patched

Protein Interactions

Evidence that Patched physically interacts with HH 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, 1996b).

Genetic and expression evidence point to a role of both Patched and Smoothened in Hedgehog receptor function. PTC appears to be 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).

During Drosophila development, cells belonging to the posterior compartment of each segment organize growth and patterning by secreting Hedgehog (Hh), a protein that induces a thin strip of adjacent cells in the anterior compartment to express the morphogens Decapentaplegic (Dpp) and Wingless (Wg). Hedgehog is bound and transduced by a receptor complex that includes Smoothened (Smo), a member of the Frizzled (Fz) family of seven-pass transmembrane receptors, as well as the multiple-pass transmembrane protein Patched (Ptc). Ptc is required for the binding of Hh to the complex as well as for the Hh-dependent activation of Smo within the complex. A likely null allele of the smo gene has been identified. It was used to determine whether Hh is bound by Ptc alone, or by Smo in concert with Ptc. Cells devoid of Smo can sequester Hh, but their ability to do so depends, as in wild-type cells, on the expression of high levels of Ptc protein. These results suggest that Ptc normally binds Hh without any help from Smo and hence favor a mechanism of signal transduction in which Hh binds specifically to Ptc and induces a conformational change leading to the release of latent Smo activity (Chen, 1998).

During wing development, cells in the posterior (P) compartment secrete Hh and cells in the anterior (A) compartment respond to Hh by turning on, or up-regulating, several genes, including those encoding Dpp and Ptc. These genes are expressed at high level only in a thin strip of A cells adjacent to the A/P compartment boundary, indicating that Hh normally moves only a short distance into the A compartment. Examined were clones of A compartment cells homozygous for the smo³ mutation, which codes for an altered protein that might not be null because it may retain the ability to insert into the cell membrane. These clones fail both to express dpp and to upregulate Ptc expression, even when adjacent to the A/P compartment boundary, indicating that they are unable to transduce Hh. Moreover, they appear unable to restrict the movement of Hh into the A compartment, as indicated by the response of wild-type cells positioned just anterior to large A compartment clones of smo³ cells that abut the A/P boundary. These smo+ cells express dpp and Ptc at high levels, even though they are located many cell diameters away from the A/P boundary, at positions that are normally too far from the boundary to be exposed to Hh. The same analysis was performed using the smo^D16 allele, certain to be a null allele because it gives rise to a truncated protein, in place of the smo³ allele, and the same result was obtained. Both Ptc expression as well as the expression of a dpp-lacZ transgene, placed in trans to the smo^D16 mutant allele, were monitored. A compartment cells lacking Smo protein not only fail to respond to Hh, but also fail to limit the further spread of Hh into the A compartment, indicating that they lack a Hh-sequestering activity (Chen, 1998).

In the case of the smo³ mutant allele, the failure of mutant cells to impede the movement of Hh into the A compartment can be attributed to their failure to up-regulate the expression of Ptc, which confers Hh-sequestering activity. Indeed, it is possible to restore the ability of smo³ mutant cells to restrict Hh movement by simultaneously eliminating the activity of Protein kinase A (PKA), a manipulation that constitutively activates the Hh signal transduction pathway causing smo³ mutant cells to express high levels of Ptc. However, it is not known whether the Hh-sequestering activity of smo³ PKA - cells is mediated solely by Ptc, or by smo³ mutant protein in conjunction with Ptc. Indeed, molecular analysis indicates the smo³ allele encodes a truncated form of Smo that includes the entire cysteine rich extracellular domain (CRD), as well as the first three transmembrane domains, consistent with the possibility that truncated Smo retains a Hh-binding activity. To resolve this uncertainty, the smo-;PKA- experiment was repeated using the smo^D16 allele in place of the smo³ allele. smo^D16;PKA- clones were generated using two genetic configurations. In the first configuration, both Ptc expression as well as the expression of a dpp-lacZ transgene were monitored. In contrast to smo^D16 clones, which express only low levels of Ptc protein, smo^D16;PKA- clones autonomously express high levels of Ptc protein throughout. Moreover, large smo^D16;PKA- clones that abut the A/P boundary differ from similarly positioned smo^D16 clones in that they appear to impede the movement of Hh through the clone. In the second genetic configuration, smo^D16;PKA- mutant cells were marked by the loss of a ubiquitously expressed arm-lacZ transgene. This configuration allows the smo PKA genotype to be assessed independently of Ptc expression. A compartment cells that are homozygous for the smo^D16 mutation and hence devoid of Smo protein are nevertheless able to sequester Hh, provided that they also express high levels of Ptc. These findings favor the proposal that Hh normally binds specifically to Ptc within the Hh receptor complex without any direct involvement of Smo (Chen, 1998).

Although the transmembrane protein encoded by the patched gene has been genetically implicated as the Hedgehog receptor, the intercellular signaling pathways involved in these inductive processes remain uncharacterized. The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-C1) is required for the correct spatial regulation of dpp expression during eye development. Loss of PKA-C1 function is sufficient to produce an ectopic morphogenetic wave marked by premature ectopic photoreceptor differentiation and non-autonomous propagation of dpp expression. PKA-C1 lies in a signaling pathway that controls the orderly temporal progression of differentiation across the eye imaginal disc (Strutt, 1995).

On the other hand, high PKA activity cannot counteract the phosphorylation of Fused that depends on HH signaling. Since the phosphorylation of Fused can be inhibited by PTC it is more likely that Fused is downstream of PTC. In this model PKA signals are integrated further downstream (Thérond, 1996 and Alcedo, 1996).

Patched is found associated with Wingless in discrete regions of the lateral plasma membrane of the embryonic epidermal cells. Preferential sites of accumulation resemble the described localization of the cell-cell adhesive junctions of the epidermal cells. Patched partially co-localizes with the Wingless protein in wingless-expressing and nearby cells, in structures that seem to be endocytic vesicles. This suggests patched protein interacts with elements of the reception complex of Wingless, as a way to control wingless expression (Capdevila, 1994a).

Current models view Patched and Smoothened as a preformed receptor complex that is activated by Hedgehog binding. Evidence is presented that Patched destabilizes Smoothened in the absence of Hedgehog. Hedgehog binding causes removal of Patched from the cell surface. In contrast, Hedgehog causes phosphorylation, stabilization, and accumulation of Smoothened at the cell surface. Comparable effects can be produced by removing Patched from cells by RNA-mediated interference. These findings raise the possibility that Patched acts indirectly to regulate Smoothened activity (Denef, 2000).

As a first step toward addressing how Smoothened activity is regulated by Ptc, an antibody to the C-terminal cytoplasmic tail of Smoothened (Smo) protein was produced and Smo expression was examined in imaginal discs. Ptc and Smo are both differentially expressed in A and P cells but in different ways. Ptc is absent from P cells, whereas Smo is expressed at relatively high levels in the P compartment. In the A compartment, Smo levels follow a profile similar to Ptc. Smo protein levels are highest in Hh-responsive anterior cells adjacent to the AP boundary. Smo expression decreases sharply near the boundary and then more gradually across the A compartment. To verify that this accurately reflects Smo protein levels, clones of cells mutant for the smo³ allele were examined. smo³ is associated with a nonsense mutation that truncates the protein after the third transmembrane domain. The C-terminal intracellular portion of the protein recognized by anti-Smo should be absent from the protein encoded by this allele. Clones of mutant cells in both compartments lack Smo antigen. Smo protein is clearly detectable in cells adjacent to the mutant clone in the middle of the A compartment. The level of Smo in wild-type cells adjacent to a more anterior clone is barely distinguishable from background. Although Smo protein expression levels differ in the A and P compartments, SMO mRNA does not appear to be spatially regulated in the wing or leg imaginal discs. In situ hybridization using sense and anti-sense RNA probes did not detect differential expression of SMO mRNA in A and P compartments of the wing disc or of the leg disc. SMO transcript is expressed in a spatially regulated pattern in the brain, which corresponds to the pattern of Smo protein expression (Denef, 2000).

A model for Ptch and Smo activity postulates that Ptc is required for Hh binding, whereas Smo is required to transduce the signal. Ptc blocks the intrinsic signaling activity of Smo, and Hh binding to Ptc alleviates this block and thereby activates Smo. The available evidence suggests that Hh does not bind to Smo in the absence of Ptc nor does Hh activity appear to be required to activate Smo in the absence of Ptc. The prevalent model suggests that Smo might be constitutively active when the inhibitory effects of Ptc are alleviated, and it has been thought that Ptc and Smo exist in a preformed complex at the cell surface that is activated by Hh binding. Three observations from the current analysis support an alternative view of the relationship between Smo and Ptc in Hh signaling: (1) Ptc acts to reduce the level of Smo protein in the absence of Hh. (2) Hh binding triggers removal of Ptc from the cell surface. (3) Hh treatment or removal of Ptc by RNA(i) induces a net increase in phosphorylation of Smo. This correlates with an increase in the level of Smo on the cell surface. These observations raise the possibility that Ptc acts indirectly to regulate Smo activity (Denef, 2000).

How does Hh treatment alter the degree of Smo phosphorylation? Hh binding to Ptc could stimulate the activity of a kinase that phosphorylates Smo. Alternatively, the constitutive activity of Ptc could be mediated by promoting dephosphorylation of Smo. If this is the case, Hh treatment might inactivate a Ptc-dependent phosphatase. The second possibility is favored because dsRNA-mediated depletion of Ptc is sufficient to increase Smo phosphorylation in S2 cells without addition of Hh. In the wing disc, most of the Smo protein comes from the posterior compartment where Ptc is not expressed. Smo from the disc is mostly in the highly phosphorylated form. Thus, in the absence of Ptc, Smo is mainly found in the highly phosphorylated form. The available evidence indicates that Smo is active in the P compartment, but the signal is nonproductive due to the absence of Ci. Taken together, these observations suggest that the highly phosphorylated form of Smo is active in signaling. It is suggested that Ptc activity is mediated by promoting dephosphorylation of Smo and that Hh blocks the ability of Ptc to regulate Smo in A cells near the AP boundary. The relatively low amount of dephosphorylated Smo seen in discs may derive from anterior cells in which Ptc is active because these anterior cells are out of the range of Hh (Denef, 2000).

These findings are consistent with the possibility that Smo is dephosphorylated by a type 2A protein phosphatase. At the concentrations used in these experiments, okadaic acid inhibits PP2A but not PP1-type phosphatases. Smo protein contains consensus sites for phosphorylation by serine/threonine protein kinases, including PKA. The serine/threonine kinase Fused is phosphorylated in response to Hh and plays a role in Hh signaling. In addition, previous work has also implicated PKA and an okadaic acid-sensitive phosphatase in the regulation of Ci phosphorylation. Thus, there appear to be several levels at which phosphorylation and dephosphorylation can regulate Hh signaling activity. The favored model suggests that the constitutive activity of Ptc stimulates activity of a phosphatase that leads to reduced phosphorylation of Smo. Hh binding to Ptc might reduce the ability of Ptc to promote phosphatase activity and allow Smo phosphorylation to increase. According to this view, the state of Smo phosphorylation reflects a balance in the activity of a kinase (which could be constitutively active) and the Ptc-dependent activity of a phosphatase (Denef, 2000).

Evidence has been presented that Hh induces distinct alterations in the subcellular localization of Ptc and Smo proteins. In cells, Hh treatment induces internalization of Ptc and accumulation of Smo at the cell surface. Double labeling studies have shown that Ptc and Hh colocalize in vesicles in the wing disc. In embryos, immunoelectronmicroscopic analysis has shown that Ptc is found in endocytic vesicles and in multivesicular bodies. Multivesicular bodies are intermediates between early and late endosome compartments. The appearance of Ptc in multivesicular bodies is consistent with the possibility that Hh-induced endocytosis targets Ptc to the lysosome for degradation. In this context, it is interesting that Ptc shows sequence similarity to the Niemann-Pick C1 protein, which has been linked to defects in recycling of vesicles in the endocytic and lysosomal pathways. Together, these observations support the view that Hh binding triggers internalization and degradation of Ptc. Under normal circumstances, Hh signaling induces new synthesis of Ptc, which serves to limit the range of Hh movement into the A compartment. These apparently opposing effects on Ptc levels may be reconciled by the idea that this mechanism is used to target Hh for degradation once it has bound Ptc and activated Smo. Clearing Hh from the system may contribute to limiting its range of movement (Denef, 2000).

