Dataset for phenotypic classification of genetic modifiers of smoothened and Hedgehog

AbstractThis data article includes supporting information for the research article entitled “The Small GTPase Rap1 is a Modulator of Hedgehog Signaling” [1]. Drosophila wing phenotypes induced by expression of a dominant negative Smoothened (Smo) mutant were cataloged into five distinct classes. Class distributions observed following expression of dominant negative Smo in control and sensitized backgrounds were quantified to serve as references for strength of phenotypic modification. Shifts in class distribution of Hedgehog (Hh) wing phenotypes resulting from introduction of loss-of-function alleles of select Ras family G protein genes and the Hh pathway regulators Fused and Suppressor of Fused are shown

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

N213 glycosylation partially compensates for N336 N-glycan loss.

<p><b>A.</b> The indicated mutants were expressed in Cl8 cells. Each of the mutant proteins migrated more quickly in SDS-PAGE than wild type Smo, but not as quickly as SmoNQ4 or NQ5. <b>B.</b> Glycosylation at N213 partially compensates for N336 glycan loss. The rescue reporter assay was performed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2A</a>. Each of the indicated double mutants, with the exception of N213Q,N336Q, was able to rescue reporter gene expression in the <i>smo</i> knockdown background to a level similar to that of N336Q. dSmoN213Q,N336Q demonstrated a level of activity similar to dSmoNQ5. Significance was determined using Student’s t-test. <b>C.</b> N336Q-containing double mutants are retained in the ER. Cl8 cells expressing the indicated Smo proteins (anti-Myc, magenta), the Cal-EGFP-KDEL marker (green) and Hh were examined by immunofluorescence microscopy. Whereas wild type Smo reached the plasma membrane, the double mutants overlapped with the ER marker. ActinRed (red) marks F-actin. DAPI (blue) marks the nucleus. Scale bar is 5 μm (upper right). <b>D.</b> Treatment of lysates from WT or N213Q,N336Q expressing cells with deglycosylating enzymes reveals that the N213,336Q mutant is present in the EndoH sensitive ER fraction, arrowhead. <b>E-E’</b>. N336Q and N213Q,N336Q mutants have disulfide bond defects. Biotin-maleimide was used to tag free thiol groups in cellular lysates prepared from Cl8 cells expressing WT, N336Q, N213Q,N336Q and C320A dSmo proteins. WT dSmo is not well captured on NeutrAvadin beads (lane 2, bound). N-glycan mutants are captured similarly to the disulfide bond mutant C320A (lanes 3–5, bound), indicating that at least one disulfide bridge is disrupted by N-glycan loss. E’ shows the ratio of bound to unbound dSmo proteins normalized to kinesin. Relative binding was determined by densitometry analysis of two independent binding assays. C320A, which has an established disulfide bond defect served as positive control. It’s binding ratio was arbitrarily set to 1.0 and other values are shown relative to it. Error bars are provided to show the standard deviation between the two experiments. <b>F.</b> dSmoN213Q,N336Q fails to rescue <i>smo</i> knockdown <i>in vivo</i>. <i>UAS-dsmoN213Q</i>, <i>N336Q</i> was co-expressed with <i>UAS-dicer</i> and <i>UAS-smo</i>^{<i>3’UTR</i>} using the <i>nubbin-Gal4</i> driver. Its expression did not modify the <i>smo</i> knockdown phenotype (compare to <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005473#pgen.1005473.g002" target="_blank">Fig 2F</a>). Multiple progeny were analyzed over two crosses and a representative wing is shown.</p