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The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking
Brief Communication 601
The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking Vero´nica Martı´n*, Graciela Carrillo*, Carlos Torroja and Isabel Guerrero The Hedgehog (Hh) family of signaling molecules function as organizers in many morphogenetic processes. Hh 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 sterolsensing domain (SSD). In the present study, we show that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. This mutant PtcSSD 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 dominantnegative activity, the mutant Ptc protein functioned like the wild-type protein in sequestering and internalizing Hh. In addition, we show that PtcSSD 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. Address: Centro de Biologı´a Molecular “Severo Ochoa,” Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´noma de Madrid, Cantoblanco, Madrid E-28049, Spain. * These authors contributed equally to this work. Correspondence: Isabel Guerrero E-mail: [email protected] Received: 2 January 2001 Revised: 6 March 2001 Accepted: 6 March 2001 Published: 17 April 2001 Current Biology 2001, 11:601–607 0960-9822/01/$ – see front matter 2001 Elsevier Science Ltd. All rights reserved.
Results and discussion The sterol-sensing domain of Ptc is required for Hh signaling
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 [1, 2]. To address the functional role of the SSD of Ptc, we engineered a G-to-A substitution that causes an Asp583 to change to Asn in the SSD (PtcSSD). 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 [3]. 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 [1] as well as in Ptc. We introduced the mutated Drosophila ptc cDNA into flies under UAS control and overexpressed the mutant protein in the ptc expression domain with ptc-GAL4. The expression of PtcSSD caused embryonic lethality and an almost complete ptc null phenotype (Figure 1a–c). Since the severity of the mutant phenotype correlated directly with the amount of PtcSSD expressed (not shown), we suggest that PtcSSD competes with the wild-type protein and has a dominant-negative effect. In fly embryos and imaginal discs, hh is expressed by posterior (P) compartment cells and signals adjacent anterior (A) compartment cells. Hh has a number of targets at the A/P compartment border. Such targets include wingless (wg) and the TGF- super-family member, decapentaplegic (dpp) [4]. To understand how PtcSSD affects Hh targets, we examined wg expression in embryos expressing PtcSSD under ptc-GAL4. In wild-type embryos, wg becomes dependent on Hh during embryogenesis. Ptc, a negative regulator of Hh signaling, acts as a repressor of wg expression, and the wg expression domain expands into the more anterior part of each segment in ptc⫺ mutants. Embryos expressing UAS-PtcSSD also showed wg expression extended into the anterior region of each segment (Figure 1d,e). We also analyzed how PtcSSD affects wing imaginal discs. In wild-type wing discs, different target genes respond to Hh at various distances from the A/P border, presumably due to the varying Hh concentration [5]. A characteristic feature of homozygous ptc⫺ clones in the A compartment of the wing discs is the autonomous activation of all Hh target genes in an apparently ligand-independent manner. We found that PtcSSD, ectopically expressed in A compartment clones, autonomously activated some but not all target genes. Genes such as the iroquois complex genes (Figure 1f,g), dpp (Figure 1h,i), and ptc (Figure 1j,k) that respond to lower Hh levels were induced by ectopic PtcSSD. However, genes such as engrailed [6] and collier [7]
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Figure 1
Ectopic PtcSSD expression induces the ptc mutant phenotype and the activation of Hh target genes. (a–c) Cuticle phenotype of (a) wild type, (b) UAS-PtcSSD/ptc-GAL4, (c) and ptcIIW109 larvae. Embryos are oriented anteriorly to the left. (c) Note that the involution of the head is not well formed (arrowhead) and the thoracic and first abdominal segment (A1) are absent in ptc⫺ larvae. (b) In UAS-PtcSSD/ptc-GAL4, these structures are still present, but remaining abdominal segments are as in ptc⫺ embryos. (d) Wg expression in the wild type is restricted to single cell stripes. (e) In UAS-PtcSSD/ptc-GAL4 embryos, Wg stripes are wider than in wild-type embryos, as observed in ptc⫺ embryos (data not shown). The phenotype of the UAS-PtcSSD/ptcGAL4 embryos shown in (b) and (e) is obtained at 29⬚C and is less severe at 18⬚C. These findings suggest that PtcSSD competes with the wild-type protein and has a dominant-negative effect. (f–m) Activation of Hh target genes by PtcSSD in the wing disc. Wild-type expression of (f) Caupolican protein of the iro complex , (h) dpp, (j) ptc, and (l) Collier in the third-instar wing imaginal disc. Ectopic PtcSSD clones induce (g) Caupolican expression (green) and (i) dpp-LacZ (red) in the A compartment of wing imaginal disc. Clones are
marked by the presence of Ptc protein (red in [g] and green in [i]). A stippled line marks the A/P compartment boundary. (k) Uniform PtcSSD expression via the c765-GAL4 line induces the expression of endogenous ptc (shown in red by ptc-LacZ reporter gene expression) and homogenous high levels of cytoplasmic Ci (green). (m) Collier protein (green) is only activated in some cells of the A compartment in a heterozygous background (ptc⫹/⫺; c765-GAL4 /UASPtcSSD). (n–p) Wing phenotypes. (n) Wild-type wing. Numbers label the veins, and a stippled line marks the A/P compartment border. (o) PtcSSD activation gives rise to overgrowth and vein disorganization of the A compartment (asterisk) in a UAS-PtcSSD/c765-GAL4 adult wing compared to a wild-type wing. (p) Ectopic PtcS2F expression via the c765-GAL4 line that contains the other mutation found in the ptcS2 allele (Val1392-to-Met substitution) gives rise to wing reduction. The phenotype is the same as that of the ectopic PtcWT. Note the wing size reduction produced by blocking the induction of the Hh targets at the A/P compartment border, and compare this with the overgrown UAS-PtcSSD/c765-GAL4 wings shown in (o).
