Smoothened

Symbol: smo	Flybase ID: FBgn0003444
Synonyms: {Name}	GadFly
Function: 7 pass transmembran protein involved in Hh signaling	LocusLink
Keywords: {Keywords}	Interactive_Fly

{Summary}

{Function/Pathway}

Smo acts downstream of hh and ptc (Alcedo, 1996)
Hh mut & PKA mut had ptc RNA; Smo mut & PKA mut had no ptc RNA -> suggest that smo has a significant basal activity in the absence of Hh (Ohlmeyer, 1997)
Little evidence implicating heterotrimeric G protein:
- In a classic assay for Galphai activation, expression of human Smo in Xenopus melanophores appears sufficient to stimulate persistent pigment aggregation in these cells, an effect that can be blocked by pertussis toxin (DeCamp et al. 2000).
- In a primary fish myoblast assay system, the effects of Shh were found to be insensitive to pertussis toxin (Norris et al. 2000), and the results of treating zebrafish embryos with the toxin are inconclusive (Hammerschmidt and McMahon 1998).
- Notably, there is to date no report of a G-protein mutation that disrupts Hh signaling in Drosophila, despite several such mutations having been isolated (e.g., Wolfgang et al. 2001).
- The M2 mutation of human Smo, isolated from a spontaneously arising basal cell carcinoma, creates a constitutively active form of Smo. This results in an amino acid substitution in the seventh transmembrane domain predicted to disrupt G-protein coupling (Xie. 1998).
- A loss-of-function mutation in Drosophila Smo removes a charged residue in the third intracellular loop; similar mutations in GPCRs have been found to abolish G-protein coupling (S. Nystedt, H. Strutt, and P.W. Ingham, in prep.).
Genetic screens in mice identified a mutation, open-brain (opb), that shows a phenotype opposite to that caused by loss of Shh activity (Gunther et al. 1994; Eggenschwiler and Anderson 2000). Significantly, floor plate and motor neurons, cell types that are absent in Shh mutants (Chiang et al. 1996), differentiate in embryos mutant for both opb and Shh, suggesting that the opb gene product acts downstream of Shh as a negative regulator of the pathway. Nevertheless, expression of some Shh target genes remains sensitive to Shh activity even in the absence of opb function, indicating that loss of opb does not result in complete derepression of the pathway (Eggenschwiler et al. 2001). Cloning of opb has revealed that it encodes RAB23, a member of a large family of small GTP-activated proteins associated with many dynamic aspects of membrane trafficking (Eggenschwiler et al. 2001). Ptc1 could negatively regulate Smo activity through a RAB23-dependent trafficking process.
smo is required for the response of cells to Hh signaling for embryo and imaginal discs (van den Heuvel, 1996)
activation of Smo might modulate PKA activity (Alcedo, 1996), there is good evidence that Hh signaling has no effect on PKA activity (Jiang, 1995). Therefore,it seems that phosphorylation by PKA is permissive for Ci cleavage, the rate-limiting step being recruitment of Ci to the microtubules. However, Hh might also contribute to cleavage regulation by promoting the dephosphorylation
of Ci.

Coimmunoprecipitates w/ ptc—unaffected by H
Co-IP studies of vertebrate family members suggest that Ptc and Smo interact directly to form a receptor complex that remains associated after ligand binding (Stone, 1996; Carpenter, 1998; Murone, 1999)
Evidence of IP Ptc/Smo complexes in Drosophila tissues is lacking (Johnson, 2000)
One model, based largely on analysis of the properties of the proteins when overexpressed in tissue culture cells (Stone, 1996; Murone et al. 1999), suggests that Smo and Ptc interact directly to form a membrane-associated receptor complex. The Smo present in this complex is postulated to be inactive in unstimulated cells, but, upon Hh binding to Ptc, the complex undergoes some conformational change that results in the activation of Smo.
It is notable, however, that in Drosophila, visualization of the two proteins suggests that most Smo does not colocalize with Ptc, at least in cells responding to Hh (Denef, 2000). Moreover, biochemical investigation of the postulated physical interaction between the two proteins in vivo has so far proved negative (Johnson et al. 2000).

