BIOLOGICAL OVERVIEW

Smoothened protein is thought to be a component of the Hedgehog receptor (Alcedo, 1996). The pathway for Hedgehog regulated induction of wingless is complex, involving both Smoothened and another membrane protein (Patched) thought to be the direct receptor for Hedgehog. Hh action alleviates the negative regulation of Hh target genes and they become transcriptionally active.

Surprisingly, a vertebrate homolog of Smoothened (vSMO), shows no direct interaction with mouse Sonic hedgehog (SHH), and Sonic hedgehog binds mPtc, a murine homolog of Patched, with high affinity. For example, epitope-tagged SHH as well as IgG-Sonic HH, both hybrid proteins containing the N terminal region of Sonic HH attached covalently to second proteins, bind to cells expressing mPTC, and no change in the affinity between SHH and PTC is observed in the presence of vSMO. mPTC can be co-immunoprecipitated with epitope-tagged SHH. Nevertheless, the three proteins can form a physical complex. In cells coexpressing mPTC and vSMO, vSMO can be co-immunoprecipitated with antibody against epitope-tagged mPTC. Thus in the end, it appears that Patched, and not SMO is the protein that directly interacts with SHH (Stone, 1996).

Confirming evidence comes from experiments in which chicken ptc was expressed in Xenopus oocytes. Binding of labelled SHH is detected in ptc transduced oocytes but not in untransduced controls. Co-immunoprecipitation experiments reveal that when transduced cells are treated with SHH and extracted, SHH can be detected in immuno-preciptates carried out with antibody against epitope tagged PTC (Marigo, 1996).

Chen and Struhl (1996) argue that PTC is positioned upstream of SMO in the HH signal transduction pathway, either as a factor that regulates SMO-transducting activity in response to HH or as a factor that facilitates the direct modulation of SMO activity by HH (Chen, 1996).

It seems likely that Patched and Smoothened function as receptor and signal transducing proteins respectively. Smoothened and Patched associate with each other on the membrane. In the absence of ligand, PTC inhibits signaling of Smoothened, while in the presence of ligand, PTC inhibition of SMO signaling is released and SMO signaling is activated.

Apparently, Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, free Ptc (unbound by Hh) has been shown to act sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumor suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule (Taipale, 2002).

Mutation of smoothened has been used to unravel the complex regulation of segmental boundary function in Drosophila. Segmental primordium is subdivided into two cell populations (the anterior (A) and posterior (P) compartments) by the selective activity of the transcription factor Engrailed (En) in P cells. Under En control, P cells secrete, but cannot respond to, the signaling protein Hedgehog (Hh). In contrast, and by default, A cells are programmed to respond to Hh by expressing other signaling molecules, such as Decapentaplegic (Dpp) and Wingless (Wg), which organize growth and patterning in both compartments. Cells of the A and P compartments do not intermix, apparently as a consequence of their having distinct cell affinities that cause them to maximize contact with cells of the same compartment, while minimizing contact with cells from the other compartment. This failure to mix has previously been ascribed to an autonomous and direct role for En in specifying a P cell affinity (as opposed to an A cell affinity). However, an alternative hypothesis is that Hh secreted by P cells induces A cells to acquire a distinct cell affinity, ensuring that a stable 'affinity boundary' forms wherever P and A cells meet. To distinguish between these two hypotheses, a mutation was used in the gene smoothened to block the ability of A-compartment cells to receive and transduce Hh. If distinct A and P cell affinities are specified autonomously by the expression state of En expression, then anterior cells should retain their A-compartment affinity and sort out from P cells, even if their ability to respond to Hh is blocked by the loss of smo activity. Conversely, if it is the Hh signal that normally induces a local affinity difference between A and P cells, then a block in the Hh signal transduction could cause the affected A cells to sort out from wild-type A cells that do receive the Hh signal, and to intermix instead with P cells on the other side of the compartment boundary. The affinity boundary that segregates A and P cells into adjacent but immiscible cell populations is to a large extent a consequence of local Hh signaling, rather than a reflection of an intrinsic affinity difference between A and P cells (Rodriguez, 1997).