that respond to maximum levels of Hh were not expressed (data not shown). The presence of wild-type Ptc protein might temper the activation of the target genes induced by ectopic PtcSSD. Consequently, ectopic PtcSSD started to activate collier when doses of the endogenous wild-type Ptc were reduced (Figure 1l,m). Wing phenotypes induced by ectopic expression of PtcSSD also mimicked the lack of function of ptc mutants (Figure 1o) and were thus consistent with a dominant-negative role. In summary, these results indicate that the mutant PtcSSD protein is incapable of repressing the Hh pathway and suggest that the SSD is essential for Ptc function.
Mutation in the SSD does not affect Hh sequestration
Ptc also has a function in circumscribing the area over which Hh acts by sequestering Hh. We subsequently determined whether this other function of Ptc was modified in PtcSSD. Ptc levels rise in A cells in response to Hh signaling [8]. These high levels of Ptc protein limit the range of Hh signaling [9]. Hh and Ptc colocalize in vesicular punctate structures in the Hh-receiving cells, and this finding suggests that Ptc internalizes Hh [10]. We examined this effect by expressing the wild-type Ptc protein in the P compartment, where Ptc is not normally present (Figure 2a,b). Hh staining is normally diffuse in P com-
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Figure 2
PtcSSD sequesters and internalizes Hh. (a–d) Hh sequestration by PtcWT and PtcSSD. Hh (detected by anti-Hh antibody in green) is sequestered and internalized by Ptc (detected by anti-Ptc antibody in red) (arrowheads). Hh sequestration occurs in ectopic posterior clones expressing either (a) PtcWT or (c) PtcSSD (arrows). Also, ectopic (b) PtcWT and (d) PtcSSD expression via c765-GAL4 show Hh sequestration (green) by Ptc (red). Detail is shown of the cellular distribution of Ptc and Hh in ectopic (bⴕ,b″) PtcWT and (dⴕ,d″) PtcSSD. Note that Hh levels (green) inside (d⬘) PtcSSD-expressing cells are comparatively higher than in (b⬘) PtcWT cells. Also note the preferential location of (d″) PtcSSD in punctate structures compared
to the more diffusely distributed (b″) PtcWT. (e,f) PtcSSD blocks the induction of Hh target genes at the A/P border. (e) Wild-type en (red), Ci (green), and dpp-LacZ (blue) expression. Note the thickness of dpp-LacZ expression (bracket 1) and the increase in cytoplasmic Ci levels (bracket 2). (f,fⴕ,f″) PtcSSD expressed in the en domain (UASPtcSSD/en-GAL4) shows (in detail in [f⬘f″]; also arrow) the lack of late en expression in the A compartment; (f,f⬘) the absence of increased cytoplasmic levels of Cubitus interruptus (Ci), the nuclear effector of the Hh pathway; and (f,f″) the decrease in dpp expression. (g) Phenotype of UAS-PtcSSD/en-GAL4 adult wings. A similar phenotype is observed in UAS-PtcWT/en-GAL4 wings (data not shown).
partment cells. However, the distribution of Hh changed in Ptc-expressing cells, where both Ptc and Hh colocalized in punctate structures (Figure 2b⬘,b″). In addition, Hh levels were elevated in Ptc-expressing clones relative to other P compartment cells (Figure 2a, arrow). Ectopic PtcSSD clones in the P compartment provoked the same changes in Hh distribution as did ectopic PtcWT (Figure 2c, arrow). However, the levels of sequestered Hh in the cells that ectopically expressed PtcSSD (Figure 2d⬘) were higher than in those expressing PtcWT (Figure 2b⬘). These findings suggest that PtcSSD maintains the ability to internalize and sequester Hh but to accumulate a higher amount of Hh than the does PtcWT protein.
A/P compartment border. This was shown by the lack of late en expression in the A compartment (Figure 2f,f⬘,f″); the absence of increased cytoplasmic levels of Cubitus interruptus (Ci), the nuclear effector of the Hh pathway (Figure 2f,f⬘); and the decrease in dpp expression (Figure 2f,f″). This low Hh response at the A/P border causes the fusion of wing veins 3 and 4 [12] (Figure 2g). Taken together, these results suggest that the SSD of Ptc is not specifically required for Hh sequestration.