Smo does not appear to follow Ptc through the endocytic pathway. In contrast, Smo accumulates on the cell surface in Hh-stimulated cells. Accumulation of Smo could reflect Hh-induced transport of Smo from an intracellular pool to the cell surface. Alternatively, Hh-induced phosphorylation might reduce Smo turnover in the membrane, leading to net accumulation. At present, these possibilities cannot be distinguished (Denef, 2000).

Whatever the mechanism for increased accumulation of Smo at the cell surface, these observations suggest that the actively signaling form of Smo is unlikely to be bound by Ptc. If Ptc acts indirectly to regulate Smo activity, the regulatory interaction between Ptc and Smo need not be stoichiometric. It is noted that levels of Smo protein significantly in excess of normal can be rendered functionally inactive by endogenous levels of Ptc. Although it is possible to exceed the capacity of Ptc to regulate Smo activity by overexpression, the results illustrate that Ptc can effectively regulate both Smo activity and Smo protein levels over a considerable range and at levels well above the endogenous level of Smo. These observations support the possibility that Ptc might act indirectly to regulate Smo phosphorylation, with concomitant effects on subcellular localization, stability, and activity (Denef, 2000).

Hedgehog signaling, mediated through its Patched-Smoothened receptor complex, is essential for pattern formation in animal development. Activating mutations within Smoothened have been associated with basal cell carcinoma, suggesting that smoothened is a protooncogene. Thus, regulation of Smoothened levels might be critical for normal development. Smoothened protein levels in Drosophila embryos are regulated posttranscriptionally by a mechanism dependent on Hedgehog signaling but not on its nuclear effector Cubitus interruptus. Hedgehog signaling upregulates Smoothened levels, which are otherwise downregulated by Patched. Demonstrating properties of a self-correcting system, the Hedgehog signaling pathway adjusts the concentrations of Smoothened and Patched to each other and to that of the Hedgehog signal, which ensures that activation of Hedgehog target genes by Smoothened signaling becomes strictly dependent on Hedgehog (Alcedo, 2000).

Posttranscriptional regulation of Smo depends on PKA, an antagonist of the Hh signaling pathway. Thus, PKA activity regulates Smo levels either by stimulating the degradation of Smo or by reducing its rate of synthesis. The mechanism by which Hh regulates the stability of the Smo protein through PKA-dependent proteolysis is favored for two reasons: there is no evidence for PKA-dependent regulation of the rate of synthesis of any protein -- in contrast, it is known that Hh signaling inhibits PKA-dependent proteolytic processing of its nuclear effector Ci. Since several consensus PKA phosphorylation sites are found in the cytoplasmic portions of Smo, PKA might also exert its effect directly on Smo. The phosphorylated form of Smo might be targeted by Slimb to the ubiquitin-ligase complex prior to its proteasome-mediated degradation, a mechanism inhibited by Hh and constitutive Smo signaling. Alternatively, PKA does not act directly on Smo but affects the stability of Smo by activating a protein that destabilizes Smo or by inhibiting a protein that stabilizes Smo (Alcedo, 2000).

A test if Smo levels are uniformly elevated after reducing or completely removing the zygotic Slimb activity in slimb mutant embryos was negative, presumably because of the presence of sufficient wild-type maternal Slimb. After reducing the maternal Slimb activity in hypomorphic slmb1 germline clones, slimb mutant embryos cease to develop by stage 6 and hence can not be tested, since Smo levels are still very low at this stage (Alcedo, 2000).

Why do Hh and Smo signaling upregulate the two Hh-receptor components, Ptc and Smo, at the transcriptional and posttranscriptional level, respectively? What are the advantages of this Hh and Smo signaling system in which Hh inhibits Ptc, which otherwise suppresses Smo signaling and hence downregulates both Smo and Ptc? For convenience, it is assumed in the following a model in which Smo signaling is activated by Hh binding to Ptc as part of a Ptc-Smo receptor complex so far only demonstrated in mammals. Yet none of the considerations presented here are affected by the assumption of such a complex because they are independent of whether Ptc inhibits Smo signaling directly or indirectly. The constitutive activation of the Hh signaling pathway in the absence of Hh is oncogenic. 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. When Hh levels decrease, Smo is destabilized 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).

Although this self-correcting Hh signaling system may appear complex, its properties are probably the simplest solution in ensuring that Smo signaling strictly depends on the concentration of Hh, as apparent from the following considerations. Since Smo signals constitutively in the absence of Ptc, 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 is observed. 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).

Hedgehog signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, it is shown that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. The mutant Ptc^SSD protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functions like the wild-type protein in sequestering and internalizing Hh. In addition, Ptc^SSD preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity (Martin, 2001).

Ptc protein has an SSD, originally identified in HMGCoA reductase and SREBP (sterol regulatory element binding protein) cleavage-activating protein (SCAP), both implicated in cholesterol homeostasis. In addition, it is structurally similar to the Niemann-Pick C1 (NPC1) protein that participates in intracellular cholesterol transport. To address the functional role of the SSD of Ptc, a G-to-A substitution was engineered that causes an Asp⁵⁸³ to change to Asn in the SSD (Ptc^SSD). This point mutation mimics an Asp-to-Asn mutation in the SSD of SCAP that causes sterol resistance in mutant Chinese hamster ovary cell lines. The Asp mutated in this line is conserved in the SSD of all six SCAP family members known in mouse, human, and C. elegans NPC1 proteins as well as in Ptc. The mutated Drosophila ptc cDNA was introduced into flies under UAS control and overexpressed the mutant protein in the ptc expression domain with ptc-GAL4. The expression of Ptc^SSD causes embryonic lethality and an almost complete ptc null phenotype. Since the severity of the mutant phenotype correlates directly with the amount of Ptc^SSD expressed, it has been suggested that Ptc^SSD competes with the wild-type protein and has a dominant-negative effect (Martin, 2001).

Ptc is located inside the cell, mainly in cytoplasmic vesicles, and when internalization is blocked in shibire mutant embryos, Ptc is associated with the plasma membrane. The large vesicles in which Ptc and Hh accumulate in Hh-responsive cells are endosomes since they colocalized with internalized Texas-red dextran. The same colocalization of Ptc and internalized Texas-red dextran also occurs in ptc^S2 mutant clones (which only express the Ptc^S2 protein. However, Smo protein is not localized with Ptc in the endocytic compartment and is mainly observed along the basolateral plasma membrane. Thus, the Ptc mutation in the SSD alters the subcellular distribution of Ptc, as also occurs upon Hh binding. This preferential location of Ptc^SSD in punctate structures is apparent when the ectopic expression of Ptc^SSD is compared with that of Ptc^WT, which shows a more generalized distribution. Interestingly, there are ptc alleles that do not show this preferential accumulation in endosomes. These alleles induce high Ptc protein levels and do not sequester Hh (Martin, 2001).

Inhibition by steroidal components of both Hh signaling and NPC1-mediated cholesterol transport suggests that Ptc and NPC1 function may have a similar molecular mechanism. A lesion in the NPC1 protein, which is normally found in cytoplasmic vesicles characteristic of late endosomes, produces a general defect in the retrieval and recycling of raft components of the endocytic pathway. Ptc and NPC1 colocalize extensively in vesicular compartments in cotransfected cells. This suggests that the function of both NPC1 and Ptc involves a common vesicular transport pathway (Martin, 2001).

To conclude, the upregulation of Smo protein and the opening of the Hh pathway in Ptc^SSD mutant cells are related to the preferential accumulation of Ptc^SSD protein in the endocytic compartment. Therefore, in wild-type cells, subcellular changes in Ptc protein distribution upon Hh binding could impede interaction with Smo and downregulation of Smo protein levels. A future challenge will be to demonstrate that cholesterol modulates the vesicular trafficking of Ptc through its SSD (Martin, 2001).

The tumor suppressor gene patched (ptc) encodes an approximately 140 kDa polytopic transmembrane protein that binds members of the Hedgehog (Hh) family of signaling proteins and regulates the activity of Smoothened (Smo), a G protein-coupled receptor-like protein essential for Hh signal transduction. Ptc contains a sterol-sensing domain (SSD), a motif found in proteins implicated in the intracellular trafficking of cholesterol, and/or other cargoes. Cholesterol plays a critical role in Hedgehog (Hh) signaling by facilitating the regulated secretion and sequestration of the Hh protein, to which it is covalently coupled. In addition, cholesterol synthesis inhibitors block the ability of cells to respond to Hh, and this finding points to an additional requirement for the lipid in regulating downstream components of the Hh signaling pathway. Although the SSD of Ptc has been linked to both the sequestration of, and the cellular response to Hh, definitive evidence for its function has so far been lacking. The identification and characterization of two missense mutations in the SSD of Drosophila Ptc is described; strikingly, while both mutations abolish Smo repression, neither affects the ability of Ptc to interact with Hh. It is speculated that Ptc may control Smo activity by regulating an intracellular trafficking process dependent upon the integrity of the SSD (Strutt, 2001).

Conventional models of Hh signaling envisage Ptc to be a ligand binding sub-unit of a Hh receptor that regulates the activity of a signaling subunit, the Smo protein, by inducing conformational changes in the latter. The results of recent studies of Smo in Drosophila have, however, challenged this view and suggested instead that Ptc regulates Smo activity by promoting its posttranslational modification and/or decreasing its stability rather than by locking it into an inactive conformational state. How and where such modifications occur is not known, but the finding that two of three antimorphic alleles of ptc are associated with lesions in the SSD indicates a critical role for this domain in the regulation of Smo. Given that other SSD-containing proteins, such as SCAP and NPC1, are known to mediate trafficking between intracellular compartments, it is tempting to speculate that Ptc may act in a similar manner by directing Smo to an intracellular compartment where it is targeted for modification/degradation. The antimorphic nature of the ptc^S2 and ptc³⁴ alleles could be explained if mutation of the SSD abolishes the putative trafficking activity of Ptc without affecting interaction with its cargo. The mutant forms of the protein would thus protect Smo from modification/degradation and leave it free to activate the downstream components of the pathway. By the same token, mutation of the C-terminal tail ( ptc¹³) might also disrupt the hypothetical trafficking activity; alternatively, it could disrupt cargo interaction. Recent studies have failed to reveal an interaction between the Ptc C-terminal tail and Smo, and this failure favors the former possibility (Strutt, 2001).

Cell pattern in the ventral neural tube is organized by Sonic hedgehog (Shh) secreted by floor plate cells. To assay the range of direct Shh action, a general method was developed for blocking transduction of Hedgehog (Hh) signals through ectopic expression of a deleted form of the Hh receptor Patched (Ptc), termed Ptc^Deltaloop2. This deleted form of Ptc appears to lack the capacity to bind Hh but retains the ability to inhibit Smo or a downstream Smo effector in the presence of Hh. This method was validated in Drosophila and mouse Ptc1^Deltaloop2 (mPtc1^Deltaloop2) was used to block Shh transduction in the chick neural tube. This Ptc protein lacks most of the second large extracellular loop. When expressed in clones of cells in the developing Drosophila wing, Ptc^Deltaloop2 autonomously blocks the ability of cells to sequester and transduce Hh, generating a phenotype that is indistinguishable from that caused by loss of Smo activity. An equivalently modified vertebrate Ptc1 protein, mPtc1^Deltaloop2, attenuates the response of chick neural cells to Shh, providing the means to test whether the long-range effects of Shh in neural tissues are direct (Briscoe, 2001).

mPtc1^Deltaloop2 expression causes cell-autonomous ventral-to-dorsal switches in progenitor identity and neuronal fate throughout the ventral neural tube, supporting a gradient mechanism whereby Shh acts directly and at long range. mPtc1^Deltaloop2 expression also causes the abnormal spread of Shh to more dorsal cells, indicating that Shh in the neural tube, like Hh in Drosophila, induces a feedback mechanism that limits its range of action (Briscoe, 2001).