Smoothened regulates alternative configurations of a regulatory complex that includes Fused, Costal, Suppressor of Fused and Cubitus interruptus

In the Drosophila wing, Hedgehog is made by cells of the posterior compartment and acts as a morphogen to pattern cells of the anterior compartment. High Hedgehog levels instruct L3/4 intervein fate, whereas lower levels instruct L3 vein fate. Transcriptional responses to Hedgehog are mediated by the balance between repressor and activator forms of Cubitus interruptus, CiR and CiA. Hedgehog regulates this balance through its receptor, Patched, which acts through Smoothened and thence a regulatory complex that includes Fused, Costal, Suppressor of Fused and Cubitus interruptus. It is not known how the Hedgehog signal is relayed from Smoothened to the regulatory complex nor how responses to different levels of Hedgehog are implemented. Chimeric and deleted forms of Smoothened were used to explore the signaling functions of Smoothened. A Frizzled/Smoothened chimera containing the Smo cytoplasmic tail (FFS) can induce the full spectrum of Hedgehog responses but is regulated by Wingless rather than Hedgehog. Smoothened whose cytoplasmic tail is replaced with that of Frizzled (SSF) mimics fused mutants, interfering with high Hedgehog responses but with no effect on low Hedgehog responses. The cytoplasmic tail of Smoothened with no transmembrane or extracellular domains (SmoC) interferes with high Hedgehog responses and allows endogenous Smoothened to constitutively initiate low responses. SmoC mimics costal mutants. Genetic interactions suggest that SSF interferes with high signaling by titrating out Smoothened, whereas SmoC drives constitutive low signaling by titrating out Costal. These data suggest that low and high signaling (1) are qualitatively different, (2) are mediated by distinct configurations of the regulatory complex and (3) are initiated by distinct activities of Smoothened. A model is presented where low signaling is initiated when a Costal inhibitory site on the Smoothened cytoplasmic tail shifts the regulatory complex to its low state. High signaling is initiated when cooperating Smoothened cytoplasmic tails activate Costal and Fused, driving the regulatory complex to its high state. Thus, two activities of Smoothened translate different levels of Hedgehog into distinct intracellular responses (Hooper, 2003).

Analyses of the activities of truncated and chimeric forms of Smo in a variety of genetic backgrounds yielded four principal observations. (1)The FFS chimera activates the full spectrum of Hh responses, but is regulated by Wg rather than Hh. From this, it is concluded that the Smo cytoplasmic tail initiates all intracellular responses to Hh, while the remainder of Smo regulates activity of the tail. (2) The SSF chimera interferes with high signaling but has no effect on low signaling. SSF mimics Class II fu mutants and is suppressed by increasing smo⁺ but not fu⁺ or cos⁺. From this, it is concluded that high Hh instructs Smo to activate Fu by a mechanism that is likely to involve dimeric/oligomeric Smo. (3) The cytoplasmic tail of Smo (SmoC) derepresses endogenous Smo activity in the absence of Hh and represses endogenous Smo activity in the presence of high Hh. That is, SmoC drives cells to the low response regardless of Hh levels. This mimics cos mutants and is suppressed by 50% increase in cos⁺. From this, it is concluded that low Hh instructs Smo to inactivate Cos, by a mechanism that may involve stoichiometric interaction between Cos and the Smo cytoplasmic tail. (4) Chimeras where the extracellular CRD and TM domains are mismatched fail to exhibit any activity. From this, it is concluded that these two domains act as an integrated functional unit. This leads to a model for signaling where Fz or Smo can adopt three distinct states, regulating two distinct activities and translating different levels of ligand into distinct responses. Many physical models are consistent with these genetic analyses (Hooper, 2003).

Two mutant forms of Smo have been identified that regulate downstream signaling through different activities. These mutant forms of Smo mimic phenotypes of mutants in other components of the Hh pathway, as well as normal responses to different levels of Hh. These data suggest a model where Smo can adopt three distinct states that instruct three distinct states of the Ci regulatory complex. The model further suggests that Smo regulates Ci through direct interactions between Fu, Cos and the cytoplasmic tail of Smo. This is consistent with the failure of numerous genetic screens to identify additional signaling intermediates, and with the exquisite sensitivity of low signaling to Cos dosage (Hooper, 2003).

The model proposes that Smo can adopt three states, a decision normally dictated by Hh, via Ptc. The Ci regulatory complex, which includes full-length Ci, Cos and Fu, likewise can adopt three states. (1) In the absence of Hh Smo is OFF. Its cytoplasmic aspect is unavailable for signaling. The Cos/Fu/Ci regulatory complex is anchored to microtubules and promotes efficient processing of Ci155 to CiR. (2) Low levels of Hh expose Cos inhibitory sites in the cytoplasmic tail of Smo. Cos interaction with these sites drives the Ci regulatory complex into the low state, which recruits Su(fu) and makes little CiR or CiA. (3) High levels of Hh drive a major change in Smo, possibly dimerization. This allows the cytoplasmic tails of Smo to cooperatively activate Fu and Cos. Fu* and Cos* (* indicates the activated state) then cooperate to inactivate Su(fu), to block CiR production, and to produce CiA at the expense of Ci155 (Hooper, 2003).