Smo is dephosphyorylated by a type 2A protein phosphatase (Denef, 2000)
Smo protein levels are upregulated posttranscriptionally by a Hh signalling dependent mechanism. Inhibition of PKA, leads to upregulation of Smo above just Hh signaling (Alcedo. 2000)
Hh signaling upregulates Smo levels, which are otherwise down regulated by Ptc (Alcedo. 2000)
"Thus, we propose that Hh and its Ptc-Smo receptor have developed the properties of a self-correcting system in which the Hh signal adjusts the concentration of its receptor to its own concentration" (Alcedo. 2000)
"Hence, it is crucial that Smo signaling strictly depends on the presence of Hh and that, in the absence of Hh, constitutive Smo signaling is restricted by Ptc below a threshold necessary for the transcriptional control of Hh target genes" (Alcedo. 2000)
"When Hh levels decrease, Smo is destabi- lized because of the inhibition of Smo signaling by Ptc. The concentration of Smo will be reduced more rapidly than that of Ptc, which continues to be translated from a decreasing concentration of its mRNA, and eventually Smo will reach a reduced steady-state concentration, which is lowest in regions where Hh is absent. When the Ptc concentration falls below a threshold, Smo signaling begins to inhibit its own degradation and to activate transcription of ptc, whose product suppresses Smo signaling and thus again downregulates itself and Smo. Hence, a new steady state is reached at which the levels of Ptc and Smo are reduced to a level corresponding to the low Hh concentration. The sequence of events are expected to be reversed, if the Hh concentration is again increased. Thus, the Hh signaling pathway has the properties of a self-correcting system, since an imbalance between Ptc and Smo or between Hh and the Ptc-Smo receptor is readjusted to equilibrium." (Alcedo. 2000)
Since Smo signals constitutively in the absence of Ptc (Hooper, 1994; Alcedo, 1996), Smo signaling must activate ptc to inhibit its constitutive activity.
"To avoid an imbalance between the two Hh- receptor moieties, Smo signaling must also upregulate Smo. If Smo levels were independent of Smo signaling, Smo would reach a uniformly high level while the concentration of Ptc would oscillate around an equilibrium since Ptc inhibits Smo signaling on which its synthesis depends. However, in this case Smo would signal even in the absence or at low levels of Hh, which is not what we observe (Figures 2C and 2D). Therefore, to ensure that Ptc and Smo reach an equilibrium at which Ptc completely inhibits Smo signaling most rapidly in the absence of Hh, Smo regulates its own breakdown." (Alcedo. 2000)
Genetic and molecular characterization of the smo gene (Alcedo, 1996)
Has structural features of G protein-coupled receptors and is homolgous to the frizzled gene—especially N term (Alcedo, 1996)
C-term contains 5 potential PKA sites, 2nd intercellular loop has a PKA site (Alcedo, 1996)
Smo has constitutive signalling activity (Alcedo, 1996)
Incubation of cells with concanamycin A, a specific vacuolar H⁺/ATPase inhibitor that blocks transport out of early endosomal compartments, protected Ptc1 from degradation

Cloned Smo (van den Heuvel, 1996)
Sequence analysis: seven putative transmembrane domain, typical of G-protein-coupled receptors, suggesting that Smo may act as a receptor for Hh (van den Heuvel, 1996)