It is known that ectopic PtcWT in the entire P compartment is able to restrain the secretion of Hh at the A compartment and thus impede the appropriate formation of the Hh gradient and the correct activation of the target gene [11]. Here, we observed that ectopic PtcSSD in the entire P compartment also blocked the response to Hh at the
The ability of some proteins to preferentially associate with membrane subdomains rich in cholesterol and sphingolipids called rafts is thought to be the basis for specific clustering events that regulate protein trafficking and signal transduction [13]. Hh modified by cholesterol is associated with rafts [14]. Thus, Ptc might require cholesterol for its association with rafts and for Hh binding. However, our results indicate that, regardless of whether Ptc is localized in the rafts or not, mutations in the SSD do not affect
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Figure 3 Upregulation of Smo protein levels by PtcSSD. (a–e) Ectopic Smo elicits similar cellular responses and a similar phenotype as PtcSSD. (a) Ectopic Smo clones (red) (flip-out clones of abx-GAL4 marked by anti–-Gal antibody) induce high levels of Ptc expression (green detected by anti-Ptc antibody) in the A compartment. (b) Uniform high levels of Smo protein (green) driven by the c765-GAL4 line activate dpp-LacZ (red) in the A compartment of the wing disc. However, genes such as en, expressed late in the A compartment, and collier that respond to maximum values of Hh are not activated (data not shown). This indicates that responses to either ectopic Smo or PtcSSD are the same. (c) Phenotype of UAS-Smo/c765-GAL4 wings. Note that the overgrowth and vein alteration in the A compartment (asterisk) of the wing is similar to that observed in the UAS-PtcSSD/c765GAL4 wings shown in Figure 1o. (d,e) Uniform high levels of both Smo (green) and PtcWT proteins driven by the c765-GAL4 line (d) normalize dpp-LacZ expression (red) and (e) rescue the wing phenotype produced by uniform levels of Smo protein driven by the same c765-GAL4 line. (f–g) PtcSSD causes the stabilization of Smo levels. (f) Ectopic PtcWT clones marked by the presence of Ptc protein (red) decrease Smo protein levels (green) in the P compartment (arrows). (fⴕ) Detail of the downregulation of Smo by Ptc. (g) Ectopic PtcSSD clones labeled by anti-Ptc antibody (red) induce higher Smo protein levels (green) in the A compartment (arrows). Note the autonomous effect of PtcSSD clones in the modulation of Smo levels in the A compartment.
the sequestering of Hh. It is therefore possible that the mutated PtcSSD still preferentially associates with rafts. The preexisting Ptc allele also has a mutation in the SSD
Next, we searched for mutations in different Ptc functional domains. For this, we performed a complementation assay of known ptc alleles with a dominant ptc gainof-function allele, ptcCon [5]. ptcCon/ptc⫺ flies died at thirdlarval instar, and the imaginal discs were small because most Hh targets were not activated. Genetic interaction between PtcCon and Hh and the localization of a point mutation in one of the extracelullar loops of Ptc suggest that this PtcCon protein affects binding to Hh [5]. One out of 15 ptc alleles, ptcS2, complemented with ptcCon (see Table S1 in the Supplementary material available with this article on the internet). Two point mutations were found in the Ptc sequence. The first point mutation was identical to the PtcSSD mutation previously characterized, and the second mutation was located in the carboxy tail of the protein (base 4174 was mutated from G to A). These mutations gave rise to a Val1392-to-Met substitution. To test if this second point mutation could also be responsible
for the ptc⫺ phenotype of ptcS2, we introduced cDNA containing this second point mutation into flies under UAS control. Flies expressing this mutant Ptc protein were viable and showed a phenotype (Figure 1p) similar to the one produced by overexpression of the wild-type protein. Therefore, we concluded that the mutation in the SSD is responsible for the lack-of-function phenotype in the ptcS2 allele. This result is consistent with previous work demonstrating that the ptcS2 allele behaves as a lack-offunction allele of ptc for blocking the Hh pathway but that the mutant protein produced is still able to sequester Hh [8]. Upregulation of Smo protein levels by PtcSSD
Hh signal transduction requires another transmembrane protein, Smoothened (Smo), but it has yet to be established whether Smo can bind Hh directly [15]. The balance between Ptc and Smo proteins seems to be fundamental in blocking or activating the Hh pathway [16, 17]. In the absence of ligand, Ptc inhibits the activity of Smo by downregulating Smo protein levels, but in the presence of Hh, this inhibition is released and the signal transduc-
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Figure 4 PtcSSD protein accumulates in the endocytic compartment. (a) Triple staining for Hh (blue and gray) internalized Texas-red dextran (red) and Ptc (green) in the A/P compartment border cells of a wild-type wing disc. The three channels show colocalization of Hh and Ptc proteins in early endosomes (arrowheads). (b) Double staining for Ptc (green) and internalized Texas-red dextran (red), which labeled early endosomes in the PtcS2/S2 clone. The white line indicates the A/P border. (bⴕ) Detail of the PtcS2/S2 clone framed in (b). Note that colocalization between PtcSSD protein and internalized Texas-red dextran is identical to that shown in (a) for Hh and Ptc at the A/P border of a wild-type disc. (c) Double staining for Ptc (red) and Smo (green) proteins in the A/P compartment border cells (Hh-receiving cells) of a wild-type wing disc. Note the different cellular distribution pattern of both proteins; Smo is mainly localized at the plasma membrane (arrows), and Ptc is preferentially observed in large punctate structures (arrowheads). (d) Double staining for internalized Texas-red dextran (red), which in this case labels early endosomes (see Materials and methods in Supplementary material) and Smo (green). Note the lack of colocalization between the dextran vesicles and Smo. White frames outline the area magnified in the individual channel panels.
