glycosylation sites induced faster mobility on SDS-PAGE. mSmoN450Q migrated similarly to SmoWT. For western blots, mSmo was detected using anti-Smo and dSmo with anti-Myc. Kinesin (Kin) and Tubulin (Tub) were blotted for loading controls.</p

N-glycosylation status correlates with signal bias.

<p>A model for modulation of mSmo signal bias. SAG (yellow) binding to N-glycosylated mSmo stabilizes a conformation that effectively signals through both canonical and non-canonical routes. SAG binding to the mSmo mutant stripped of N-glycans (red) silences non-canonical signaling, but is highly permissive for canonical signaling.</p

N336Q impacts dSmo trafficking and activity.

<p><b>A.</b> N336 possesses an essential glycan modification. The rescue reporter assay was performed in <i>smo</i> knockdown Cl8 cells as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2</a>. Each of the single N to Q mutants was able to rescue reporter gene activity in the <i>smo</i> knockdown background with the exception of N336Q. This mutant rescued to ~50% of the wild type activity. <b>B.</b> dSmoN336Q is mislocalized. Cl8 cells expressing Hh and the indicated dSmo proteins were imaged by immunofluorescence microscopy. Whereas each of the active single glycosylation site N to Q mutants (anti-Myc, magenta) reached the plasma membrane (indicated by F-actin stain, green) in Hh-expressing cells, dSmoN336Q failed to do so. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). <b>C.</b> SmoN336Q is retained in the ER. Myc-SmoN336Q expressed in Cl8 cells overlaps with the ER marker protein Calreticulin-KDEL-GFP. Scale bar is 5 μm (upper right). <b>D.</b> Treatment with deglycosylating enzymes demonstrates that the majority of dSmoN336Q is present in the EndoH sensitive, ER resident fraction (black arrowhead). WT and N213Q proteins are equally distributed between ER and post-ER fractions (white arrowhead).</p