In Drosophila, the upregulation of Ptc in response to Hh has a crucial role in restricting the range of Hh action. The results presented here suggest that Ptc1 acts similarly to restrict the action of Shh derived from the floor plate. Expression of mPtc1^Deltaloop2 in ventral neural cells results in a cell nonautonomous dorsal-to-ventral shift in the identity of progenitor cells positioned dorsal to cells that express mPtc1^Deltaloop2. This change in fate is most easily explained by the idea that cells dorsal to mPtc1^Deltaloop2 cell clusters have been exposed to a higher level of Shh activity. Normally then, exposure of cells to Shh may limit the level of Shh activity available to cells positioned further from a source of Shh. Consistent with a net ventral-to-dorsal movement of Shh through the ventral neuroepithelium, only those cells positioned dorsally to the mPtc1^Deltaloop2 cell clusters appear to be exposed to elevated Shh signaling. In Drosophila tissues, Ptc itself is responsible for the sequestration of Hh and thus for the restriction in Hh movement. By extension, it is likely that the inability of neural cells that express mPtc1^Deltaloop2 to sequester Shh results from the reduced level of expression of endogenous Ptc1. However, Shh signaling also induces other proteins such as hedgehog interacting protein (HIP) and vitronectin, which can themselves bind to Shh and could potentially participate in restricting the movement of Shh. Independent of the respective contributions of these proteins to the sequestration of Shh, these data indicate that Shh signaling in the neural tube, as in Drosophila, initiates a feedback system that limits the range of Shh movement and signaling activity (Briscoe, 2001).

The membrane protein Patched (Ptc) is a critical regulator of Hedgehog signaling. Ptc is among a family of proteins that contain a sterol sensor motif. The function of this domain is poorly understood, but some proteins that contain sterol sensors are involved in cholesterol homeostasis. In the SREBP cleavage-activating protein (SCAP), sterols inhibit the protein’s activity through this domain. Mutations in two highly conserved residues in the SCAP sterol sensor have been identified that confer resistance to sterol regulation. The analogous mutations were introduced into the sterol sensor motif of fly Ptc and mouse Ptc1 and their effect on protein activity was examined. In contrast to SCAP, the sterol sensor mutations had different effects on Drosophila Ptc; Ptc Y442C retains function, while Ptc D584N confers dominant negative activity. In the wing imaginal disc, Ptc D584N overexpression induces Hedgehog targets by stabilizing Cubitus interruptus and inducing decapentaplegic. However, Ptc D584N does not induce collier, a gene that requires high levels of Hedgehog signaling. In mouse Ptc1, the Y438C and D585N mutations do not stimulate signaling in Shh-responsive cell lines but complement murine ptc1^-/- cells. The results suggest that mutations in sterol sensor motifs alter function differently between sterol sensor family members (Johnson, 2002).

Patched and the kinesin-related protein Costal2 cause internalization of Smo

Hedgehog (Hh) signaling is critical for many developmental events and must be restrained to prevent cancer. A transmembrane protein, Smoothened (Smo), is necessary to transcriptionally activate Hh target genes. Smo activity is blocked by the Hh transmembrane receptor Patched (Ptc). The reception of a Hh signal overcomes Ptc inhibition of Smo, activating transcription of target genes. Using Drosophila salivary gland cells in vivo and in vitro as a new assay for Hh signal transduction, the regulation of Hh-triggered Smo stabilization and relocalization was investigated. Hh causes Smo (GFP-Smo) to move from internal membranes to the cell surface. Relocalization is protein synthesis-independent and occurs within 30 min of Hh treatment. Ptc and the kinesin-related protein Costal2 (Cos2) cause internalization of Smo, a process that is dependent on both actin and microtubules. Disruption of endocytosis by dominant negative dynamin or Rab5 prevents Smo internalization. Fly versions of Smo mutants associated with human tumors are constitutively present at the cell surface. Forced localization of Smo at the plasma membrane activates Hh target gene transcription. Conversely, trapping of activated Smo mutants in the ER prevents Hh target gene activation. Control of Smo localization appears to be a crucial step in Hh signaling in Drosophila (Zhu, 2003).

The salivary gland experiments show that Smo is normally present in a meshwork of organelles in the cytoplasm. Upon reception of a Hh signal, Smo protein moves quickly to the surface. This change in subcellular localization could be due to release from a tether and movement, or to a change in the net flow of protein cycling through membrane compartments. If Smo, for example, normally circulates to and from the surface, Ptc could facilitate the inward movement. When Hh binds to Ptc and inactivates it, Smo would cycle to the surface and remain there. This idea is consistent with the increased surface Smo observed when endocytosis is blocked with the shibire or Rab5 mutation (Zhu, 2003).

Surface Smo localization correlates fully with Hh target gene transcription but the amount of Smo protein does not. The apparent increase in the amount of Smo at the cell surface that occurs when Hh signal is received may be caused by sequestration of Smo away from proteases in internal membrane compartments. Smo moves to the surface on addition of Hh or removal of Ptc, conditions that activate target gene transcription (Zhu, 2003).

The mutant forms of fly Smo designed to mimic human oncogenic SMO alleles are also located at the surface. The basis for the tumors is hypothesized to be inappropriate activation of Hh (perhaps Shh) target genes in the skin and cerebellum, targets that are insufficiently restrained by Ptc1 or other regulators. The oncogenic forms of Smo appear to be resistant to inhibition by Ptc and at least one of them is clearly resistant to teratogenic drugs that bind Smo and inhibit responses to Hh signaling. These experiments suggest that the fly versions of the oncogenic proteins are also at least partially resistant to Ptc and are refractory to the Ptc-imposed internalization of Smo protein. Overexpression of the oncogenic mutants causes dramatic changes in wing patterning and anterior outgrowth. Conversely, tagging oncogenic Smo mutants with an ER retrieval motif, KKDE at the C terminus prevents Hh signaling. This addition of only three amino acids (one K is already present at the C terminus of fly Smo) drastically changes the activities and localization of the protein (Zhu, 2003).

Flies lacking smo function are unable to activate target genes in response to Hh signaling, in contrast to ptc mutants, which activate target genes inappropriately even in the absence of Hh signal. Double mutants that lack both smo and ptc function fail to activate Hh target gene transcription, indicating that the failure of repression by Ptc is irrelevant if Smo is not present to allow activation. On this basis, Ptc has been viewed as an opponent of Smo function (Zhu, 2003).

The regulation of Smo by Ptc remains a mystery. Hh could inactivate Ptc by binding to it either on the surface or in internal vesicles. If Ptc cycles between surface and interior regions of the cell, the binding by Hh could change Ptc so that it is less likely to travel to the surface. In contrast to Hh inactivation of Ptc, mutational inactivation of Ptc does not lead to an internal location, at least in the case of the dominant-negative form that accumulates at the surface. Ptc, possibly through a transporter function, could change organelle contents or composition so that Smo changes conformation to its active form. Alternatively Ptc could either detach Smo from an intracellular tether or alter the movements of vesicles bearing Smo protein. Smo, previously cycling to and from the surface, would accumulate on the surface and increase in amount (Zhu, 2003).

The dominant-negative form of Ptc is present at the cell surface and could compete for an association between Ptc and Smo, compete for an association between Ptc and another protein, or associate with wild-type Ptc and inactivate its activity. The distinct locations of Ptc and Smo in fly imaginal disc cells and in salivary gland cells, suggest that the bulk of each protein is not in association with the other protein. There could nonetheless be some of the proteins in association, below the level of detection by staining techniques. Although little or no Ptc-Smo association was seen by immunoprecipitation from cultured cells, transient associations would not be seen, particularly if the arrival of Ptc or Hh/Ptc in a Smo-containing organelle immediately caused the departure of Smo (Zhu, 2003 and references therein).

Ptc contains a sequence related to 'sterol-sensing domains (SSDs)' that have been implicated in altered functions or stability of proteins involved in lipid metabolism. Mutations in the Ptc SSD reduce the ability of Ptc to repress Smo function. It is possible that Ptc regulates Hh signaling through effects on membrane trafficking. Analysis of mice with mutations in the open brain (opb) gene lend further support for the potential involvement of protein trafficking in Hh signal regulation. opb encodes Rab23, which negatively regulates Hh signal transduction. Rab GTPases coordinate the budding, fission, transport, docking, and fusion of vesicles as they move from one cellular location to a target compartment. The shuttling of Smo and Ptc between internal membrane compartments and the cell surface presumably requires Rab activity. Disruption of endocytosis by dominant-negative Shibire and by Rab5 manipulation prevents both Smo and Ptc internalization (Zhu, 2003).

Movement of Smo to the surface requires actin and tubulin components of the cytoskeleton, though the relevant motors are unknown. Cos2 is an unusual member of the kinesin family, with sequence features at odds with conventional ATPase binding site structure. Cos2 could be either a motor or a tether. Cos2 could have a role in controlling movements of vesicles that contain Smo. Overproduction of Cos2 alters GFP-Smo localization, and furthermore, prevents Hh from bringing much GFP-Smo to the surface, and the GFP-Smo that does reach the surface is located in discreet dots. Ptc also blocked Hh from bringing GFP-Smo to the surface, but no such dots were observed. Overexpression of a presumably irrelevant other motor protein, Nod, has no effect on localization of GFP-Smo. Cos2 production may therefore specifically cause the movement of Smo-containing organelles to discreet locations on the membrane, either tethering them to the cytoskeleton at specific locations or causing a coalescence effect at random locations. Cos2 has been envisioned as functioning as part of a cytoplasmic complex whose activity in processing the Ci transcription factor is controlled by Smo. The present data suggest a new function in which the complex (oralternatively, Cos2 independently of the complex), feeds back to alter Smo activity. It is interesting that both GFP-Smo (when Cos2 and Hh were coexpressed) and PtcDN-YFP exhibits a similar punctate cell surface localization pattern. PtcDN may function through competing with endogenous Ptc, raising an intriguing alternative possibility that Cos2 may interact directly with Smo to control Smo subcellular localization (Zhu, 2003).

Human oncogenesis by activated Smo and the importance of the Hh pathway in numerous developmental events in all animals makes understanding Hh signal transduction critical. The present approach has identified new interactions between components of the pathway. The causal link between surface location and activity during Hh signaling, with Ptc inactivation, with Smo oncogenic mutants and with mislocalization of Smo add strong evidence that the localization of Smo is a critical regulatory step in Hh signaling (Zhu, 2003).

Patched function as revealed by the inhibition of Smoothened by cyclopamine

Plants of the genus Veratrum have a long history of use in the folk remedies of many cultures, and members of the jervine family of alkaloids, constituting a majority of Veratrum secondary metabolites, have been used for the treatment of hypertension and cardiac disease. The association of Veratrum californicum with an epidemic of sheep congenital deformities during the 1950s raised the possibility that jervine alkaloids are also potent teratogens. Extensive investigations by the U.S. Department of Agriculture subsequently confirmed that jervine and cyclopamine (11-deoxojervine) given during gestation can directly induce cephalic defects in lambs, including cyclopia in the most severe cases. It is now known that the teratogenic effects of jervine and cyclopamine are due to their specific inhibition of vertebrate cellular responses to the Hedgehog (Hh) family of secreted growth factors, as first suggested by similarities between the Vertarum-induced developmental malformations and holoprosencephaly-like abnormalities associated with loss of Sonic hedgehog (Shh) function. In accordance with this general mechanism, cyclopamine also has shown some promise in the treatment of medulloblastoma tumors caused by inappropriate Hh pathway activation (Chen, 2002 and references therein).

Using photoaffinity and fluorescent derivatives, it has now been shown that this inhibitory effect is mediated by direct binding of cyclopamine to the heptahelical bundle of Smoothened (Smo). Cyclopamine also can reverse the retention of partially misfolded Smo in the endoplasmic reticulum, presumably through binding-mediated effects on protein conformation. These observations reveal the mechanism of cyclopamine's teratogenic and antitumor activities and further suggest a role for small molecules in the physiological regulation of Smo (Chen, 2002).

