In Vivo Functions of the Patched Protein

Molecular Cell, Vol. 6, 467–478, August, 2000, Copyright 2000 by Cell Press

In Vivo Functions of the Patched Protein: Requirement of the C Terminus for Target Gene Inactivation but Not Hedgehog Sequestration Ronald L. Johnson,*‡ Ljiljana Milenkovic,† and Matthew P. Scott† * Departments of Cell Biology and Neurobiology University of Alabama at Birmingham Birmingham, Alabama 35294 † Departments of Developmental Biology and Genetics Howard Hughes Medical Institute Stanford University School of Medicine Stanford, California 94305

Summary The membrane protein Patched (Ptc) is a key regulator of Hedgehog (Hh) signaling in development and is mutated in human tumors. Ptc opposes Hh-induced gene transcription and sequesters Hh protein. To dissect these functions, we tested partially deleted forms of Ptc in Drosophila. Deletion of either half of Ptc abolishes all function while coexpression of the halves restores nearly full activity. Deletion of the final 156 residues of Ptc permits Hh sequestration but abolishes inhibition of Hh targets. This deletion has dominantnegative activity, promoting target gene activation in a ligand-independent manner. We observe little or no association of full-length or partially deleted Ptc with the membrane protein Smoothened in Drosophila cultured cells. Introduction The Hedgehog (Hh) signaling pathway patterns developing tissues in many animals (reviewed in Ingham, 1998). The transmembrane proteins Patched (Ptc) and Smoothened (Smo) are critical in receiving and regulating Hh signals. Ptc is predicted to contain twelve transmembrane domains (Hooper and Scott, 1989; Nakano et al., 1989; Goodrich et al., 1996) and inhibits the activation of target gene transcription (Ingham et al., 1991). Smo is a seven transmembrane domain protein that is required to transduce the Hh signal (Alcedo et al., 1996; van den Heuvel and Ingham, 1996). In the absence of Hh, Ptc is proposed to inhibit Smo to prevent target gene activation. Hh activates signaling by relieving the inhibition of Smo, presumably by binding to and inactivating Ptc (Marigo et al., 1996; Stone et al., 1996; Fuse et al., 1999). Hh regulates gene transcription by controlling the activation and proteolytic processing of Cubitus interruptus (Ci), a homolog of the vertebrate Gli transcription factors (Orenic et al., 1990). In the absence of signal, full-length Ci protein is cleaved into a smaller form that acts as a transcriptional repressor (Aza-Blanc et al., 1997). In cells that receive Hh, Ci cleavage is inhibited, and the protein is converted into a transcriptional activator (Ohlmeyer and Kalderon, 1998; Chen et al., 1999a; Methot and ‡ To whom correspondence should be addressed (e-mail: rlj@

uab.edu).