tion cascade is activated. Smo levels are high in the P compartment and in Hh-responsive A cells. Smo expression decreases sharply near the boundary and then more gradually across the A compartment ([16]; Figure 3f). Previous observations indicate that the ectopic expression of relatively large amounts of smo mRNA fails to induce ectopic Hh signal transduction. This prompted the suggestion that Ptc may act catalytically rather than stoichiometrically repressing Smo activity [16, 18]. However, we found that Smo overexpression induced the activation of the Hh pathway. This indicates that high Smo levels cannot be downregulated by Ptc. Ectopic Smo expression produced the same activation of the target genes (Figure 3a,b) and a phenotype (Figure 3c) similar to that of ectopic PtcSSD. Consistently, the phenotype obtained by Smo overexpression was reversed when wild-type Ptc and Smo proteins were coexpressed (Figure 3d,e). To determine how PtcSSD modulates Smo protein levels compared to the PtcWT protein, we examined the Smo staining pattern in ectopic clones of these two Ptc protein forms. Ectopic PtcWT, but not PtcSSD, clones negatively modulated Smo levels in the P compartment (Figure 3f, arrows). Ectopic PtcSSD, but not PtcWT, clones increased Smo levels in the
A compartment (Figure 3g, arrows). These results indicate that Ptc is a modulator of Smo levels according to the results recently reported by Denef et al. [16]. The opposite modulation of Smo levels in cells that ectopically express either PtcSSD or PtcWT proteins indicates that PtcSSD causes the stabilization of Smo levels, probably because it cannot interact with Smo. The negative and positive regulation of Ptc and Smo in Hh signaling has also been described in tumorogenic processes in vertebrates, where Ptc acts as a tumor suppressor gene by repressing the oncogenic activity of Smo during transduction of the Hh signal [19]. In addition, steroidal alkaloids analogous to cholesterol, such as cyclopamine [20], can reverse oncogenic mutations in Smo and Ptc. This steroidal alkaloid appears to be a specific antagonist of Shh signal transduction in mouse and chick and produces a phenocopy of the Shh loss-of-function mutation [21, 22]. Cyclopamine inhibits the Shh pathway by antagonizing Smo and may act by affecting the balance between active and inactive forms of Smo [20]. Herein, we show that mutations in the SSD of Ptc exert the opposite effect to wild-type Ptc and thus give rise to higher Smo activity. This effect is similar to that produced
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by increasing Smo protein levels. These results indicate that the SSD domain of Ptc is required to negatively regulate Smo activity. Like cyclopamine, Ptc activity shifts the Smo balance toward the inactive state, and our finding suggests that Ptc could be the direct or indirect target of cyclopamine in the Hh signaling pathway.
PtcSSD protein accumulates in endosomes
We previously reported that Ptc is located inside the cell, mainly in cytoplasmic vesicles, and that when internalization is blocked in shibire mutant embryos, Ptc is associated with the plasma membrane [23]. Here, we were able to identify the large vesicles in which Ptc and Hh accumulate in Hh-responsive cells as endosomes (Figure 4a) since they colocalized with internalized Texas-red dextran [24]. The same colocalization of Ptc and internalized Texasred dextran also occurred in ptcS2 mutant clones (which only expressed the PtcS2 protein; Figure 4b,b⬘). However, Smo protein was not localized with Ptc (Figure 4c) in the endocytic compartment (Figure 4d) and was mainly observed along the basolateral plasma membrane (Figure 4c,d), as also shown by Denef et al. [16]. Thus, the Ptc mutation in the SSD alters the subcellular distribution of Ptc, as also occurs upon Hh binding. We also observed this preferential location of PtcSSD in punctate structures when we compared the ectopic expression of PtcSSD with that of PtcWT (which showed a more generalized distribution; Figure 2b″,d″). 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 (C. T., V. M. and I. G., unpublished data).
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 [22, 25]. It has been reported that a lesion in the NPC1 protein, which is normally found in cytoplasmic vesicles characteristic of late endosomes [26], produces a general defect in the retrieval and recycling of raft components of the endocytic pathway [27]. Ptc and NPC1 colocalize extensively in vesicular compartments in cotransfected cells [25]. This suggests that the function of both NPC1 and Ptc involves a common vesicular transport pathway.
To conclude, the upregulation of Smo protein and the opening of the Hh pathway in PtcSSD mutant cells are related to the preferential accumulation of PtcSSD 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.
Supplementary material Supplementary materials and methods are available with the electronic version of this article at http://images.cellpress.com/supmat/supmatin.htm.
Acknowledgements The authors are very grateful to Carmen Iban˜ez and Marı´a Sanz for their excellent technical assistance and to Jose´ Luis Mullor for his help in the initial stages of this work. We are indebted to Marcos Gonza´lez-Gaita´n and Eugeni Entchev for their help in setting up the endocytosis assays, to Ignacio Sandoval, Tom Kornberg, and Nicole Gorfinkiel for useful discussions and for comments on the manuscript. We thank S. Cohen for the anti-Smo antibody, J. Modolell for the anti-Caup antibody, A.Vincent for the anti-Col antibody, R. Holmgren for the anti-Ci antibody, and F. de Celis, T. Kornberg, G. Struhl, and S. L. Zipurski for fly strains. This work was financed by grant PB98-0680 of the Direccio´n General de Investigacio´n Cientifica y Te´cnica (DGICYT), grant 08.9/0001/1998 of the Comunidad Auto´noma de Madrid, and an institutional grant from the Fundacio´n Areces. V. M., G. C., and C. T. were financially supported by fellowships awarded by the Comunidad Auto´noma de Madrid.