Since both cyclopamine and Ptch negatively regulate Smo activity, how Ptch activity influences the ability of Smo to bind cyclopamine was investigated. Increased levels of mouse Ptch expression in COS-1 cells dramatically enhances the photoaffinity cross-linking of post-ER Smo by 125I-labeled photoaffinity reagent-tagged cyclopamine (PA-cyclopamine). In contrast, the labeling of ER-localized Smo was not affected, and cellular concentrations of either Smo form were not altered by Ptch expression. Treatment of the Smo- and Ptch-expressing cells with the N-terminal domain of Shh (ShhN) is able to reverse the effect of Ptch expression on PA-cyclopamine/Smo cross-linking, confirming its dependence on Ptch activity (Chen, 2002).

These results provide some insights into the regulation of Smo by Ptch. (1) Ptch appears to act only on post-ER Smo, since the PA-cyclopamine cross-linking of ER-localized Smo is independent of Ptch expression levels. This subcellular compartmentalization of Ptch action is consistent with previous observations that Ptch is primarily localized to endosomal/lysosomal vesicles and the plasma membrane. (2) The ability of Ptch expression to significantly increase post-ER Smo labeling by PA-cyclopamine without influencing overall protein levels suggests that the effect of Ptch activity alters Smo conformation and that Ptch and cyclopamine promote inactive Smo states that may be structurally related (Chen, 2002).

How Ptch influences Smo conformation remains enigmatic, despite extensive genetic analyses of the Hh pathway. Although it was initially proposed that Ptch and Smo form a heteromeric receptor, it is now believed that Smo activity is modulated by Ptch in an indirect, nonstoichiometric manner (Taipale, 2002). In the case of the Frizzled family of seven-TM receptors, which are closely related to Smo in structure, receptor activation involves the binding of Wnt ligands to the Frizzled CRD and recruitment of an LDL receptor-related protein. No analogous protein interactions have been associated with Smo activation, and removal of the Smo CRD does not appear to significantly alter Smo function or its suppression by Ptch (Chen, 2002 and references therein).

These observations coupled with the susceptibility of Smo to cyclopamine suggest that Smo regulation may involve endogenous small molecules rather than direct protein-protein interactions. Consistent with this model, Ptch is structurally related to the resistance-nodulation-cell division family of prokaryotic permeases and to the Niemann-Pick C1 protein, which are both capable of transporting hydrophobic molecules. Ptch action might similarly affect the subcellular and/or intramembrane distribution of endogenous molecules, thus influencing Smo activity by altering the localization of a Smo ligand. Alternatively, this Ptch activity could influence membrane structure and Smo trafficking; a shift in Smo localization might then be accompanied by activity-modulating changes in the molecular composition of specific subcellular compartments (Chen, 2002 and references therein).

The demonstration of cyclopamine binding to Smo establishes the mechanism of action for this plant-derived teratogen. These studies show that cyclopamine interacts with the Smo heptahelical bundle, thereby promoting a protein conformation that is structurally similar to that induced by Ptch activity. Equally important, these studies reveal the molecular basis for cyclopamine's antitumor activity and validate Smo as a therapeutic target in the treatment of Hh-related diseases. Aberrant Hh pathway activation has been associated with several cancers, such as medulloblastoma and basal cell carcinoma, and many of these tumors involve mutations in Ptch or Smo. As a specific Smo antagonist, cyclopamine may be generally useful in the treatment of such cancers, a therapeutic strategy further supported by the absence of observable toxicity in cyclopamine-treated animals. Additional Smo antagonists might also be discovered through small molecule screens for specific Hh pathway inhibitors, thus comprising a class of pharmacological agents with possible utility in the treatment of Hh-related oncogenesis (Chen, 2002).

Hrs mediates downregulation of Patched and other signalling receptors in Drosophila

Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-ß signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).

Monoubiquitination of membrane proteins has an important role in regulating their internalization and sorting to lysosomal degradation. The ubiquitin tag is recognized by proteins containing a ubiquitin interaction motif (UIM), such as epsins, Hse1p/STAM and Eps15. Hrs and its budding yeast homolog, Vps27p, also have a UIM and bind to ubiquitin. The ubiquitin-binding ability of Hrs and Vps27p is required for the efficient sorting of ubiquitinated transferrin receptors in mammalian cells and Fth1p in yeast (Jékely, 2003 and references therein).

To determine whether Hrs is generally required for sorting and degradation of ubiquitinated proteins in Drosophila tissues, clones of cells mutant for hrs were generated within an epithelium using somatic recombination. Follicle cells of the Drosophila ovary and wing imaginal disc cells from third instar larvae were examined. Follicular cells form a simple monolayer epithelium surrounding the germline cells and are large enough to detect subcellular localization of protein. The imaginal disc cells are smaller and form a pseudo-stratified epithelium. The mosaic tissues were stained with an antibody that recognizes mono- and poly-ubiquitinated proteins. Both follicle cells and wing disc cells lacking Hrs show a dramatic accumulation of ubiquitinated proteins. Most of the signal localizes to intracellular structures. In some cases accumulation at the cell cortex could also be observed. Thus, Hrs is required for the efficient removal of ubiquitinated proteins from the cell (Jékely, 2003).

An enlarged vesicular structure, the 'class E' compartment, has been observed in yeast cells mutant for VPS27. Genetic studies in mice and Drosophila (Lloyd, 2002) have also shown that cells mutant for hrs have enlarged endosomes, possibly due to impaired membrane invagination and multivesicular body (MVB) formation (Lloyd, 2002). To determine whether ubiquitinated proteins accumulate in the endosomal compartment in hrs mutant cells, GFP-Rab5 or GFP-2xFYVE fusion proteins were expressed in hrs mutant cells. Rab5, a small GTPase regulating endosome fusion, is a marker of early endosomes. FYVE domains bind to phosphatidylinositol-3-phosphate, which is enriched in endosomal membranes, and can also be used to specifically label endosomes. The ubiquitinated protein signal and the GFP-2xFYVE signal show extensive overlap in hrs mutant follicle cells. GFP-Rab5 and ubiquitinated proteins also show significant, although not complete, overlap. These data indicate that nondegraded ubiquitinated proteins accumulate in the endosomal compartment. Additionally, when the GFP-2xFYVE signal in hrs mutant and nonmutant cells is compared, an enlargement of FYVE-positive structures is observed in mutant cells, consistent with an enlargment of the endosomal compartment (Jékely, 2003).

Hrs affect degradation of receptor tyrosine kinases (RTKs). Indeed the two RTKs that were analysed in follicle cells, EGFR and PVR (PDGF/VEGF receptor), accumulate within hrs mutant cells, mostly in intracellular structures. These structures were also positive for the ubiquitinated protein signal, indicating that the receptors accumulate in endosomes (Jékely, 2003).

To test whether the requirement for Hrs was limited to RTKs, other types of signalling receptors were analysed. The Hedgehog receptor Patched and the Hedgehog signal transducer Smoothened are multi- and seven-pass transmembrane proteins, respectively. Thickveins (Tkv) is a type-I serine-threonine kinase receptor for the TGF-ß family ligand Dpp. Notch is a single-pass transmembrane protein that undergoes specific proteolytic cleavage upon activation. Interestingly, hrs mutant follicle cells show a marked accumulation of each of these receptors. As for RTKs, most of the receptor molecules accumulate intracellularly and show significant colocalization with the ubiquitinated protein signal. Thus, Hrs has a general role in regulating the sorting and degradation of diverse classes of signalling receptors. The homotypic adhesion molecule Shotgun is not affected visibly in hrs mutant cells. The latter observation is in agreement with observations that nonsignalling transmembrane proteins are not upregulated in hrs mutant animals (Lloyd, 2002). Either the trafficking of these proteins is independent of Hrs function or they have a low turnover rate in the examined tissues (Jékely, 2003).

The high degree of overlap between the signal for each of the receptors and the signal for ubiquitinated proteins means that the receptors accumulate in roughly the same endosomal compartment. This, together with the increase in receptor levels in hrs mutant cells, suggests that these receptors are degraded through the same Hrs-dependent pathway. Ubiquitination of the inhibitory Smad7 by the E3 ubiquitin ligase Smurf2 has been shown to target the Smad7-TGF-ß receptor complex for lysosomal degradation. In follicle cells, a similar complex may be sorted for degradation in an Hrs-dependent manner. It has been argued that the turnover of Hedgehog receptors is strongly regulated and may be critical for signalling, but a role of ubiquitination in this event has not been reported. The observation that Patched and Smoothened accumulate in compartments highly enriched in ubiquitinated proteins in hrs mutant cells suggests that trafficking of Patched and Smoothened is also regulated by ubiquitination (Jékely, 2003).

When analysing hrs mutant clones, an increase of ubiquitinated proteins at the cell cortex was occasionally noticed in addition to the intracellular accumulation. Some cortical accumulation could also be observed directly for the signalling receptors, in particular for Tkv. This accumulation could be due to inefficient endocytosis from the plasma membrane or increased recycling of endocytosed proteins. Hrs does not appear to be required directly for endocytosis (Lloyd, 2002), but downstream defects may 'clog up' the endocytosis machinery. Hrs can also affect receptor recycling. Overexpression of Hrs in tissue culture cells increases the retention of ubiquitinated transferrin receptors. The strong intracellular accumulation of receptors in hrs mutant cells could therefore either be due to defective sorting towards lysosomal degradation or due to defective post-endocytic retention, a concomitant general increase in the steady-state levels of the receptors at the plasma membrane, and therefore in endosomes. The first explanation is favored because increased surface levels of receptors or ubiquitinated proteins were often not detected even when strong intracellular accumulation was evident. Receptors therefore seem to be retained intracellularly, rather than recycled, in hrs mutant cells. Hrs is most likely not the only factor responsible for the post-endocytic retention of receptors. Redundancy in sorting to the vacuole has been reported for the yeast alpha-factor receptor Ste3p. In this case, Vps27p and Hse1 have overlapping roles to sort Ste3p to the vacuolar lumen (Jékely, 2003).

The bulk of Hrs-dependent downregulation of signalling receptors appears to be constitutive as well. Hedgehog acts very early in egg chamber development, and patched-lacZ, which reflects Hedgehog activity, has very restricted expression. Downregulation of Patched in the stage 10 egg chamber should therefore be ligand independent. Smoothened protein is, in turn, controlled by Patched. EGFR ligands are highly enriched and active at the dorsal side of the egg chamber, whereas a PVR ligand is present throughout the oocyte. However, for both receptors, the level of receptor accumulation in hrs mutant cells is similar throughout the follicular epithelium. Signal-induced endocytosis is well established for acute stimulation of signalling receptors, in particular RTKs. Yet signalling does not appear to control the bulk of receptor turnover in follicle cells. The physiological levels of stimulatory ligands may be relatively low compared to the levels used for acute stimulation experiments (Jékely, 2003).

Precise regulation of signalling strength is essential for interpreting morphogen gradients and thus for correct patterning during development. The control of signalling receptor levels at the cell membrane is an important aspect of this regulation. It is therefore of interest to know how receptor levels are regulated under physiological conditions. The results presented here indicate that diverse classes of signalling receptors undergo constitutive (ligand-independent) ubiquitination, endocytosis and Hrs-dependent degradation. The efficiency of this traffic affects the responsiveness of cells to patterning signals: blockage of trafficking in hrs mutants can sensitize cells to a low level of signalling molecules, whether RTK ligands (Lloyd, 2002) or Dpp (this study). However, it does not lead to ligand-independent signalling, supporting the conclusion that most endocytosed receptor molecules are not activated. Ligand-induced endocytosis may also occur, but affects only a minority of receptor molecules in this in vivo context. Constitutive turnover of receptors may serve as quality control by removing damaged receptors or receptors in partially formed signalling complexes. A constant flux of all receptor molecules may also facilitate the efficient clearance of activated receptors (Jékely, 2003).

Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment

The morphogenetic gradient of Hh is tightly regulated for correct patterning in Drosophila and vertebrates. The Patched (Ptc) receptor is required for restricting Hh long-range activity in the imaginal discs. In this study, the different types of Hh accretion that can be observed in the Drosophila embryonic epithelial cells were investigated. In receiving cells, large apical punctate structures of Hh (Hh-LPSs) are not depending on the Ptc receptor-dependent internalization of Hh but rather reflect Hh gradient. By analyzing the dynamic of the Hh-LPS gradient formation, it was demonstrated that Hh distribution is strongly restricted during late embryonic stages compared to earlier stages. The up-regulation of Ptc is required for the temporal regulation of the Hh gradient. Dynamin-dependent internalization of Hh does not regulate Hh spreading but is involved in shaping Hh gradient. Hh gradient modulation is directly related to the dynamic expression of the ventral Hh target gene serrate (ser) and with the Hh-dependent dorsal cell fate determination. Finally, this study shows that, in vivo, the Hh/Ptc complex is internalized in the Rab7-enriched lysosomal compartment in a Ptc-dependent manner without the co-receptor Smoothened (Smo). It is proposed that controlled degradation is an active mechanism important for Hh gradient formation (Gallet, 2005).

To distinguish the several types of Hh accretion that can be observed in the ectodermal cells, different markers were used. In the secretory cells [engrailed (en) expressing cells], cytosolic punctate staining of Hh colocalizes with a secreted Green Fluorescent Protein at least initially in the cell secretory machinery. Hh accretions are also distributed very apically in expressing cells. Indeed, their position is more apical to Crumbs, which is localized in the subapical complex close to the top of the cells. These accretions are referred to as Hh-LPSs. Hh-LPS formation depends on Dispatched (Disp) activity and on the presence of the cholesterol modification on Hh and that Hh-LPS movement depends on specific proteoglycans at the cell surface. Similar apical localization of Hh-LPS is also observed in the receiving cells. Additionally, more basal cytosolic Hh accretions are observed in receiving cells and are frequently associated with Ptc. In ptc mutant embryos, these accretions disappear, while apical accumulation of Hh-LPS is observed. This suggests that large cytoplasmic Hh accretions reflect internalization of the Hh/Ptc complex after ligand-receptor binding. It also suggests that the presence of Hh-LPS is not consequent to Ptc binding or to internalization with Ptc. Also, one has to notice that small cytosolic Hh staining is still visible in the absence of Ptc in receiving cells (Gallet, 2005).

Altogether, the data show that it is possible to distinguish three different types of Hh accretions: (1) apical Hh-LPSs representing the state of Hh before internalization; (2) more basal cytosolic accretions that are strictly depending on the presence of Ptc and represent internalization of the ligand/receptor complex; (3) Ptc-independent cytosolic Hh particles that might correspond to internalization of Hh independently of its known receptor. It is proposed that LPSs reflect Hh functional gradient since their range correlates with the expression of target genes and because their assembly and movement depends, respectively, on two genes, disp and tout velu, necessary for Hh activity (Gallet, 2005).

During Drosophila embryonic development, hh is activated by the selector gene en in two rows of cells in the posterior compartment of each segment. In the embryonic ectoderm, Hh is thought to elicit pattern formation through short-range activity by inducing two different signal relays anterior and posterior to its source of expression. In addition, Hh movement encompasses more than one or two cells from the source of Hh. In order to study Hh gradient, the distribution of the apical Hh-LPS was analyzed. Secreted Hh-LPSs are easily detectable at a distance from its source and cover apically the entire segmental field during stages 9 and 10 of embryogenesis. Interestingly, it was observed that although the level of hh mRNA expression is similar in stages 10 and 11, the Hh gradient appears to be spatially restricted from stage 11 onward, Hh-LPSs being more restricted to the vicinity of the secreting cells. This observation was confirmed with a plot analysis of Hh-LPS apical distribution within the segment. At stage 11 and later, the slope of the Hh-LPS gradient is steeper compared to earlier stages suggesting that Hh movement becomes restricted. Alternatively, this could be due to a decrease in Hh protein stability. Moreover, it is not thought that the restriction of Hh distribution observed at stage 11 could be consequent to a decrease in the rate of Hh-LPS formation nor to a different apicobasal localization of Hh-LPS at this stage. Indeed, Hh-LPSs in the secreting cells at stages 10 and 11 seem similar in their density and subcellular localization (Gallet, 2005).

To confirm that Ptc is involved in the temporal restriction of Hh movement, Hh distribution was analyzed in ptc mutant embryos and in embryos expressing ptc in en/hh cells. In ptc null embryos, the Hh gradient is impaired and Hh-LPSs distribution was found to be extended throughout the entire segment without restriction. In such embryos, ser expression is totally repressed in a manner similar to that seen under ubiquitous Hh expression. Note that the ectopic hh expressing source present in ptc mutant might also contribute to the broad distribution of Hh. When Ptc is expressed in en/hh expressing cells, the range of Hh-LPSs movement is limited to the vicinity of the producing cells. This effect is not due to a diminution of hh expression since it has been shown that Ptc does not affect hh transcription. Hence, Ptc might directly affect Hh-LPS range of action. Indeed, the slope of the Hh-LPSs gradient decreased sharply compared to wild-type stage 11 embryos. Interestingly, in these embryos, ser expression was extended correlating with the absence of Hh-LPS away from the source (Gallet, 2005).

Temporal regulation of Hh gradient is necessary because signaling requirements for Hh change in a time-dependent manner. One can suggest that, during early development, Hh acts at long range due to moderate levels of Ptc. Hh would easily overcome repression by the low concentrations of Ptc protein to prime a subset of ectodermal cells at a long distance to make them competent to respond to other signals. At later stages, Hh distribution is restricted and allows expression of ser and acts over a short range to induce rhomboid, both genes being necessary for ventral denticle specification (Gallet, 2005).

It appears that temporal regulation of Hh movement is an evolutionarily conserved mechanism since similar observations to these have been reported for vertebrate development. Altogether these data suggest that endocytosis of Hh and Ptc is needed to restrict Hh distribution in the ectodermal field and to shape Hh gradient. It is hypothesized that regulation of Ptc turn-over at the plasma membrane is important in this process (Gallet, 2005).

From these data, several observations were made that are worthy of mention. (1) It is interesting to note that even in the absence of Ptc, some Hh staining is still detected in receiving cells, suggesting that some Hh internalization still occurs in the absence of Ptc. These and the few percentage of Ptc/Hh colocalizations suggest that another Hh receptor might be involved in Hh internalization. (2) The presence of Ptc was observed in Rab7 vesicles without Hh. These Ptc-Rab7 vesicles were present in cells located close or away from Hh source, suggesting that Ptc might be internalized and targeted for degradation independently of Hh. Accordingly, analysis of the subcellular localization of Ptc showed that Ptc was mostly present in internal punctate structures, suggesting that turnover of Ptc at the plasma membrane is very high (Gallet, 2005).

Taken together these results suggest that after Hh binding to Ptc, the Hh/Ptc complexes are targeted to a lysosomal compartment. Moreover Ptc appears to be constitutively internalized and degraded independently of its binding to Hh. Likewise, in vertebrate cultured cell, Ptc is constitutively transported to lysosomes and degraded. In conclusion, the data suggest that, as in the case of Wingless gradient, controlled degradation is an active mechanism for Hh gradient formation and Ptc plays a major role in this process (Gallet, 2005).

It has been reported that in Hh receiving cells, the co-receptor Smo is stabilized at the plasma membrane while, in the absence of Hh, Ptc exerts its repressive activity on Smo by destabilizing it. However, no clear mechanism has been demonstrated for Smo destabilization. The possibility that Ptc might target Smo to lysosomal compartments was explored. Thus, the possible presence of Smo in Rab7-containing vesicles was analyzed. Very little colocalization was found between Rab7 and Smo in cells close to hh source, or further away. Thus, these data suggest that the Hh/Ptc-dependent regulation of Smo stability does not principally involve a lysosomal targeting mechanism (Gallet, 2005).

Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila: Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active

The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. This study identifies domains essential for Smo activity and investigates the sub-cellular distribution of the wild type protein in vivo. Deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins it was shown that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. It was further demonstrated that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction (Nakano, 2004).

The Smo protein is an essential component of the Hh signal transduction pathway: in all contexts analysed to date, inactivation of Smo renders cells incapable of responding to Hh. Reciprocally, gain of function mutations have been isolated in human Smo that are sufficient to activate the Hh pathway in the absence of ligand (Nakano, 2004).

Hh signalling regulates Smo activity via Ptc. In the absence of Hh ligand, Ptc represses Smo activity whereas when Hh binds to Ptc, Smo becomes active. Immunoprecipitation studies of vertebrate Smo from tissue culture cells co-expressing Ptc and Smo have suggested that this Ptc-dependent modulation of Smo activity is mediated by a direct interaction between the two proteins, a view consistent with the effects of high level Smo expression in cultured cells carrying a Shh-responsive reporter gene. Studies in Drosophila, however, have suggested that Ptc suppresses Smo activity by regulating the sub-cellular distribution and stability of the protein in a non-stoichiometric manner. The relatively weak effects of ectopic Smo expression in the wing imaginal disc, presumably reflect this sub-stoichiometric regulation of the exogenously supplied protein by endogenous Ptc activity. Consistent with this, it was found that ectopically expressed Smo protein is clearly subject to Ptc-mediated destabilisation, as revealed by the distribution of the GFP and YFP tagged forms of Smo. Nevertheless, when expressed at very high levels, as occurs for instance using the 30A GAL4 driver line, the Smo protein can escape the effects of Ptc activity and accumulate in anterior compartment cells where it activates Hh target gene activity. Even under these circumstances, however, the phenotypic effects of its over-expression are relatively mild (Nakano, 2004).

Smo shares limited sequence homology with members of the Frizzled family of Wnt receptors, in particular in the Cys rich domain (CRD) of the putative N-terminal ECD that in Fz has been shown to be necessary and sufficient for Wnt binding. By contrast with Fz, there is no evidence that the ECD of Smo binds Wg (or other Wnts) or indeed Hh, consistent with the finding that Smo activity is independent of Hh in the absence of the inhibitory effects of Ptc. Nevertheless, of the five Smo point mutants identified in this study, three affect residues in the N-terminal ECD, including two Cys residues that are highly conserved in all Fz family members, suggesting ECD and the CRD in particular play some critical role in Smo function. In line with this, it was found that deletion of the ECD results in a loss of function of the protein when assayed both in mutant rescue and imaginal disc ectopic expression systems. One explanation for this requirement could be that the N-terminus binds an as yet unidentified ligand that is required even in the absence of Ptc repressive activity, or indeed the secretion of which is inhibited by Ptc. Alternatively, the ECD may itself function as an activating ligand. The protease-activated G-protein coupled receptors (PARs) provide a precedent for such intramolecular receptor activation. PARs are activated by proteolytic cleavage of the ECD, the tip of the new amino terminus created by this cleavage acting as a tethered ligand by binding to the receptor core. It is emphasised, however, that there is no evidence that Smo undergoes a similar proteolytic activation. In contrast to these findings, it has previously been demonstrated that an N-terminally deleted form of human Smo retains activity when over expressed in 10T1/2 cells. The disparity between this result and the current findings may reflect differences in the levels of over-expression achieved in the in vivo versus the in vitro systems; it may be that at high enough levels, the mutant form of Smo can saturate the repressive activity of endogenous Ptc in the 10T1/2 cells, thus leading to the activation of the endogenous wild type Smo protein (Nakano, 2004).

Since no mutations have been identified in the CTD of Smo, the requirement for this region was also investigated by deletion analysis. In contrast to the results of in vitro studies of human Smo suggesting that the terminal domain is dispensable, this study found that deletion of the entire domain or even 80% of it completely inactivates the protein. Only deletions that preserve the juxtamembrane half of the C-terminal tail retain any activity; notably it is this part of the C-terminus that has been highly conserved between Drosophila and vertebrates (Nakano, 2004).