The OFF state is normally found deep in the anterior compartment where cells express no Hh target genes (except basal levels of Ptc). In this state, the Ci regulatory complex consists of Fu/Cos/Ci155. Cos and Fu contribute to efficient processing of Ci155 to the repressor form, CiR, presumably because the complex promotes access of PKA and the processing machinery to Ci155, correlating with microtubule binding of the complex. This state is universal in hh or smo mutants, indicating that intracellular responses to Hh cannot be activated without Smo. Therefore Smo can adopt an OFF state where it exerts no influence on downstream signaling components and the OFF state of the Ci regulatory complex is its default state (Hooper, 2003).

The low state is normally found approximately five to seven cells from the compartment border, where cells are exposed to lower levels of Hh. These cells express Iro, moderate levels of dpp, no Collier and basal levels of Ptc. They accumulate Ci155, indicating that little CiA or CiR is made. Ci155 can enter nuclei but is insufficient to activate high responses. The physical state of the Ci regulatory complex in the low state has not been investigated. Cells take on the low state regardless of Hh levels when Ci is absent or when SmoC is expressed, and are strongly biased towards that state in fu(classII); Su(fu) double mutants. This state normally requires input from Smo, which becomes constitutive in the presence of SmoC. Because SmoC drives only low responses and cannot activate high responses, this identifies a low state of Smo that is distinct from both OFF and high. It is proposed that the low state is normally achieved when Smo inactivates Cos, perhaps by direct binding of Cos to Smo and dissociation of Cos from Ci155. Neither CiR nor CiA is made efficiently, and target gene expression is similar to that of ci null mutants (Hooper, 2003).

The high state is normally found in the two or three cells immediately adjacent to the compartment border where there are high levels of Hh. These cells express En, Collier, high levels of Ptc and moderate levels of Dpp. They make CiA rather than CiR, and Ci155 can enter nuclei. In this state a cytoplasmic Ci regulatory complex consists of phosphorylated Cos, phosphorylated Fu, Ci155 and Su(fu). Dissociation of Ci from the complex may not precede nuclear entry, since Cos, Fu, and Sufu are all detected in nuclei along with Ci155. Sufu favors the low state, whereas Cos and Fu cooperate to allow the high state by repressing Sufu, and also by a process independent of Sufu. This high state is the universal state in ptc mutants and requires input from Smo. As this state is specifically lost in fu mutants, Fu may be a primary target through which Smo activates the high state. SSF specifically interferes with the high state by a mechanism that is most sensitive to dosage of Smo. This suggests SSF interferes with the high activity of Smo itself. It is suggested that dimeric/oligomeric Smo is necessary for the high state, and that Smo:SSF dimers are non-productive. Cooperation between Smo cytoplasmic tails activates Fu and thence Cos. The activities of the resulting Fu* and Cos* are entirely different from their activities in the OFF state, and mediate downstream effects on Sufu and Ci (Hooper, 2003).

The cytoplasmic tail of Smo is sufficient to activate all Hh responses, and its activity is regulated through the extracellular and TM domains. This is exemplified by the FFS chimera, which retains the full range of Smo activities, but is regulated by Wg rather than Hh. The extracellular and transmembrane domains act as an integrated unit to activate the cytoplasmic tail, since all chimeras interrupting this unit fail to activate any Hh responses, despite expression levels and subcellular localization similar to those of active SSF or FFS. As is true of other serpentine receptors, a global rearrangement of the TM helices is likely to expose 'active' (Cos regulatory?) sites on the cytoplasmic face of Smo. The extracellular domain of Smo must stabilize this conformation and Ptc must destabilize it. But how? Ptc may regulate Smo through export of a small molecule, which inhibits Smo when presented at its extracellular face. Hh binding to Ptc stimulates its endocytosis and degradation, leaving Smo behind at the cell surface. Thus, Hh would separate the source of the inhibitor (Ptc) from Smo, allowing Smo to adopt the low state. Transition from low to high might require Smo hyperphosphorylation. The high state, which is likely to involve Smo oligomers, might be favored by cell surface accumulation if aggregation begins at some threshold concentration of low Smo. Alternatively, these biochemical changes may all be unnecessary for either the low or high states of Smo (Hooper, 2003).