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The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking Vero´nica Martı´n*, Graciela Carrillo*, Carlos Torroja and Isabel Guerrero The Hedgehog (Hh) family of signaling molecules function as organizers in many morphogenetic processes. Hh 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 sterolsensing domain (SSD). In the present study, we show that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. This mutant PtcSSD 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 dominantnegative activity, the mutant Ptc protein functioned like the wild-type protein in sequestering and internalizing Hh. In addition, we show that PtcSSD 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. Address: Centro de Biologı´a Molecular “Severo Ochoa,” Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´noma de Madrid, Cantoblanco, Madrid E-28049, Spain. * These authors contributed equally to this work. Correspondence: Isabel Guerrero E-mail: [email protected] Received: 2 January 2001 Revised: 6 March 2001 Accepted: 6 March 2001 Published: 17 April 2001 Current Biology 2001, 11:601–607 0960-9822/01/$ – see front matter 2001 Elsevier Science Ltd. All rights reserved.
Results and discussion The sterol-sensing domain of Ptc is required for Hh signaling
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 [1, 2]. To address the functional role of the SSD of Ptc, we engineered a G-to-A substitution that causes an Asp583 to change to Asn in the SSD (PtcSSD). 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 [3]. 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 [1] as well as in Ptc. We introduced the mutated Drosophila ptc cDNA into flies under UAS control and overexpressed the mutant protein in the ptc expression domain with ptc-GAL4. The expression of PtcSSD caused embryonic lethality and an almost complete ptc null phenotype (Figure 1a–c). Since the severity of the mutant phenotype correlated directly with the amount of PtcSSD expressed (not shown), we suggest that PtcSSD competes with the wild-type protein and has a dominant-negative effect. In fly embryos and imaginal discs, hh is expressed by posterior (P) compartment cells and signals adjacent anterior (A) compartment cells. Hh has a number of targets at the A/P compartment border. Such targets include wingless (wg) and the TGF- super-family member, decapentaplegic (dpp) [4]. To understand how PtcSSD affects Hh targets, we examined wg expression in embryos expressing PtcSSD under ptc-GAL4. In wild-type embryos, wg becomes dependent on Hh during embryogenesis. Ptc, a negative regulator of Hh signaling, acts as a repressor of wg expression, and the wg expression domain expands into the more anterior part of each segment in ptc⫺ mutants. Embryos expressing UAS-PtcSSD also showed wg expression extended into the anterior region of each segment (Figure 1d,e). We also analyzed how PtcSSD affects wing imaginal discs. In wild-type wing discs, different target genes respond to Hh at various distances from the A/P border, presumably due to the varying Hh concentration [5]. A characteristic feature of homozygous ptc⫺ clones in the A compartment of the wing discs is the autonomous activation of all Hh target genes in an apparently ligand-independent manner. We found that PtcSSD, ectopically expressed in A compartment clones, autonomously activated some but not all target genes. Genes such as the iroquois complex genes (Figure 1f,g), dpp (Figure 1h,i), and ptc (Figure 1j,k) that respond to lower Hh levels were induced by ectopic PtcSSD. However, genes such as engrailed [6] and collier [7]
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Figure 1
Ectopic PtcSSD expression induces the ptc mutant phenotype and the activation of Hh target genes. (a–c) Cuticle phenotype of (a) wild type, (b) UAS-PtcSSD/ptc-GAL4, (c) and ptcIIW109 larvae. Embryos are oriented anteriorly to the left. (c) Note that the involution of the head is not well formed (arrowhead) and the thoracic and first abdominal segment (A1) are absent in ptc⫺ larvae. (b) In UAS-PtcSSD/ptc-GAL4, these structures are still present, but remaining abdominal segments are as in ptc⫺ embryos. (d) Wg expression in the wild type is restricted to single cell stripes. (e) In UAS-PtcSSD/ptc-GAL4 embryos, Wg stripes are wider than in wild-type embryos, as observed in ptc⫺ embryos (data not shown). The phenotype of the UAS-PtcSSD/ptcGAL4 embryos shown in (b) and (e) is obtained at 29⬚C and is less severe at 18⬚C. These findings suggest that PtcSSD competes with the wild-type protein and has a dominant-negative effect. (f–m) Activation of Hh target genes by PtcSSD in the wing disc. Wild-type expression of (f) Caupolican protein of the iro complex , (h) dpp, (j) ptc, and (l) Collier in the third-instar wing imaginal disc. Ectopic PtcSSD clones induce (g) Caupolican expression (green) and (i) dpp-LacZ (red) in the A compartment of wing imaginal disc. Clones are
marked by the presence of Ptc protein (red in [g] and green in [i]). A stippled line marks the A/P compartment boundary. (k) Uniform PtcSSD expression via the c765-GAL4 line induces the expression of endogenous ptc (shown in red by ptc-LacZ reporter gene expression) and homogenous high levels of cytoplasmic Ci (green). (m) Collier protein (green) is only activated in some cells of the A compartment in a heterozygous background (ptc⫹/⫺; c765-GAL4 /UASPtcSSD). (n–p) Wing phenotypes. (n) Wild-type wing. Numbers label the veins, and a stippled line marks the A/P compartment border. (o) PtcSSD activation gives rise to overgrowth and vein disorganization of the A compartment (asterisk) in a UAS-PtcSSD/c765-GAL4 adult wing compared to a wild-type wing. (p) Ectopic PtcS2F expression via the c765-GAL4 line that contains the other mutation found in the ptcS2 allele (Val1392-to-Met substitution) gives rise to wing reduction. The phenotype is the same as that of the ectopic PtcWT. Note the wing size reduction produced by blocking the induction of the Hh targets at the A/P compartment border, and compare this with the overgrown UAS-PtcSSD/c765-GAL4 wings shown in (o).