Although these results demonstrate a requirement for carboxy terminal regions of the protein, they do not give any direct indications as to their molecular function. For example, structures in the carboxy terminus could be needed to keep other parts of Smo in the correct conformation. Alternatively, the carboxy terminus could bind directly to another protein or proteins that transduce the Hh signal to the multimeric protein complex that regulates Ci stability and activation, or indeed to the multimeric complex itself. Amongst the new smo mutant alleles that were isolated, one, smo^2A, is a missense mutation that converts a highly conserved arginine to a cysteine at amino acid position 474 in the third intracellular loop. Residues at the C-terminal end of i3 in GPCRs have been shown to be critical for appropriate coupling to heterotrimeric G-proteins: loss of charged residues in this region of the angiotensin II receptor type I abolishes such coupling. While it has been shown that Frizzled proteins can transduce Wnt signals via heterotrimeric G-proteins, the evidence for G-protein involvement in Smo activity remain equivocal (Nakano, 2004).

Both smo^4DI and smo^2A hypomorphic mutations reside in the heptahelical domain of the protein, the structural integrity of which has been implicated in Smo activity. Small molecule agonists and antagonists of vertebrate Smo have been shown to bind to this domain, implying its involvement in regulating the activity of the protein. Exactly how these small molecules influence Smo activity is unclear, but one suggestion is that they may alter the conformation of the protein, respectively, towards a more active or inactive state. In this view, the normal regulation of Smo by Ptc might involve the transport of a cellular small molecule agonist/antagonist that similarly binds to Smo, altering its conformation. Alternatively, Ptc might act to redistribute Smo to a membrane compartment where it is susceptible to such an antagonist ligand or inaccessible to an agonist (Nakano, 2004).

Analysis of the sub-cellular distribution of tagged forms of the Smo protein in Drosophila tissues is consistent with a role for Ptc in the intracellular trafficking of Smo. Specifically it was found that Smo accumulates in the plasma membrane of cells that lack Ptc activity, whereas when co-expressed with Ptc, the same protein localises exclusively intracellularly, accumulating in distinct punctate structures. Similar results using the salivary gland as an assay system have been reported. Significantly, electron microscopy analysis of the HRP::Smo fusion proteins reveals high levels of HRP activity in the lysosomes of cells in which Ptc is active whereas in Ptc negative cells, the protein accumulates predominantly in the plasma membrane as well as in endosomes and unidentified presumptive transport vesicles (Nakano, 2004).

Analysis of the localisation and activity of putative hypermorphic forms of Smo might afford an insight into the mechanism of Smo function. Interestingly, when driven with ap-GAL4, the putative hypermorphic form of Smo, GFP::SmoA479, accumulates in the plasma membrane irrespective of Ptc activity and yields a stronger phenotype than GFP::Smo expressed under the same conditions, as assayed by dpp-lacZ expression. Surprisingly, however, the resulting adult phenotype appears to be no more severe in the case of the putative hypermorph than the wild type form. Furthermore, the over-expression of SmoA479Y as well as two other putative gain of function forms of Smo, SmoK580Q and SmoW553L, directed by the 30A and 71B GAL4 drivers result in lower levels of ectopic dpp-lacZ activation than those induced by wild type Smo. Consistent with this, the resulting adult phenotypes are also weaker in the case of the mutant forms of Smo than their wild type counterpart. It has been reported, however, that the over-expression of certain putative hypermorphic forms of Smo results in stronger phenotypes than wild type Smo. The reason for these disparities is currently unclear (Nakano, 2004).

Finally, observations that the cellular distribution of Smo is unaffected in imaginal disc clones lacking the activity of PKA or Cos2 (and hence constitutively transducing the Hh signal) has suggested Hh signalling is not sufficient to stabilise Smo and that Smo stabilisation is therefore more likely to be a cause, rather than an effect, of Hh signal transduction. However, it has been observed that over-expression of Cos2 in salivary gland cells can result in Smo destabilisation, suggesting that Cos2 activity is capable of influencing Smo levels. Taken together with the current data, this suggests that antagonising Cos2 activity might be a necessary, but not sufficient, prerequisite for Smo stabilisation at the cell surface (Nakano, 2004).

In conclusion, the data favour a mechanism whereby Ptc regulates Smo activity through inhibition of its accumulation in the plasma membrane, targeting it instead to the lysosomal pathway. In this view, Ptc might regulate Smo activity simply by modulating the levels of protein present in the cell. Alternatively, it may be that the sub-cellular location of Smo is critical for its activation, the plasma membrane perhaps providing an environment in which Smo is accessible to an intracellular agonist (Nakano, 2004).

The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover

Patched is a membrane protein whose function in Hedgehog (Hh) signal transduction has been conserved among metazoans and whose malfunction has been implicated in human cancers. Genetic analysis has shown that Ptc negatively regulates Hh signal transduction, but its activity and structure are not known. This study investigated the functional and structural properties of Drosophila Ptc and its C-terminal domain (CTD), 183 residues that are predicted to reside in the cytoplasm. The results show that Ptc, as well as truncated Ptc deleted of its CTD, forms a stable trimer. This observation is consistent with the proposal that Ptc is structurally similar to trimeric transporters. The CTD itself trimerizes and is required for both Ptc internalization and turnover. Two mutant forms of the CTD, one that disrupts trimerization and the other that mutates the target sequence of the Nedd4 ubiquitin ligase, stabilize Ptc but do not prevent internalization and sequestration of Hh. Ptc deleted of its CTD is stable and localizes to the plasma membrane. These data show that degradation of Ptc is regulated at a step subsequent to endocytosis, although endocytosis is a likely prerequisite. It was also shown that the CTD of mouse Ptc regulates turnover (Lu, 2006).

Analysis of CTD mutants revealed that the CTD controls Ptc localization and half-life. Both functions mapped to the CTD's 106 C-terminal residues. Deletion of this CTD reduced the levels of internal Ptc and stabilized the protein. Note that assays of Ptc localization measured its steady-state distribution and did not distinguish between effects on the rate of internalization or on recycling of internalized protein to the cell surface. Therefore, it is not known whether Hh directly affects the removal of Ptc from the cell surface, or if it affects a process that sorts internalized protein. Since internalized Ptc^WT has been observed to colocalize with Hh in multivesicular bodies, which are late endosomes that ferry cargo to lysosomes, it seems reasonable to propose that internalized Hh-bound Ptc is programmed for degradation, and that internalization is a requisite step in the pathway toward that fate. The observation that the instability conferred by the CTD is sensitive to NH₄Cl, an inhibitor of lysosomal proteolysis, is consistent with this model. These experiments do not reveal whether unbound Ptc cycles between early endosomes and the cell surface in the absence of Hh, and so these experiments do not implicate Hh in the regulation of Ptc endocytosis per se (Lu, 2006).

The phenotypes of the ptc^3P and ptc^PPAA missense mutants add to the understanding of the Ptc degradation pathway. Both the Ptc^3P and the Ptc^PPAA mutant proteins are processed by the degradation pathway less efficiently than Ptc^WT. Yet, both Ptc^3P and Ptc^PPAA internalize in the presence of Hh. Since Ptc^PPAA mutates a PPXY motif in the CTD that is a recognition site for Nedd4, these results suggest that mono-ubiquitination in the CTD is a signal that targets Ptc to lysosomes, but mono-ubiquitination is not required for movement to early endosomes. Ptc^3P retains the PPXY motif, but in contrast to CTD^WT and CTD^PPAA, its CTD cannot multimerize. This behavior suggests that the process that marks Ptc for sorting to late endosomes may require both the PPXY motif and a conformation that is generated by the trimerized CTD. Since both Ptc^3P and the Ptc^PPAA proteins can sequester Hh, and both internalize and colocalize with Hh, these functions are apparently required for sorting, not for Hh binding or internalization. The inability of Ptc^Δ1/2C to internalize indicates that the 106 C-terminal residues also include a domain that targets Ptc to early endosomes (Lu, 2006).

The importance of regulated turnover to the proper function of signaling pathways has recently been illuminated by the isolation and analysis of the Drosophila vps25 and erupted genes. Both genes encode proteins that function in endosomal sorting, and mutants have impressive phenotypes characterized by unregulated growth and defective patterning. Endocytic defects in mutant clones result in accumulation of signaling receptors such as Notch and Thickveins as well as other signaling components, highlighting the critical role that endocytic sorting plays in regulating signaling. The multiple functions of the Ptc CTD that are necessary for proper trafficking and turnover testify to the many steps in this complex (Lu, 2006).

Cells that express a mouse Ptc CTD deletion (Ptc^ΔCTD) have more than five times the number of binding sites for Shh as do cells expressing wild-type Ptc. The mouse Ptc^ΔCTD mutant protein, like the Drosophila Ptc^ΔCTD, has an increased half-life. It is noted that both mouse and human Ptc have a PPXY motif in their respective CTDs at a location that is comparable to that of the Drosophila PPAY sequence. Although the role of the PPXY motif in mouse Ptc was not investiated, it seems reasonable to propose that the functions of the CTD are generally conserved in the vertebrate and invertebrate proteins, that the increased stability of mouse Ptc^ΔCTD derives in part from the absence of the PPXY sequence, that mouse Ptc^ΔCTD is not internalized efficiently, and that these properties contribute to the increased binding of Shh to Ptc^ΔCTD-expressing cells (Lu, 2006).

Patched, the receptor of Hedgehog, is a lipoprotein receptor

The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. This study examined the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. The up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, was used to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. Similar results were obtained after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, it was also shown that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, it was shown that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation (Callejo, 2008).

At least two models have been proposed to explain how the lipophilic Hh can spread through an aqueous tissue. Fractionation studies of the supernatant of Hh-expressing cells showed that Hh participates in high molecular weight structures that probably represent multimeric complexes, and cholesterol and palmitic acid seems to mediate this multimerization. The lipid moieties are thought to be embedded in the core of these complexes, in analogy to micelles. Recently, a second model was proposed: it suggests that lipoprotein particles could carry lipid-modified ligands such as Hh and Wingless, acting as vehicles for long-range transport. Vertebrate lipoprotein particles are scaffolded by apolipoproteins and consist of a phospholipid monolayer surrounding a core of esterified cholesterol and triglycerides. Insects form similar particles that are called Lipophorins (Lp) and contain Apolipophorins I and II (ApoLI and ApoLII). These proteins are produced in the fat body by cleavage of the precursor pro-Apolipophorin, and are not synthesized by imaginal disk cells but receive them through the hemolymph. Panakova (2005) described that a systemic reduction of lipoprotein levels in the hemolymph, by expression of Lp (ApoLI-II) RNAi in the fat body, affects long-range but not short-range Hh signaling. That study also found that Wnt and Hh proteins copurify with lipoproteins from tissue homogenates and colocalize with lipoprotein particles in the developing wing epithelium. More recently, an interaction between Lp and the glypicans, Dally and Dally-like, has been found (Callejo, 2008).

This study has tested the role of lipoproteins in Hh signaling. To this aim, the lipoprotein gene was knocked down by RNA interference, reducing Lp supply in the hemolymph. In addition, the amount of extracellular Lp was locally reduced in the wing imaginal disk cells by overexpressing Lipophorin receptor 2 (LpR2), which increases Lp endocytosis. Under both experimental conditions a decrease was observed in extracellular Hh. These results suggest an important role of lipoproteins in Hh anchoring and spreading through the extracellular matrix. Moreover, this study has observed that Ptc actively internalizes Lipophorins, effectively acting as a Lipoprotein receptor, and that its over-expression can alter intracellular lipid homeostasis. Collectively, these results are consistent with the model of lipoprotein particles acting as vehicles for Hh transport (Callejo, 2008).