Basler, 1999). Recent evidence suggests that Ci phosphorylation state and nuclear translocation are important regulatory events (Chen et al., 1999a, 1999b; Wang and Holmgren, 2000). Misregulation of Hh signaling has been implicated in human disease. Inactivating mutations in a human homolog of ptc, PTCH1, occur in sporadic and inherited forms of the common skin tumor, basal cell carcinoma (BCC) (Gailani et al., 1996; Hahn et al., 1996; Johnson et al., 1996), and the brain tumor, medulloblastoma (Pietsch et al., 1997; Raffel et al., 1997; Rorke et al., 1997; Vorechovsky et al., 1997; Xie et al., 1997). Missense mutations in human SMO also occur in these tumor types and are proposed to constitutively activate Smo by uncoupling the protein from Ptc regulation (Reifenberger et al., 1998; Xie et al., 1998; Lam et al., 1999). In Drosophila, ptc and hh specify cell fate in the developing embryo and along the anterior/posterior (A/P) axis of the adult wing. In the wing imaginal disc, signaling between two groups of cells, an anterior and posterior compartment, activates gene expression at the compartment border (reviewed in Lawrence and Struhl, 1996). Hh is secreted by the posterior cells and diffuses several cell diameters into the anterior compartment to induce the transcription of specific genes such as decapentaplegic (dpp) and ptc itself (Tabata and Kornberg, 1994). The induction of ptc results in an accumulation of Ptc protein that sequesters Hh to limit its range of signaling (Chen and Struhl, 1996). In tissues lacking Ptc, Hh diffuses farther (Taylor et al., 1993) and activates target genes at a greater distance from its source (Chen and Struhl, 1996). Perturbations in Hh signaling during wing development result in inappropriate gene regulation and wing patterning. Misexpression of Hh in anterior cells induces ectopic dpp and ptc expression and mirror-image duplications of the anterior wing blade (Basler and Struhl, 1994; Kojima et al., 1994; Tabata and Kornberg, 1994; Felsenfeld and Kennison, 1995). ptc mutant clones in the anterior have a similar effect, indicating that Hh signaling is inappropriately activated (Phillips et al., 1990; Capdevila et al., 1994). In contrast, ptc overexpression inhibits Hh signaling to reduce gene expression and wing pattern at the A/P boundary (Johnson et al., 1995). While the role of Ptc in regulating Hh signaling is well established, little is known at the molecular level about what parts of Ptc mediate these functions. We have deleted different regions of Ptc and assessed the ability of the mutant proteins to cause defects in adult wings and to complement homozygous ptc embryos. Our studies indicate that the ligand binding and target gene inactivation functions of Ptc are separable and identify the poorly conserved C terminus as an important regulatory region. Results Reconstitution of Ptc Function from Two Separate Polypeptides Hydropathy analyses of Ptc sequences suggest that Ptc is composed of 12 membrane spanning helices like members of the transporter superfamily (Hooper and

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Figure 1. Deletions of Patched and Their Effects on Ci Levels and Wing Pattern (A) The Drosophila Ptc sequence is depicted schematically containing hydrophilic (unshaded) and hydrophobic (shaded) regions. Ptc-C and Ptc-C⌬ were fused in-frame to the first nine amino acids of Ptc, and Ptc-C⌬ has 12 unrelated amino acids at the C terminus (shaded but not hydrophobic). (B–M) Wild-type wing disc stained with Ci antibody (B) and wild-type wing with longitudinal veins marked 1–5 (C) are shown. UAS ptc expression in the 71B Gal4 pattern causes a decrease in full-length Ci levels in the wing pouch (E) and mild (F) to severe (G) loss of pattern from the central portion of the wing blade. Expression of UAS ptc-N⫹C (H–J) and UAS ptc-N⫹C⌬ (K–M) by 71B Gal4 cause similar phenotypes. UAS ptc-C⌬ causes anterior cross-vein defects (arrow) and wing notching (D). White and black arrows indicate decreased Ci levels and proximal vein defects, respectively.

Scott, 1989; Nakano et al., 1989; Goodrich et al., 1996). Ptc appears to be topologically duplicated as it is composed of a tandemly repeated motif of six hydrophobic domains and one large hydrophilic loop, each corresponding to roughly one half of the protein (Goodrich et al., 1996). Transporter proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) have a similarly duplicated structure. In the case of CFTR, multimerization of the N-terminal half of the protein suffices for channel activity (Sheppard et al., 1994). The structure of Ptc suggests that, as for CFTR, expression of one half of the protein alone might provide function by forming a homodimer or multimer. To test this idea, UAS constructs were made to produce either the amino terminal (amino acids 1–676, UAS ptc-N) or carboxyl terminal (amino acids 676–1286, UAS ptc-C) half of Ptc (Figure 1A). To provide an initiating methionine, the C-terminal half was fused in-frame to the starting nine amino acids of Ptc. These UAS vectors were injected into embryos and several independent transgenic fly lines were recovered for each. The separate halves of Ptc were tested for function by expressing each in the developing wing under the control of 71B Gal4. In the wing imaginal disc, 71B Gal4 is expressed in the anterior and posterior compartments of the wing pouch but not in the notum (Brand and Perrimon, 1993). When full-length Ptc is expressed in the 71B pattern, Ci proteolysis is enhanced and Hhinduced gene expression is inhibited at the compartment boundary (Johnson et al., 1995). Changes in Ci proteolysis can be detected in situ with an antibody