that respond to maximum levels of Hh were not expressed (data not shown). The presence of wild-type Ptc protein might temper the activation of the target genes induced by ectopic PtcSSD. Consequently, ectopic PtcSSD started to activate collier when doses of the endogenous wild-type Ptc were reduced (Figure 1l,m). Wing phenotypes induced by ectopic expression of PtcSSD also mimicked the lack of function of ptc mutants (Figure 1o) and were thus consistent with a dominant-negative role. In summary, these results indicate that the mutant PtcSSD protein is incapable of repressing the Hh pathway and suggest that the SSD is essential for Ptc function.
Mutation in the SSD does not affect Hh sequestration
Ptc also has a function in circumscribing the area over which Hh acts by sequestering Hh. We subsequently determined whether this other function of Ptc was modified in PtcSSD. Ptc levels rise in A cells in response to Hh signaling [8]. These high levels of Ptc protein limit the range of Hh signaling [9]. Hh and Ptc colocalize in vesicular punctate structures in the Hh-receiving cells, and this finding suggests that Ptc internalizes Hh [10]. We examined this effect by expressing the wild-type Ptc protein in the P compartment, where Ptc is not normally present (Figure 2a,b). Hh staining is normally diffuse in P com-
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Figure 2
PtcSSD sequesters and internalizes Hh. (a–d) Hh sequestration by PtcWT and PtcSSD. Hh (detected by anti-Hh antibody in green) is sequestered and internalized by Ptc (detected by anti-Ptc antibody in red) (arrowheads). Hh sequestration occurs in ectopic posterior clones expressing either (a) PtcWT or (c) PtcSSD (arrows). Also, ectopic (b) PtcWT and (d) PtcSSD expression via c765-GAL4 show Hh sequestration (green) by Ptc (red). Detail is shown of the cellular distribution of Ptc and Hh in ectopic (bⴕ,b″) PtcWT and (dⴕ,d″) PtcSSD. Note that Hh levels (green) inside (d⬘) PtcSSD-expressing cells are comparatively higher than in (b⬘) PtcWT cells. Also note the preferential location of (d″) PtcSSD in punctate structures compared
to the more diffusely distributed (b″) PtcWT. (e,f) PtcSSD blocks the induction of Hh target genes at the A/P border. (e) Wild-type en (red), Ci (green), and dpp-LacZ (blue) expression. Note the thickness of dpp-LacZ expression (bracket 1) and the increase in cytoplasmic Ci levels (bracket 2). (f,fⴕ,f″) PtcSSD expressed in the en domain (UASPtcSSD/en-GAL4) shows (in detail in [f⬘f″]; also arrow) the lack of late en expression in the A compartment; (f,f⬘) the absence of increased cytoplasmic levels of Cubitus interruptus (Ci), the nuclear effector of the Hh pathway; and (f,f″) the decrease in dpp expression. (g) Phenotype of UAS-PtcSSD/en-GAL4 adult wings. A similar phenotype is observed in UAS-PtcWT/en-GAL4 wings (data not shown).
partment cells. However, the distribution of Hh changed in Ptc-expressing cells, where both Ptc and Hh colocalized in punctate structures (Figure 2b⬘,b″). In addition, Hh levels were elevated in Ptc-expressing clones relative to other P compartment cells (Figure 2a, arrow). Ectopic PtcSSD clones in the P compartment provoked the same changes in Hh distribution as did ectopic PtcWT (Figure 2c, arrow). However, the levels of sequestered Hh in the cells that ectopically expressed PtcSSD (Figure 2d⬘) were higher than in those expressing PtcWT (Figure 2b⬘). These findings suggest that PtcSSD maintains the ability to internalize and sequester Hh but to accumulate a higher amount of Hh than the does PtcWT protein.
A/P compartment border. This was shown by the lack of late en expression in the A compartment (Figure 2f,f⬘,f″); the absence of increased cytoplasmic levels of Cubitus interruptus (Ci), the nuclear effector of the Hh pathway (Figure 2f,f⬘); and the decrease in dpp expression (Figure 2f,f″). This low Hh response at the A/P border causes the fusion of wing veins 3 and 4 [12] (Figure 2g). Taken together, these results suggest that the SSD of Ptc is not specifically required for Hh sequestration.