There is no suggestion that Ptc has multiple states in response to different levels of Hh. Ptc mutants that fail to derepress signaling, or that constitutively derepress signaling coordinately affect both high (e.g. En) and low (e.g. Iro) responses. Thus, it is suggested that Smo and not Ptc is the first step in which the Hh pathway adopts three distinct states (Hooper, 2003).

Both Hhs and Wnts act as morphogens, with different levels of ligand dictating different intracellular responses. Those intracellular responses are respectively initiated by Smo and Fz. Fz and Smo have a high degree of sequence similarity in their extracellular and transmembrane domains. The similarity must extend to function, since graded levels of Wg acting through the FFS chimera drive low and then high signaling by the Smo cytoplasmic tail. This suggests unanticipated complexity in Fz function, where low levels of Wnts 'low-activate' Fz while higher levels trigger oligomerization-dependent 'high activation'. Fz8 CRD crystallizes as a dimer, suggesting a physical basis for Fz family oligomerization (Hooper, 2003).

There is precedent within the serpentine receptor superfamily for dimerization/oligomerization and for multiple signaling states. ß2-adrenergic receptor (ß2AR), the archetypical serpentine receptor has at least three states. In the absence of ligand, ß2AR is OFF. The agonist-occupied state favors a global conformational change that allows the cytoplasmic loops and tail to activate heterotrimeric G proteins as well as the receptor kinase, GRK2. GRK2 then phosphorylates the cytoplasmic tail of ß2AR. In the phosphorylated state, ß2AR binds ß-arrestin. ß2AR + ß-arrestin1 then assemble novel trafficking and signaling complexes that mediate endocytosis, Src binding and ERK activation. Complementation between two inactive ß2AR mutant forms demonstrates that adjacent molecules can exchange helices to reconstitute a functional receptor; that is, ß2AR can homodimerize. Moreover, a peptide derived from the sixth TM domain simultaneously blocks dimerization and activation. There are substantial parallels between this model of ß2AR signaling and the current model of Smo signaling. Each recruits and activates a kinase when the receptor is stimulated. Each stimulated receptor then becomes a substrate for assembly of a new signaling complex. It is suggested that multiple signaling states could be a general mechanism by which serpentine receptors translate different levels and/or kinetics of ligand exposure into distinct responses (Hooper, 2003 and references therein).

Evidence for a novel feedback loop in the Hedgehog pathway involving Smoothened and Fused

Hedgehog (HH) is a major secreted morphogen involved in development, stem cell maintenance and oncogenesis. In Drosophila wing imaginal discs, Hh produced in the posterior compartment diffuses into the anterior compartment to control target gene transcription via the transcription factor Cubitus interruptus (Ci). The first steps in the reception and transduction of the Hh signal are mediated by its receptor Patched (Ptc) and the seven-transmembrane-domain protein Smoothened (Smo). Ptc and Hh control Smo by regulating its stability, trafficking, and phosphorylation. Smo interacts directly with the Ser-Thr protein kinase Fused (Fu) and the kinesin-related protein Costal2 (Cos2), which interact with each other and with Ci in an intracellular Hedgehog transducing complex. Hh induces Fu targeting to the plasma membrane in a Smo-dependent fashion and, reciprocally, Fu controls Smo stability and phosphorylation. Fu anchorage to the membrane is sufficient to make it a potent Smo-dependent, Ptc-resistant activator of the pathway. These findings reveal a novel positive-feedback loop in Hh transduction and are consistent with a model in which Fu and Smo, by mutually enhancing each other's activities, sustain high levels of signaling and render the pathway robust to Ptc level fluctuations (Claret, 2007).

This work provides new information about (1) the mechanisms by which the activation of Smo is transduced to its cytoplasmic effector Fu, (2) the mechanisms of Fu activation, and (3) a novel positive-feedback loop between Fu and Smo. Evidence is provided that Smo controls the subcellular distribution of two of its physical partners, Fu and Cos2, recruiting them to the plasma membrane in response to Hh. The data also suggest that Fu might link Cos2 to Smo in a vesicle-associated complex in the absence of Hh, whereas Fu and Cos2 might independently bind Smo at the plasma membrane in the presence of Hh. Thus, Hh might not only promote, via Smo, the recruitment of Fu and Cos2 to the plasma membrane; it might also modulate the nature of interactions between these three proteins (Claret, 2007).