generated against the C-terminal part of Ci. The antibody specifically recognizes the uncleaved form (Motzny and Holmgren, 1995; Figure 1B). The changes caused by Ptc overexpression in the wing imaginal disc result in defects at the A/P boundary of the adult wing. Phenotypes range from a weak effect, the partial fusion of veins 3 and 4 (Figure 1F), to a stronger effect, the deletion of the region between veins 2 and 4 (Figure 1G). In contrast to full-length Ptc, expression of either UAS ptc-N or UAS ptc-C alone caused no detectable wing phenotype (data not shown). Indeed no lethality or phenotypes were seen with either Ptc half when 71B or other Gal4 lines were used, including 69B Gal4, a line that causes lethality when used to express UAS ptc (data not shown). To confirm that the proteins were produced in the Gal4 experiments, UAS ptc-N or UAS ptc-C was expressed throughout the embryo using 69B Gal4 and protein extracts were immunoblotted with antibodies to Ptc. Proteins of the predicted size were detected for both halves, indicating that the proteins were produced and stable when expressed alone (data not shown). Ptc function was reconstituted almost completely when the two halves were produced together. UAS ptc-N and UAS ptc-C transgenes were recombined onto a single chromosome (UAS ptc-N⫹C) and expressed using 71B Gal4. Like intact ptc, several UAS ptc-N⫹C lines (derived from independent UAS ptc-N and UAS ptc-C chromosomes) enhanced Ci proteolysis at the compartment border (Figure 1H). UAS ptc-N⫹C caused both mild (Figure 1I) and severe wing phenotypes like

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Table 1. Complementation of ptc Embryos by Altered Forms of Ptca ptc/wg lacZ CyO; da Gal4 ⫻

Wild-Type

ptc

1. 2. 3. 4. 5.

60% 70% 70% 62% 80%

20% 1% 1% 25% 20%

ptc/wg lacZ CyO; da Gal4 ptc/wg lacZ CyO; UAS ptc ptc/wg lacZ CyO; UAS ptc-N⫹C ptc/wg lacZ CyO; UAS ptc-N⫹C⌬ b ptc/CyO; UAS ptc1130X

ptc Partial 3% 10%

wg 20% 25% 19% 12% –

Unidentified 1% 1%

# 92 202 92 232 169

a Flies of the genotype ptcW109/wg lacZ CyO; da Gal4 were crossed with ptcB98/wg lacZ CyO; UAS ptc or UAS ptc⌬ stocks. Homozygous wg lacZ CyO embryos show a strong wg phenotype and serve as an internal control for recovery of mutant cuticular phenotypes. Experiments were repeated twice for each of two independent lines for crosses 3–5. One line each of UAS ptc-N and UAS ptc-C⌬ was also tested, and both failed to complement ptc embryos (data not shown). b This genotype was analyzed in a separate experiment and did not carry a wg lacZ CyO chromosome. The negative (1) and positive (2) controls yielded results similar to above but are not shown.

those caused by the intact protein (Figure 1J). The wing defects were fully penetrant, and the severity depended on the particular combination of UAS ptc-N and UAS ptc-C chromosomes used. The differences in expressivity between lines may be caused by varied levels of transgene expression. We next carried out a more stringent test of UAS ptcN⫹C function, the ability to complement ptc mutant embryos. In ptc embryos, the central portion of each cuticular segment is deleted and replaced by mirrorimage duplications of the segment ends (Nusslein-Volhard and Wieschaus, 1980). When ptc is expressed ubiquitously in ptc embryos using a heat shock promoter, the normal cuticular pattern is restored (Ingham et al., 1991; Sampedro and Guerrero, 1991). Ubiquitous expression of intact UAS ptc or UAS ptc-N⫹C under the control of da Gal4 complemented the cuticle defects of ptc mutant embryos (Table 1). Expression of intact ptc resulted in 1% of the embryos having the ptc cuticular phenotype (compared to the expected 25%) and in 3% having a mixture of wild-type and ptc denticle belts. Expression of UAS ptc-N alone failed to complement the ptc cuticle phenotype (data not shown). However, UAS ptc-N⫹C rescued ptc embryos almost as well as the intact protein with 1% and 10% of the embryos having a ptc and partial ptc phenotype, respectively. The results indicate that the two halves of Ptc can work together through a noncovalent linkage, even in the absence of intact endogenous Ptc protein. Deletion of the Ptc-C Terminus Compromises Target Gene Repression but Not Hh Sequestration A comparison of Ptc homologs from vertebrate and invertebrate species reveals a lack of amino acid sequence conservation in the C-terminal region, which is predicted to be intracellular (Johnson and Scott, 1997). To test whether the C terminus is required for Ptc function, a deletion was made following amino acid 1130 in both the full-length protein (UAS ptc1130X) and the C-terminal half (UAS ptc-C⌬, Figure 1A). This truncation occurs 21 amino acids after proposed transmembrane domain 12, and the protein retains all the conserved residues found in the C terminus. In contrast to UAS ptc-C, UAS ptc-C⌬ caused wing notching and disrupted anterior cross-vein formation when expressed alone under the control of 71B Gal4 (Figure 1D). These phenotypes occurred in several different fly lines and had variable penetrance. The wing notch phenotype caused by UAS ptc-C⌬ was distinct