It is known that ectopic PtcWT in the entire P compartment is able to restrain the secretion of Hh at the A compartment and thus impede the appropriate formation of the Hh gradient and the correct activation of the target gene [11]. Here, we observed that ectopic PtcSSD in the entire P compartment also blocked the response to Hh at the
The ability of some proteins to preferentially associate with membrane subdomains rich in cholesterol and sphingolipids called rafts is thought to be the basis for specific clustering events that regulate protein trafficking and signal transduction [13]. Hh modified by cholesterol is associated with rafts [14]. Thus, Ptc might require cholesterol for its association with rafts and for Hh binding. However, our results indicate that, regardless of whether Ptc is localized in the rafts or not, mutations in the SSD do not affect
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Figure 3 Upregulation of Smo protein levels by PtcSSD. (a–e) Ectopic Smo elicits similar cellular responses and a similar phenotype as PtcSSD. (a) Ectopic Smo clones (red) (flip-out clones of abx-GAL4 marked by anti–-Gal antibody) induce high levels of Ptc expression (green detected by anti-Ptc antibody) in the A compartment. (b) Uniform high levels of Smo protein (green) driven by the c765-GAL4 line activate dpp-LacZ (red) in the A compartment of the wing disc. However, genes such as en, expressed late in the A compartment, and collier that respond to maximum values of Hh are not activated (data not shown). This indicates that responses to either ectopic Smo or PtcSSD are the same. (c) Phenotype of UAS-Smo/c765-GAL4 wings. Note that the overgrowth and vein alteration in the A compartment (asterisk) of the wing is similar to that observed in the UAS-PtcSSD/c765GAL4 wings shown in Figure 1o. (d,e) Uniform high levels of both Smo (green) and PtcWT proteins driven by the c765-GAL4 line (d) normalize dpp-LacZ expression (red) and (e) rescue the wing phenotype produced by uniform levels of Smo protein driven by the same c765-GAL4 line. (f–g) PtcSSD causes the stabilization of Smo levels. (f) Ectopic PtcWT clones marked by the presence of Ptc protein (red) decrease Smo protein levels (green) in the P compartment (arrows). (fⴕ) Detail of the downregulation of Smo by Ptc. (g) Ectopic PtcSSD clones labeled by anti-Ptc antibody (red) induce higher Smo protein levels (green) in the A compartment (arrows). Note the autonomous effect of PtcSSD clones in the modulation of Smo levels in the A compartment.
the sequestering of Hh. It is therefore possible that the mutated PtcSSD still preferentially associates with rafts. The preexisting Ptc allele also has a mutation in the SSD
Next, we searched for mutations in different Ptc functional domains. For this, we performed a complementation assay of known ptc alleles with a dominant ptc gainof-function allele, ptcCon [5]. ptcCon/ptc⫺ flies died at thirdlarval instar, and the imaginal discs were small because most Hh targets were not activated. Genetic interaction between PtcCon and Hh and the localization of a point mutation in one of the extracelullar loops of Ptc suggest that this PtcCon protein affects binding to Hh [5]. One out of 15 ptc alleles, ptcS2, complemented with ptcCon (see Table S1 in the Supplementary material available with this article on the internet). Two point mutations were found in the Ptc sequence. The first point mutation was identical to the PtcSSD mutation previously characterized, and the second mutation was located in the carboxy tail of the protein (base 4174 was mutated from G to A). These mutations gave rise to a Val1392-to-Met substitution. To test if this second point mutation could also be responsible
for the ptc⫺ phenotype of ptcS2, we introduced cDNA containing this second point mutation into flies under UAS control. Flies expressing this mutant Ptc protein were viable and showed a phenotype (Figure 1p) similar to the one produced by overexpression of the wild-type protein. Therefore, we concluded that the mutation in the SSD is responsible for the lack-of-function phenotype in the ptcS2 allele. This result is consistent with previous work demonstrating that the ptcS2 allele behaves as a lack-offunction allele of ptc for blocking the Hh pathway but that the mutant protein produced is still able to sequester Hh [8]. Upregulation of Smo protein levels by PtcSSD
Hh signal transduction requires another transmembrane protein, Smoothened (Smo), but it has yet to be established whether Smo can bind Hh directly [15]. The balance between Ptc and Smo proteins seems to be fundamental in blocking or activating the Hh pathway [16, 17]. In the absence of ligand, Ptc inhibits the activity of Smo by downregulating Smo protein levels, but in the presence of Hh, this inhibition is released and the signal transduc-
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Figure 4 PtcSSD protein accumulates in the endocytic compartment. (a) Triple staining for Hh (blue and gray) internalized Texas-red dextran (red) and Ptc (green) in the A/P compartment border cells of a wild-type wing disc. The three channels show colocalization of Hh and Ptc proteins in early endosomes (arrowheads). (b) Double staining for Ptc (green) and internalized Texas-red dextran (red), which labeled early endosomes in the PtcS2/S2 clone. The white line indicates the A/P border. (bⴕ) Detail of the PtcS2/S2 clone framed in (b). Note that colocalization between PtcSSD protein and internalized Texas-red dextran is identical to that shown in (a) for Hh and Ptc at the A/P border of a wild-type disc. (c) Double staining for Ptc (red) and Smo (green) proteins in the A/P compartment border cells (Hh-receiving cells) of a wild-type wing disc. Note the different cellular distribution pattern of both proteins; Smo is mainly localized at the plasma membrane (arrows), and Ptc is preferentially observed in large punctate structures (arrowheads). (d) Double staining for internalized Texas-red dextran (red), which in this case labels early endosomes (see Materials and methods in Supplementary material) and Smo (green). Note the lack of colocalization between the dextran vesicles and Smo. White frames outline the area magnified in the individual channel panels.