Smo may be limited to a more basolateral domain (Denef, 2000)
Expression location in embryos see (Alcedo. 2000)
Smo protein accumulates specifically in cells in which Ptc activity is absent or abrogated by Hh signaling, a process that seems to involve the redistribution of a hyperphosphorylated form of the protein to the cell surface (Denef, 2000) and may also be accompanied by a conformational change (Ingham, 2000)
In KNRK cells in the absence of Ptc1, Smo accumulates at the cell surface and in early endosomal compartments (Incardona, 2002)
Internalization of Smo N-terminal antibodies by live cells resulted in the labeling of structures indistinguishable from those labeled by C-terminal anti-Flag in fixed, permeabilized cells (Figure 2B), suggesting that intracellular Smo is derived by endocytosis (Incardona, 2002)
juxtanuclear Smo showed no colocalization with the TGN marker TGN38 (Figure 3G) and instead colocalized with the transferrin receptor (Figure 2E). Finally, Smo did not colocalize with LBPA⁺ or LAMP-1⁺ late endosomes/lysosomes (Figures 2F and 2G), even in the presence of leupeptin (Figure 2H). (Incardona, 2002)
Treatment of cells with chloroquine showed differences between the distributions of Ptc1 and Smo
- Ptc1 appeared in endosomes marked with fluid-phase tracer within 30–60 min of chloroquine addition (Figures 3B and 3C), prior to the appearance of TGN38 in endosomes. By 90 min, Ptc1 and TGN38 colocalized extensively in endosomes (Figure 3D). The size and number of Ptc1⁺/TGN38⁺ vesicles gradually increased and were maximal between 2 and 3 hr of treatment (Figures 3E and 3F). In contrast, while TGN38 accumulated in endosomes, chloroquine had little effect on the distribution of Smo (Figures 3G–3L). However, after 6 hr in chloroquine, large, ring-shaped Smo⁺ vesicular structures were observed (Figure 4). Therefore, Smo either undergoes much slower internalization than Ptc1 or is sorted from the endocytic pathway at a relatively chloroquine-insensitive step. ShhN had no effect on Smo distribution (data not shown). Shh had no influence on the kinetics with which Ptc1⁺ vesicles appeared in chloroquinetreated cells, and the colocalization of Ptc and Shh in these vesicles was retained (data not shown). These results confirm that Ptc1 reaches endosomes via the cell surface rather than by a direct Golgi-endosome route, consistent with the effects of concanamycin A. (Incardona, 2002)

{Modifications}

Resemblance to G protein-coupled receptor and members of the Frizzled family of serpetine proteins
belongs to the superfamily of G-protein-coupled receptor (GPCR) polytopic membrane-spanning proteins, being most closely related to the Frizzled family of Wnt receptors (Wodarz and Nusse 1998; Dann et al. 2001

mutant clones in the posterior compartment or far from the A/P boundary develop normally (Chen, 1996)
Two activated Smo mutants, SmoM1 and SmoM2, were isolated from human basal cell carcinomas and are resistant to Ptc1 inhibition.
- M1 represents a change of Arg562 to Gln in the cytoplasmic tail and was 50% inhibited by Ptc1 coexpression.
  - SmoM1 showed a distribution that appeared to be a combination of the wtSmo and SmoM2 patterns,with SmoM1⁺ juxtanuclear structures superretained imposed on the ER pattern. (Incardona, 2002)
- M2 represents a change of Trp535 to Leu in the seventh transmembrane segment and was uninhibited by Ptc1.
  - distributions: differes most significantly from wild-type Smo, appearing in a pattern virtually identical to an ER marker and absent from the juxtanuclear region, where most of the intracellular wtSmo resides. (Incardona, 2002)
- Drug treatment of the SmoM1 and SmoM2 mutants:
  - Brefeldin A blocks ER-to-Golgi transport: Treatment of Smo mutants: SmoM2-no effect. SmoM1- intensification of the juxtanuclear component wtSmo-had a small degree of increase in the juxtanuclear component. (Incardona, 2002)
  - 6-hr chloroquine treatment: wtSmo-in large, swollen vesicular structures (Figure 4G). SmoM2-retained its ER distribution (Figure 4I). SmoM1-showed a distribution intermediate to wtSmo and SmoM2 (Figure 4H), with a smaller fraction appearing in swollen vesicles in addition to a large ER component. (Incardona, 2002)

overexpression of Smo is not sufficient to activate the pathway, casting doubt on the proposed stoichiometric relationship between Ptc and Smo (Alcedo. 2000; Denef, 2000; Ingham, 2000).
Increasing levels of Smo (4x UAS-Smo and UAS-Smo^high driven with MS1096) leads to activation of low (Iro) and then intermediate (dpp & ptc) Hh responses (Figure 2) (Hooper, 2003)

{Reagents}