from the wing defects caused by full-length Ptc. The defects caused by UAS ptc interact genetically with alleles of the pathway components, ci and hh (Johnson et al., 1995), but the UAS ptc-C⌬ phenotypes do not (data not shown). This suggests that UAS ptc-C⌬ interacts with components not related to Hh signaling. Wing notching can be caused by perturbations in the Notch (N) pathway that reduce wg expression at the presumptive wing margin (Diaz-Benjumea and Cohen, 1995). However, the UAS ptc-C⌬ phenotypes were not modified by alleles of wg, dsh, Ser, and N (data not shown). In addition, UAS ptc-C⌬ did not rescue ptc embryos (data not shown). When UAS ptc-N and UAS ptc-C⌬ (UAS ptc-N⫹C⌬) were expressed together using 71B Gal4, the resulting phenotypes were similar to those caused by either the intact or Ptc-N⫹C proteins. UAS ptc-N⫹C⌬ reduced full-length Ci levels at the compartment boundary (Figure 1K) and caused wing defects that varied from mild (Figure 1L) to severe (Figure 1M) depending on the combinations of independent UAS ptc-N and UAS ptc-C⌬ chromosomes. The wing notching that is caused by UAS ptc-C⌬ was greatly suppressed in combination with UAS ptc-N (Figure 1M). While the expression of UAS ptcN⫹C⌬ in the developing wing reconstituted the dominant effects of intact Ptc, it did not rescue the cuticle pattern of ptc embryos (Table 1). These results indicate that UAS ptc-N⫹C⌬ can phenocopy the dominant wing phenotypes of the full-length protein but cannot block target gene expression in the absence of endogenous Ptc. Ptc 1130X Activates Hh Signaling in the Anterior Compartment The failure of UAS ptc-N⫹C⌬ to complement ptc embryos could have been caused by expressing the protein as two separate halves. To control for this variable, we produced the Ptc C-terminal deletion as a single polypeptide (UAS ptc1130X). When expressed ubiquitously using da Gal4, UAS ptc1130X also did not complement ptc mutant embryos (Table 1). This indicates that the C terminus is required for appropriate regulation of the Hh signaling pathway. To examine the effects on wing development, UAS ptc1130X was expressed using 71B Gal4. Surprisingly, instead of causing loss of pattern in both compartments like UAS ptc, UAS ptc1130X increased the venation and size of the anterior compartment while leaving the posterior region unaffected (Figure 2D). This phenotype is like that of the hhMrt allele (Figure 2B), where ectopic hh

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Figure 2. UAS ptc1130X Expression Causes Activation of Hh Signaling Ectopic hh expression in hhMrt stabilizes full-length Ci throughout the anterior compartment of wing discs (A) and causes overgrowth and mispatterning of anterior structures in the wing (B). UAS ptc1130X expression in the 71B Gal4 pattern shows similar effects in both the wing imaginal disc (C) and adult wing (D). Clones that overexpress UAS ptc1130X under the control of act⬍CD4⬍Gal4 (as marked by B-gal staining from UAS lacZ [E]) specifically stabilize full-length Ci ([E], B-gal, Ci; [F], Ci alone) and induce dpp (G) in the anterior compartment (arrows).