tion cascade is activated. Smo levels are high in the P compartment and in Hh-responsive A cells. Smo expression decreases sharply near the boundary and then more gradually across the A compartment ([16]; Figure 3f). Previous observations indicate that the ectopic expression of relatively large amounts of smo mRNA fails to induce ectopic Hh signal transduction. This prompted the suggestion that Ptc may act catalytically rather than stoichiometrically repressing Smo activity [16, 18]. However, we found that Smo overexpression induced the activation of the Hh pathway. This indicates that high Smo levels cannot be downregulated by Ptc. Ectopic Smo expression produced the same activation of the target genes (Figure 3a,b) and a phenotype (Figure 3c) similar to that of ectopic PtcSSD. Consistently, the phenotype obtained by Smo overexpression was reversed when wild-type Ptc and Smo proteins were coexpressed (Figure 3d,e). To determine how PtcSSD modulates Smo protein levels compared to the PtcWT protein, we examined the Smo staining pattern in ectopic clones of these two Ptc protein forms. Ectopic PtcWT, but not PtcSSD, clones negatively modulated Smo levels in the P compartment (Figure 3f, arrows). Ectopic PtcSSD, but not PtcWT, clones increased Smo levels in the
A compartment (Figure 3g, arrows). These results indicate that Ptc is a modulator of Smo levels according to the results recently reported by Denef et al. [16]. The opposite modulation of Smo levels in cells that ectopically express either PtcSSD or PtcWT proteins indicates that PtcSSD causes the stabilization of Smo levels, probably because it cannot interact with Smo. The negative and positive regulation of Ptc and Smo in Hh signaling has also been described in tumorogenic processes in vertebrates, where Ptc acts as a tumor suppressor gene by repressing the oncogenic activity of Smo during transduction of the Hh signal [19]. In addition, steroidal alkaloids analogous to cholesterol, such as cyclopamine [20], can reverse oncogenic mutations in Smo and Ptc. This steroidal alkaloid appears to be a specific antagonist of Shh signal transduction in mouse and chick and produces a phenocopy of the Shh loss-of-function mutation [21, 22]. Cyclopamine inhibits the Shh pathway by antagonizing Smo and may act by affecting the balance between active and inactive forms of Smo [20]. Herein, we show that mutations in the SSD of Ptc exert the opposite effect to wild-type Ptc and thus give rise to higher Smo activity. This effect is similar to that produced
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by increasing Smo protein levels. These results indicate that the SSD domain of Ptc is required to negatively regulate Smo activity. Like cyclopamine, Ptc activity shifts the Smo balance toward the inactive state, and our finding suggests that Ptc could be the direct or indirect target of cyclopamine in the Hh signaling pathway.
PtcSSD protein accumulates in endosomes
We previously reported that Ptc is located inside the cell, mainly in cytoplasmic vesicles, and that when internalization is blocked in shibire mutant embryos, Ptc is associated with the plasma membrane [23]. Here, we were able to identify the large vesicles in which Ptc and Hh accumulate in Hh-responsive cells as endosomes (Figure 4a) since they colocalized with internalized Texas-red dextran [24]. The same colocalization of Ptc and internalized Texasred dextran also occurred in ptcS2 mutant clones (which only expressed the PtcS2 protein; Figure 4b,b⬘). However, Smo protein was not localized with Ptc (Figure 4c) in the endocytic compartment (Figure 4d) and was mainly observed along the basolateral plasma membrane (Figure 4c,d), as also shown by Denef et al. [16]. Thus, the Ptc mutation in the SSD alters the subcellular distribution of Ptc, as also occurs upon Hh binding. We also observed this preferential location of PtcSSD in punctate structures when we compared the ectopic expression of PtcSSD with that of PtcWT (which showed a more generalized distribution; Figure 2b″,d″). 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 (C. T., V. M. and I. G., unpublished data).
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 [22, 25]. It has been reported that a lesion in the NPC1 protein, which is normally found in cytoplasmic vesicles characteristic of late endosomes [26], produces a general defect in the retrieval and recycling of raft components of the endocytic pathway [27]. Ptc and NPC1 colocalize extensively in vesicular compartments in cotransfected cells [25]. This suggests that the function of both NPC1 and Ptc involves a common vesicular transport pathway.
To conclude, the upregulation of Smo protein and the opening of the Hh pathway in PtcSSD mutant cells are related to the preferential accumulation of PtcSSD 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.
Supplementary material Supplementary materials and methods are available with the electronic version of this article at http://images.cellpress.com/supmat/supmatin.htm.
Acknowledgements The authors are very grateful to Carmen Iban˜ez and Marı´a Sanz for their excellent technical assistance and to Jose´ Luis Mullor for his help in the initial stages of this work. We are indebted to Marcos Gonza´lez-Gaita´n and Eugeni Entchev for their help in setting up the endocytosis assays, to Ignacio Sandoval, Tom Kornberg, and Nicole Gorfinkiel for useful discussions and for comments on the manuscript. We thank S. Cohen for the anti-Smo antibody, J. Modolell for the anti-Caup antibody, A.Vincent for the anti-Col antibody, R. Holmgren for the anti-Ci antibody, and F. de Celis, T. Kornberg, G. Struhl, and S. L. Zipurski for fly strains. This work was financed by grant PB98-0680 of the Direccio´n General de Investigacio´n Cientifica y Te´cnica (DGICYT), grant 08.9/0001/1998 of the Comunidad Auto´noma de Madrid, and an institutional grant from the Fundacio´n Areces. V. M., G. C., and C. T. were financially supported by fellowships awarded by the Comunidad Auto´noma de Madrid.
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