Several nonexclusive mechanisms seem to be involved in controlling Fu activity. (1) The forced localization of Fu at the membrane induces strong Smo-dependent activation of the pathway in the wing. This study is the first to report a dominant active form of this type of kinase. (2) This study shows that the presence of a conserved Thr in the activating loop is important for the promotion of full Smo phosphorylation and for the activating effects of GAP-Fu. Thus, because Fu is known to be phosphorylated in response to Hh, the phosphorylation of this loop (by autophosphorylation or by other kinases) might be a key element in Fu regulation. (3) Hh might regulate Fu by controlling its dimerization or its interaction with potential regulatory proteins. Possible Fu dimerization is consistent with (1) the reported interaction between the regulatory domain of Fu and its kinase domain, (2) the recruitment to the plasma membrane of wild-type Fu by the wild-type and mutant forms of GAP-Fu, and (3) the dominant negative effects of Fu mutants with modified kinase domains (Claret, 2007).

Evidence is presented for of a novel, positive-feedback loop in which Smo and Fu enhance each other's activities. Indeed, Smo promotes the relocalization of Fu to the plasma membrane and is required for the activating effects of GAP-Fu, whereas both GAP-Fu and Fu control Smo stability and phosphorylation. Fu kinase activity is required for Smo phosphorylation and for the activating effects of GAP-Fu, but not for its association with Smo. Fu might phosphorylate Smo directly or might act on other substrates, indirectly facilitating Smo phosphorylation, inhibiting phosphatases, or stabilizing phosphorylated Smo. Both Fu activity and its interaction with Smo seem to be required for full hyperphosphorylation of Smo in response to Hh (Claret, 2007).

In the wing imaginal disc, Fu is required principally for responses to the highest levels of Hh present at the anteroposterior border, where Smo is active despite the strong upregulation of ptc. The effects of GAP-Fu and Fu on Smo provide the first clues to a putative mechanism (Fu-dependent phosphorylation and stabilization of Smo), potentially accounting for the resistance of Smo to the high level of Ptc induced by Hh in responding cells (Claret, 2007).

The following model is proposed: (1) The Hh-induced relocalization of Smo to the plasma membrane leads to the recruitment of Fu and Cos2 at this membrane. (2) Fu, in turn, acts on Smo, probably by further enhancing its phosphorylation, to stabilize it further and prevent its inhibition by Ptc. It is not yet possible to determine whether Fu regulates Smo directly or indirectly. The kinesin Cos2 may be also part of this regulatory loop. (3) The stabilized, activated Smo/Fu/Cos2 complex at the plasma membrane then promotes the accumulation and activation of Ci-FL, leading to the activation of Hh target genes, including ptc (Claret, 2007).

Smo^δFu, which does not bind Fu, is constitutively active, suggesting that Fu might also act as a negative regulator of Smo in the absence of the Hh signal. Thus, Fu might act as a switch, sensing the level of Hh, inhibiting Smo in the absence of Hh or activating the pathway in response to high levels of Hh. Interestingly, the existence of such regulatory loops might account for the bistability properties of signaling pathways and explain how graded levels of signal might act as morphogens, leading to differential cell responses (Claret, 2007).

In conclusion, Fu was found to be recruited by Smo at the plasma membrane in response to Hh and this recruitment was found to be directly dependent on the physical interaction of Fu with Smo. The expression of a membrane-anchored form of Fu (GAP-Fu) constitutively activates the Hh pathway, indicating that Fu activity might be regulated by its subcellular location. Surprisingly, the activating effects of GAP-Fu require a wild-type dose of endogenous Smo. Evidence is reported that (1) Fu and GAP-Fu induce the phosphorylation of Smo, (2) GAP-Fu recruits Smo to the plasma membrane, (3) GAP-Fu renders Smo resistant to the destabilizing effects of Ptc, and (4) Fu controls the level of accumulation of Smo in the wing imaginal disc. Thus, these data demonstrate that Fu, which is generally considered to be an effector of Smo, can also act on Smo (Claret, 2007).

GENE STRUCTURE

mRNA length - 4005 bases

Exons - 6

Bases in 3' UTR - 654

PROTEIN STRUCTURE

Amino Acids - 1028

Structural Domains

SMO is an integral membrane protein with seven membrane spanning alpha helices and a long hydrophilic C-terminal tail. An additional hydrophobic segment near the N-terminus is a signal peptide. Its cleavage generates an N-terminal extracellular domain with five potential N-linked glycosylation sites. The protein has the characteristics of a G protein coupled receptor. The C-terminal domain of SMO is unusually large (481 amino acids). It includes five potential phosphorylation sites for PKA that together with the PKA site of the second intracellular loop, might serve to desensitize SMO by uncoupling SMO from the G alpha protein subunit (Alcedo, 1996 and van den Heuval, 1996).

date revised: 10 August 2003

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