expression in the anterior compartment stabilizes fulllength Ci (Johnson et al., 1995; Figure 2A) and induces dpp and ptc expression in the anterior (Tabata and Kornberg, 1994; Felsenfeld and Kennison, 1995). Likewise, UAS ptc 1130X caused an expansion of full-length Ci in much of the anterior wing pouch (Figure 2C). To see whether the stabilization of Ci is cell autonomous, clones of cells overexpressing UAS ptc1130X under the control of actin Gal4 were induced in wing discs. Clones in the anterior compartment (as marked by ␤-gal staining) elevated Ci levels autonomously while clones in the posterior had no effect (Figures 2E and 2F). Since Hh has both long- and short-range effects in patterning the A/P axis of the developing wing, we tested whether Ptc1130X stimulated both effects. In the wing disc, long-distance Hh signaling is mediated largely through the induction of dpp, a signal that patterns both compartments over a distance (Capdevila and Guerrero, 1994; Zecca et al., 1995). Clones that overexpressed Ptc1130X induced ectopic dpp expression at positions throughout the anterior (Figure 2G) like clones overexpressing Hh (Basler and Struhl, 1994). Hh also has short-

range effects that are independent of dpp, such as regulating the number of campaniform sensilla in vein 3 and the size of the central intervein region between veins 3 and 4 (Mullor et al., 1997; Strigini and Cohen, 1997). Elevated Hh activity increases the number of campaniform sensilla and the intervein distance (Ingham and Fietz, 1995; Porter et al., 1996; Mullor et al., 1997) while Ptc overexpression has the opposite effect (Johnson et al., 1995; Figure 3C). Ptc1130X caused the same effects as short-range Hh signaling. Under the control of 71B Gal4, UAS ptc1130X increased the number of campaniform sensilla and expanded the central intervein region, as measured by the number of double row bristles at the distal wing margin (Figure 3E). To examine how Ptc1130X affects the prospective central intervein region in wing discs, we examined the expression pattern of collier (col; allelic to knot), which encodes a COE transcription factor (Crozatier et al., 1996). col transcription at the A/P border is controlled by Hh (Vervoort et al., 1999). When expressed in the 71B Gal4 pattern, UAS ptc eliminated the A/P stripe of col expression. col expression flanking the wing pouch,

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Figure 3. UAS ptc and UAS ptc1130X Have Opposite Effects on the Patterning of the Central Intervein Region In the wild-type wing, campaniform sensilla occur at stereotyped positions along vein 3 (black arrows), and the double row of bristles in the wing margin appear below vein 2 and end near vein 3 (white double-headed arrow [A]). col is expressed at the compartment boundary of the wing disc under the control of hh and is important in patterning the region around vein 3 (B). UAS ptc expression in the 71B Gal4 pattern decreases campaniform sensilla and double row bristle number (C) as well as inhibits col expression at the A/P border (D). UAS ptc1130X expression increases the number of campaniform sensilla, the length of the double row bristle region (E), and expands col (F) into the anterior region.

which is independent of Hh control (Vervoort et al., 1999), was unaffected (Figure 3D). In contrast, production of UAS ptc1130X expanded the A/P stripe of col expression further into the anterior (Figure 3F). Ptc1130X also repressed the stripe of A/P expression near the dorsal/ ventral (D/V) border. While the reason for this suppression is unclear, Ptc1130X affected ptc-lacZ expression in a similar manner (data not shown), and overexposure of the staining showed weak ptc-lacZ expression at the D/V boundary. The ectopic induction of dpp and col suggests that Ptc1130X stimulates targets of long- and short-range Hh signaling. Since Ptc1130X stabilizes Ci protein and increases dpp transcription levels at positions far from the sources of Hh (Figures 2F and 2G), it appears that Ptc1130X stimulates signaling in a ligand-independent manner. This idea was further tested by examining the effect of Ptc1130X in wing discs where hh function was inactivated. UAS ptc1130X was expressed under the control of 71B Gal4 in hhts2 wing imaginal discs that were heteroallelic for a temperature sensitive (hhts2) and a null allele (hhAC) of hh. Crosses were incubated at the permissive temperature (17.5⬚) until the third instar larval stage, shifted to the restrictive temperature (29⬚) for about 12 hr, and then wing imaginal discs were stained with a Ci antibody. Wing imaginal discs heterozygous for hh had