Patched regulates Smoothened trafficking using lipoprotein-derived lipids

Hedgehog (Hh) is a lipoprotein-borne ligand that regulates both patterning and proliferation in a wide variety of vertebrate and invertebrate tissues. When Hh is absent, its receptor Patched (Ptc) represses Smoothened (Smo) signaling by an unknown catalytic mechanism that correlates with reduced Smo levels on the basolateral membrane. Ptc contains a sterol-sensing domain (SSD) and is similar to the Niemann-Pick type C-1 protein, suggesting that Ptc might regulate lipid trafficking to repress Smo. However, no endogenous lipid regulators of Smo have yet been identified, nor has it ever been shown that Ptc actually controls lipid trafficking. This study shows that Drosophila Ptc recruits internalized lipoproteins to Ptc-positive endosomes and that its sterol-sensing domain regulates trafficking of both lipids and Smo from this compartment. Ptc utilizes lipids derived from lipoproteins to destabilize Smo on the basolateral membrane. It is proposed that Ptc normally regulates Smo degradation by changing the lipid composition of endosomes through which Smo passes, and that the presence of Hh on lipoproteins inhibits utilization of their lipids by Ptc (Khaliullina, 2009).

A central feature of the Hh pathway is repression of Smo signaling by Ptc when Hh is absent. How this repression functions at a mechanistic level is not understood. Ptc represses Smo at substoichiometric levels and the two proteins do not appear to be enriched at similar subcellular locations in the steady state in vivo. Repression of Smo by Ptc correlates with changes in Smo subcellular localization and decreased stability (Khaliullina, 2009). Models for Ptc-mediated repression have been proposed based on its SSD and on its sequence similarity to RND (resistance nodulation division) family of proton-driven transmembrane transporters. Membrane sterol levels can alter trafficking and/or the stability of other proteins with SSDs, thus it has been proposed that lipids might control the repressive activity of Ptc. Alternatively, based on its similarity to RND transporters, it has been proposed that Ptc might regulate Smo by modulating lipid trafficking. This is an attractive idea because Smo activity can be regulated by small lipophilic molecules. Although these are plausible models, there has as yet been no evidence to suggest that Ptc alters lipid trafficking or that any lipid alters Ptc function. Furthermore, if Ptc did regulate lipid transport to repress Smo, it is not clear where contact between Smo and Ptc-mobilized lipids would occur (Khaliullina, 2009).

This study shows that one or more lipids derived from the Drosophila lipoprotein particle Lipophorin (Lpp) are required for normal Ptc-mediated Smo destabilization. Ptc sequesters a small fraction of internalized Lpp in the Ptc-positive endosomal compartment and regulates lipid trafficking from this compartment via its SSD. Mutation of the Ptc SSD causes at least one Lpp-derived lipid, sterol, to accumulate in Ptc endosomes. Given that RND permeases have rather broad substrate specificities, Ptc SSD mutation may also perturb the trafficking of additional lipids from this compartment. Similar mutations in NPC1 alter endosomal trafficking of sphingolipids, as well as sterols (Khaliullina, 2009).

Although Ptc-dependent sterol trafficking is clearly dependent on its SSD, Ptc has been reported to reduce the accumulation of neutral lipid in a manner that does not depend on its SSD. Rather, this effect of Ptc appears to depend on its ability to sequester Lpp. This may suggest that Ptc-mediated sequestration of Lpp diverts these particles away from trafficking pathways that promote neutral lipid storage (Khaliullina, 2009).

Ptc is normally present in a small fraction of endosomes and, unlike NPC1, does not generally perturb cellular sterol trafficking when mutated. How could altered trafficking in the small subset of endosomes that contain Ptc affect Smo? This study show that mutation of the Ptc SSD not only alters lipid trafficking in Ptc endosomes, but also causes Smo to accumulate in this compartment (as well as on the basolateral membrane). Similar endosomal colocalization between Ptc and Smo has been observed in vertebrate tissue culture cells. This suggests that Smo normally traffics through Ptc endosomes, where it can be exposed to lipids that are mobilized by the Ptc SSD. Thus, Ptc may control the level of basolateral Smo by changing the balance of Smo degradation and recycling from Ptc endosomes. Lipid accumulation in endosomes of NPC1 mutant cells influences the activity of Rab7, Rab9 and Rab4, perturbing degradation and recycling. The lipid composition of Ptc-positive endosomes may exert similar, but much more specific, effects on Smo, altering the total levels of Smo protein (Khaliullina, 2009).

Which Lpp lipids does Ptc use to regulate Smo trafficking? Although sterols are present in Lpp and the Ptc SSD does regulate sterol trafficking from Ptc endosomes, the data do not support a role for bulk membrane sterol in Smo regulation. Membrane sterol can be reduced sixfold by dietary depletion without changing Smo levels on the basolateral membrane or other aspects of Hh signaling. Furthermore, liposome-mediated addition of ergosterol, the most abundant membrane sterol in Drosophila, does not reverse Smo accumulation caused by LppRNAi. However, the data do not rule out the possibility that signaling sterol derivatives that act at low concentrations might be responsible. Lpp lipid extracts are now being fractionated to identify this active molecule (Khaliullina, 2009).

Loss of Lpp reproduces only a subset of the effects of Ptc mutation on Smo signaling -- although it stabilizes Smo and causes Smo-dependent accumulation of full-length Ci₁₅₅, it does not allow target gene activation. Similar uncoupling of Ci₁₅₅ stability and target gene activation is seen in fused and dally mutants. It seems unlikely that 'activation' of Ci₁₅₅ simply requires higher levels of Smo than Ci₁₅₅ stabilization; in LppRNAi discs, Smo levels reach those that result in target gene activation in wild-type discs over a broader region than in wild-type discs -- nevertheless, the range of target gene activation is narrowed. Rather, these data suggest that, although Ci₁₅₅ stabilization is regulated by the level of Smo on the basolateral membrane, its activation as a transcription factor requires a separate Smo-dependent signal. Similarly, in vertebrate cells, where Ptc regulates Smo trafficking to the primary cilium, it is clear that ciliary localization is insufficient for Smo signaling to activate transcription of target genes. Other G-protein-coupled receptors activate multiple signal transduction pathways in response to different ligands. Thus, Smo trafficking and Smo activation may also be controlled by different ligands. Although Lpp lipids regulate Smo trafficking, other lipids mobilized by Ptc could have additional effects on Smo signaling activity (Khaliullina, 2009).

The following model would be consistent with what is already known of Ptc-dependent Smo regulation and with the new observations. Ptc sequesters Lpp into Ptc-positive endosomes and regulates lipid trafficking in this compartment via its SSD. Smo that passes through Ptc endosomes can be targeted either for degradation or recycling, depending on Ptc-dependent modulation of endosomal lipid composition, and on other functions of the Ptc protein. Lipids derived from Lpp particles bias Smo trafficking towards degradation. The balance of degradation versus recycling affects total Smo levels on the basolateral membrane and its ability to stabilize Ci₁₅₅ (Khaliullina, 2009).

The association of Hh with Lpp may do more than simply promote the release of Hh from the membrane. Hh is thought to bind to the extracellular loops of Ptc -- a region that is important for conferring substrate specificity in RND family transporters. The presence of Hh on lipoproteins may block Ptc-mediated mobilization of their lipids. Alternatively, increased Ptc degradation upon Hh binding may prevent Lpp sequestration and lipid mobilization. In either case, association with Lpp particles efficiently positions Hh to interfere with, or alter, the mobilization of Lpp lipid contents by Ptc, helping to alleviate Ptc-mediated Smo repression. Thus, Hh may signal, in part, by influencing the utilization of the lipoprotein particles on which it is carried (Khaliullina, 2009).

Genetic and biochemical definition of the Hedgehog receptor

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. This study shows that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. This study shows that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, it is concluded that Ihog and Ptc together constitute the Drosophila Hh receptor (Zheng, 2010).

Using the targeted alleles of ihog and boi developed in this study, evidence is provided that Ihog proteins are an essential component required for all biological responses to the Hh signal, including target gene induction and patterning in the embryonic segment and in the wing imaginal disc. The central role of Ihog proteins in Hh response was not noted previously because of the functionally overlapping expression of Ihog and Boi in embryos and imaginal discs, which complicates genetic screens and analysis and accounts for the observation that neither the ihog nor boi targeted alleles are lethal in homozygous form. The cl-8 cells used in the genome-scale RNAi screen, in contrast, do not express Boi, and this exposed a critical role for Ihog and facilitated initial discovery of this essential component. In addition to functional overlap, analysis of these functions has been complicated by the required removal of all maternal function for fully penetrant expression of embryonic phenotypes, although maternal expression is neither necessary nor sufficient for Hh response (Zheng, 2010).

The interaction of Ihog Fn2 (the second FNIII domain) with Ptc is essential for presentation of wild-type Ptc on the cell surface. It is not possible, at present, to distinguish between the possibilities that Ihog-mediated surface presentation of Ptc is due to an increased rate of transport to the surface or to an increased duration of residence on the surface. Whatever the mechanism, Fn2 can interact with Ptc in vitro and promote surface presentation of Ptc in cells, even in the absence of the first FNIII domain (Fn1). Similarly, Fn1 alone can interact with the cleavage and cholesterol modified Hh protein HhN in vitro, and Fn1 and Fn2 thus have demonstrably independent functions. Neither domain alone, however, suffices for formation of a high-affinity complex, and the presence of both domains is required for Hh signal reception and transduction and participation in signaling in vivo (Zheng, 2010).

In addition to surface presentation of Ptcour evidence indicates that Ihog proteins also play a direct role in binding the Hh ligand in a multimolecular receptor complex that is critical for transduction. It was thus found that Hh ligand is bound to the surface of cultured cells expressing a variant of Ptc (Ptc1130) with increased localization on the surface. It was also found, with the use of quantitative assays, that endogenous Ihog expressed in these cultured cells contributes critically to binding, and that additional Ihog expression can dramatically enhance binding. In addition, expression of Ptc1130 in the wing imaginal disc clearly produces visible accumulation of the Hh protein on what appears to be the surface of anterior cells at the compartment boundary; this accumulation depends critically on the expression of Ihog/Boi (Zheng, 2010).

Consistent with the role of Ihog in binding, a striking contribution was noted of Ihog to binding in membrane vesicle preparations when present in combination with Ptc. In addition, purified, immobilized HhN and detergent-solubilized extracts containing Ptc and Ihog could be used to demonstrate Ihog-dependent, enhanced precipitation of Ptc. In these biochemical experiments, it was observed that immobilized HhN fails to precipitate detergent-solubilized Ptc alone, but does so in the presence of detergent-solubilized Ihog, and that Ihog alone precipitates Ptc much less efficiently than when HhN is present. This enhancement of Ptc precipitation was dependent on the presence of both the HhN-binding Fn1 domain and the Ptc-binding Fn2 domain of Ihog, consistent with the formation of a multimolecular complex involving HhN, Ptc, and Ihog. Similar results were noted for (Zheng, 2010).

It is interesting to note that little interaction between HhN and Ptc was observed in the absence of Ihog. Formally, it is possible that the interaction of Ptc with HhN is indirect and mediated through enhanced Ihog interaction due to Ptc-induced multimerization or allosteric effects on Ihog. This is thought to be unlikely, however, because Ihog is capable of dimerization in the absence of Ptc, and because the HhN-interacting surface of Ihog is located on the Fn1 domain, which folds independently and is quite distinct from the Ptc-interacting Fn2 domain, thus making allostery unlikely. Thus the interpretation is favored that favorable energetic contributions in the multimolecular receptor/ligand complex derive from Ptc-HhN contacts as well as contacts between Ihog-Ptc and Ihog-HhN (Zheng, 2010).

It is important to note that, despite a direct physical interaction of Ihog and Ptc and their mutual contributions to formation and surface presentation of receptor, and to ligand binding, these two pathway components have opposing roles in pathway regulation. Ihog proteins are thus absolutely required for pathway activation in response to Hh ligand, whereas Ptc alone suffices for suppression of Smo activity in the absence of ligands (Zheng, 2010).