the normal pattern of elevated Ci at the compartment boundary (Figure 4A). In imaginal discs where hh function was inactivated at the restrictive temperature, the stripe of full-length Ci at the A/P border was no longer maintained (Figure 4B). Under these same conditions, Ptc1130X elevated Ci levels throughout the anterior wing pouch of hhts2 wing imaginal discs while in the notum, where Ptc1130X is not expressed, Ci levels remained low (Figure 4C). These results demonstrate that Ptc1130X stimulation of the Hh pathway is ligand independent. Ptc1130X may exert its effects by interfering with the ability of endogenous Ptc to inhibit target gene transcription. If Ptc1130X is a dominant-negative protein, the phenotypes that it causes should be changed by varying the dose of wild-type ptc. We tested several UAS ptc1130X lines in genetic backgrounds heterozygous for either a null allele of ptc (ptcB98) or a UAS ptc transgene. Crosses with 71B Gal4 flies were made at 25⬚ C to lower the activity of Gal4. At this temperature, the UAS ptc 1130X-D line caused slight disorganization of the vein 3 region and proximal fusion of veins 3 and 4 (Figure 4D). However, in the absence of one copy of ptc, this phenotype was enhanced, as is indicated by the expansion of the vein 3 region (note the increased venation and larger double row of bristles at the wing margin;

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Figure 4. UAS ptc1130X Activity Is Ligand Independent and Modulated by ptc Dosage The elevated levels of Ci at the A/P border of wing imaginal discs (A) are not maintained upon inactivation of hh (B). Expression of UAS ptc1130X by 71B Gal4 elevates Ci levels in the anterior wing pouch independent of hh function (C). The wing phenotypes caused by UAS ptc 1130X (D) are enhanced in the absence of one dose of ptc (E) and suppressed by coexpression with UAS ptc (F).

Figure 4E). Conversely, production of full-length Ptc from a UAS transgene suppressed the ptc1130X phenotypes by suppressing the venation defects along vein 3 (Figure 4F). A second ptc1130X line was suppressed by UAS ptc in a similar manner, but the loss of one ptc copy did not noticeably enhance the phenotype (data not shown). The sensitivity of the phenotypes to the dose

of wild-type ptc suggests that ptc1130X has dominantnegative or antimorphic behavior. Ptc1130X Produced Near Sources of Hh Opposes Signaling In addition to its dominant-negative properties, Ptc1130X causes wing defects like those of full-length

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Figure 5. Expression of UAS ptc1130X in the Posterior Compartment Opposes Hh Signaling in a Manner Similar to Full-Length Ptc UAS hh expression in the en Gal4 pattern stabilizes Ci throughout the anterior of the wing disc (C) and increases the central intervein region of the wing (D) compared to that of wild-type (A and B). Ci is destabilized (arrows), and central wing pattern is deleted by expression of UAS ptc (E and F), UAS ptcN⫹C (G and H), and UAS ptc1130X (I and J) in the en Gal4 pattern.

Ptc. When expressed by 71B Gal4, some UAS ptc1130X lines lost pattern elements between veins 3 and 4, as with UAS ptc (data not shown). This suggested that Ptc1130X causes phenotypes by both inhibiting and stimulating the Hh signaling pathway. Since 71B Gal4 is expressed in both the anterior and posterior compartments of the wing pouch (Brand and Perrimon, 1993), Ptc1130X may oppose signaling near Hh sources by sequestering the ligand and stimulate signaling far away from Hh by competing with endogenous Ptc. To test whether Ptc1130X can oppose Hh signaling, we expressed altered Ptc forms in the posterior compartment of the developing wing disc using engrailed (en) Gal4. Overexpressing hh in the posterior compartment of the wing disc has effects opposite to those of overexpressing ptc with respect to Ci stability and wing patterning.