Functional genetic analyses of the mammalian Ihog proteins Cdo and Boc have revealed roles in Hh signaling. Cdo mutant mice thus display mild to intermediate forms of holoprosencephaly, a classic manifestation of Hh signaling deficiency, with the severity of the effect depending on strain background and subject to modifying effects of mutations in other Hh pathway components. Boc mutant mice also show defects in Hh signal-dependent axonal pathfinding by dorsal neurons with ventral commissural projections in the developing neural tube. Neither of these mutants displays phenotypes as severe as those seen in the Shh mutant mouse, or in the Smo mutant, which affects all aspects of Hh signaling. It is possible, however, that a systematic analysis of the double mutant Cdo; Boc animals might reveal more severe phenotypes, as is noted in this study for ihog; boi in Drosophila. In addition, phenotypic characterization of ihog and boi mutants was not designed to reveal defects in axonal pathfinding functions like that of murine Boc, and the possibility of such a function in Drosophila remains to be explored (Zheng, 2010).

Role of lipid metabolism in smoothened derepression in hedgehog signaling

The binding of Hedgehog (Hh) to its receptor Patched causes derepression of Smoothened (Smo), resulting in the activation of the Hh pathway. This study shows 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, these 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 (Yavari, 2010).

A major regulatory step in the modulation of Hedgehog signaling occurs at the level of the two multipass transmembrane proteins, Patched and Smoothened. Genetic and biochemical studies suggest that the ligand Hh binds Ptc and functions in its inactivation. This inhibitory step is critical for the activation of Smo, which transduces the signal intracelluarly to promote Hh target gene activation. The importance of this regulatory step is further underscored by the observation that the Ptc/Smo interaction is the most commonly disrupted step in cancers caused upon aberrant Hh signaling (Yavari, 2010).

This article shows that phospholipid metabolism plays an important role in the modulation of Hh signaling at the level of Ptc/Smo interaction. In particular, the results show that an increase in the level of PI4P by the inactivation of Sac1 phosphatase leads to Smo protein relocalization to the membrane and an increase in Hh signaling in multiple tissues during Drosophila development. Furthermore the kinase (STT4), which is required for the generation of PI4P, is also required for the proper transduction of Hh signaling as indicated by its effects on Hh target gene expression. PI4P accumulation in the cell is a hallmark of sac1 mutations and is also seen upon loss of ptc activity. Furthermore, in sac1 mutant tissue, both increased membrane localization of Smo and accumulation of PI4P were found, whereas reduction in the PI4P kinase function leads to an hh-like loss of function phenotype. These results establish that phospholipid metabolism provides a critical regulatory input in the modulation of Hh signaling (Yavari, 2010).

Recent studies have proposed that Smo activation requires an input from a nonprotein small molecule. Cholesterol and its derivatives (oxysterols) are likely candidates for the small molecules required directly or indirectly for Ptc inhibition or Smo activation, because they also promote the translocation of Smo to the cilium. Because oxysterols are known to bind to vesicular transport proteins that also interact with phospholipids, further studies on possible cooperation between these two lipid types could further shed light the mechanism of Smo activation (Yavari, 2010).

Inactivation of Smo by Ptc occurs in a catalytic fashion in that a small number of Ptc molecules can inactivate many more Smo molecules. The current results provide an explanation for this nonstoichiometric inhibitory mechanism. The finding that inactivation of Ptc increases PI4P suggests that Ptc normally functions in keeping PI4P levels low within a cell. This could be achieved either by the down-regulation of the STT4 kinase or by the up-regulation of the Sac1 phosphatase. It is less likely that Ptc modulates Sac1 activity because in vivo localization studies in multiple models system have shown that Sac1 is predominantly localized to the Golgi and, as a result of proximity arguments alone, it seems a more likely possibility that Ptc modulates PI4P levels by down-regulating the lipid kinase. In this model, during normal Hh signaling, binding of Hh to Ptc will relieve repression of the kinase by Ptc and cause an increase in PI4P. As with all genetic analysis in Drosophila, the results do not imply direct protein interactions; currently unknown transduction components could exist, and future biochemical analyses will reveal which, if any, of the interactions is direct. However, the genetic analysis does allow a proposal of how an increase in the levels of this lipid can activate Hh signaling. Studies from both flies and vertebrate model system have suggested that the localization of Smo protein to the plasma membrane is essential for the activation of the pathway, and studies in multiple model systems have shown that PI4P function is essential in the vesicular transport of cargo proteins from the Golgi to the plasma membrane (Skwarek, 2009). It is therefore proposed that Hh binding to Ptc releases inhibition of a lipid kinase such as STT4, resulting in high PI4P levels. This aids vesicular transport of Smo to the membrane and causes its activation. A schematic representing the genetic model that is consistent with past and present data is shown in the graphical abstract. The results using Shh-responsive mouse fibroblasts indicate that mammalian Hh signal transduction is dependent on the activity of the murine STT4 ortholog. Previous localization studies suggest the STT4 ortholog contributes to plasma membrane PI4P pools, an observation consistent with a conserved role for PI4P metabolites in the control of Smo by mammalian Ptc1 (Balla, 2005, Wong, 1997). The observation that RNAi against the mammalian PIK1 homolog, PI4III kinase α, also reduces Hh signal transduction could suggest it has diverged in function between flies and mammals. Alternatively, PI4P pools could be exchanged more readily between membrane-bound subcellular compartments and the cell surface in mammalian cells, making the removal of either of the PI4III kinases affect global availability of PI4P derivatives. In mammalian cells, Smo activation is associated with translocation of the molecule to the primary cilium, a ubiquitous microtubule-based cell surface protrusion. Given that Drosophila cells appear to lack primary cilia, it will be of interest to determine whether PI4III kinase activity is required for Smo translocation (Yavari, 2010).

Ihog and Boi elicit Hh signaling via Ptc but do not aid Ptc in sequestering the Hh ligand

Hedgehog (Hh) proteins are secreted molecules essential for tissue development in vertebrates and invertebrates. Hh reception via the 12-pass transmembrane protein Patched (Ptc) elicits intracellular signaling through Smoothened (Smo). Hh binding to Ptc is also proposed to sequester the ligand, limiting its spatial range of activity. In Drosophila, Interference hedgehog (Ihog) and Brother of ihog (Boi) are two conserved and redundant transmembrane proteins that are essential for Hh pathway activation. How Ihog and Boi activate signaling in response to Hh remains unknown; each can bind both Hh and Ptc and so it has been proposed that they are essential for both Hh reception and sequestration. Using genetic epistasis this study established that Ihog and Boi, and their orthologs in mice, act upstream or at the level of Ptc to allow Hh signal transduction. In the Drosophila developing wing model it was found that through Hh pathway activation Ihog and Boi maintain the boundary between the anterior and posterior compartments. The contributions of Ptc was dissociated from those of Ihog/Boi, and, surprisingly, it was found that cells expressing Ptc can retain and sequester the Hh ligand without Ihog and Boi, but that Ihog and Boi cannot do so without Ptc. Together, these results reinforce the central role for Ptc in Hh binding in vivo and demonstrate that, although Ihog and Boi are dispensable for Hh sequestration, they are essential for pathway activation because they allow Hh to inhibit Ptc and thereby relieve its repression of Smo (Camp, 2014).

Ihog and Boi have been shown to be absolutely essential within Hh-responding cells for activation of the Hh signaling pathway, acting upstream of Smo. The current experiments advance understanding of Ihog and Boi function by drawing three major conclusions: First, in genetic epistasis experiments it was found that Ihog and Boi also act upstream or at the level of Ptc, supporting the idea that they function through Ptc to relieve suppression of Smo. This epistatic relationship appears conserved in evolution, as it was found that Cdon and Boc also function upstream or at the level of Ptch1 for Hh signal transduction in mice. These genetic findings establish this relationship unequivocally, and so have profound implications for future studies to further clarify how these co-receptors participate in Hh reception and pathway activation (Camp, 2014).

Second, based on experiments to dissect the relative contributions of Ihog and Boi in processes involving Ptc, it is concluded that it is through their essential roles in Hh signal transduction that Ihog, Boi and Ptc contribute to anterior-posterior compartment segregation: once the pathway is activated, all three proteins are dispensable for maintenance of the compartment boundary, implicating other, yet unidentified, cell surface recognition molecules in compartment-specific cell affinity and adhesion (Camp, 2014).

Third, it is concluded that Ihog and Boi, unlike Ptc, are completely dispensable for the sequestration and retention of Hh. Cells lacking Ihog and Boi can sequester and retain the Hh signal if the pathway is activated and Ptc is upregulated. They do so via physiological levels of endogenous Ptc induced either by pathway activation in ^ptcS2 mutants or by expression of Smo^SD123. Incidentally, it was also found that Hh sequestration was rescued in boi;ihog double mutant clones overexpressing Ptc¹¹³⁰, a dominant-negative that fully activates the Hh pathway and upregulates endogenous, wild-type Ptc. This third conclusion is not consistent with the view that Ihog and Boi aid in addressing Ptc to the cell surface and that, once there, they are required for Ptc to bind and sequester Hh. This view is based primarily on Ptc and Ihog overexpression in cultured cells, and on an experiment that failed to restore Hh sequestration to boi;ihog double mutant cells with mutation of cAMP-dependent protein kinase 1 (Pka-C1), which upregulates Ptc and other target genes because loss of Pka-C1 disinhibits the activity of the transcription factor Ci. It is unclear why Ptc upregulation in Pka-C1 mutants was unable to rescue Hh sequestration in boi;ihog double mutants, whereas Ptc upregulation in the current experiments was able to do so. In cells lacking Ihog and Boi, perhaps the level to which Ptc is upregulated in Pka-C1 mutants is inadequate. Regardless, the current data indicate that Ptc has a central role in the binding and sequestration of Hh, whereas Ihog and Boi are dispensable, despite their requirement for Hh signal transduction (Camp, 2014).

The results are consistent with vertebrate systems, in which current models strongly favor direct contacts between Hh and Ptc, primarily because: (1) expression of the Ptc ortholog Ptch1 promotes binding of Shh to transfected cells, (2) radiolabeled Shh can be chemically cross-linked to Ptch1 expressed on the cell surface and (3) Ptch1 can reach the cell surface in the absence of the Ihog/Boi-related proteins Cdon and Boc. Whether Ptc is sufficient on its own to bind Hh remains an important question that awaits technically challenging studies using purified proteins. An alternative possibility is that Hh could have additional receptor(s), with candidates including the proteoglycans Dally and Dally-like (Dlp), and Shifted, a secreted protein of the Wnt inhibitory factor 1 (WIF1) family (Camp, 2014).

The results clearly distinguish a role for Ptc that relies on Ihog/Boi (Hh reception/signal transduction) from one that does not (Hh sequestration), and so they contribute to an emerging view of the function of Ihog, Boi and related proteins. In non-responding cells, others have shown that Ihog and Boi are involved in restricting the movement of Hh, and so may contribute to its overall distribution. Within Hh-responding cells, where Ptc is co-expressed with Ihog and Boi, it was found that Ihog and Boi are essential for Hh signal transduction, but not Hh sequestration and retention. As Ihog and Boi act upstream or at the level of Ptc, they must mediate a crucial, rate-limiting step in the inhibition of Ptc in response to Hh. However, as they are not essential for Ptc to bind Hh, how they affect Ptc function remains to be elucidated. Whereas the precise molecular mechanism remains elusive, several lines of evidence provide important clues. First, an Ihog variant lacking the cytoplasmic tail can rescue boi;ihog double mutants. Second, the second Fn3 domain of Ihog or Boi interacts physically with Ptc and is quite distinct from the first Fn3 domain that harbors the Hh-interacting surface. Third, the presence of Ihog or Boi potentiates co-immunoprecipitation of Hh and Ptc. Together, these results suggest that the primary role of Ihog and Boi in Hh signaling involves the ability of their ectodomains to form favorable protein complexes with Ptc or Hh, or with both simultaneously. Although the data indicate that Ptc does not need Ihog and Boi to bind Hh in vivo, it is surmised that it is through these multimolecular complexes that Ihog and Boi allow Hh to inhibit Ptc and thereby relieve its suppression of Smo and the Hh signaling cascade (Camp, 2014).

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