UAS hh expression in the en Gal4 pattern maintained full-length Ci throughout the anterior compartment of the wing disc (Figure 5C) and enlarged the area between veins 3 and 4 (Porter et al., 1996). In contrast, UAS ptc slightly narrowed the Ci protein stripe at the compartment border of the wing imaginal disc (Figure 5E) and partially fused veins 3 and 4 in the adult wing (Johnson et al., 1995; Figure 5F). Expression of either UAS ptcN⫹C (Figure 5G and 5H) or UAS ptc-N⫹C⌬ (data not shown) in the posterior caused a similar reduction of Ci levels and a more drastic loss of central wing pattern. UAS ptc1130X expression in the posterior did not augment Hh signaling but instead markedly inhibited it. Ci levels at the A/P border was lowered (Figure 5I) and much of the pattern between veins 1 and 5 was deleted (Figure 5J). This effect was seen using multiple lines.

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Figure 6. Association between the Two Halves of Ptc (A) Extracts of S2 cells overexpressing Ptc-C and Fz1 (1), Ptc-N and Ptc-C (2), Ptc-N and Ptc (3), and Ptc-N and Fz1 (4) were immunoblotted with antisera recognizing an N-terminal loop (loop) and the C terminus of Ptc (C-term) and myc-tagged Fz1 (myc, arrowhead). The asterisks mark the positions of full-length Ptc (145 kDa), Ptc-N (65 kDa), and Ptc-C (65 kDa). (B) (Left panel) Ptc-C, Ptc, or Fz1 was immunoprecipitated and immunoblotted to detect associations with Ptc-N. Ptc-N associates strongly with Ptc-C, weakly with Ptc, and is not detectable with Fz1. (Right panel) Ptc-C does not associate with Fz1, and Fz1 is immunoprecipitated (arrowhead) but does not associate with Ptc-N. All extracts were also immunoprecipitated with a control isotype monoclonal antibody directed against the HA epitope. Molecular weight sizes are 116, 97, 66, 45, and 31 kDa.

We next examined whether intact Ptc or fragments of Ptc interact with Smo in S2 cells. While the association between Ptc and Smo has been demonstrated in mammalian cell lines (Stone et al., 1996; Carpenter et al., 1998; Murone et al., 1999), it has not been reported in Drosophila. The association of Ptc with a CFP-tagged form of Smoothened (Smo CFP) was compared to that with a myc-tagged form of Fz1 (Rulifson et al., 2000). Both tagged proteins have activity when expressed in Drosophila (L. Zheng and M. Scott, unpublished data; Rulifson et al., 2000). Fz1 provides a good control for specificity because its sequence is related to Smo (Alcedo et al., 1996; van den Heuvel and Ingham, 1996), but Fz1 acts in signaling pathways unrelated to Hh (reviewed in Wodarz and Nusse, 1998). When full-length Ptc and Smo CFP were coexpressed and Smo CFP was immunoprecipitated, a minor amount of Ptc was associated. Little or no Ptc associated with immunoprecipitated Fz1 or GFP (data not shown). The small amount of Ptc and Smo association may be due to the high levels of expression, since SmoCFP did not co-immunoprecipitate with the low level of endogenous Ptc present in S2 cells (data not shown). We also examined whether Ptc fragments associate with either full-length Smo, Fz1, or Ptc. When Ptc-N, Ptc-C, or Ptc1130X were coexpressed with either Smo CFP or Fz1, a small amount (⬍5%) of the Ptc fragments were found to interact (data not shown). While more of the Ptc fragments associate with Smo than with Fz1, the interactions did not appear specific because minor amounts of Ptc-N or Ptc 1130X also coimmunoprecipitated with full-length Ptc (Figure 6B; data not shown). Furthermore, immunoprecipitated endogenous Ptc did not associate with Ptc 1130X. Because of the lack of specificity and low levels of interaction, we were unable to confirm whether Ptc and Smo associate in Drosophila cells or identify regions of Ptc that mediate such an association. Discussion

Hence, Ptc 1130X antagonizes signaling when expressed in cells that are producing Hh, in contrast to its dominant-negative effect in anterior cells. Interactions between the Ptc Halves Based on experiments in mammalian cultured cells, Ptc is proposed to inactivate target gene regulation by associating with Smo and blocking its activity (Stone et al., 1996; Murone et al., 1999). To understand the molecular basis of Ptc action, we examined associations between the two halves of Ptc and between each deleted form of Ptc and Smo. Proteins were overexpressed in Drosophila S2 cells by transiently transfecting combinations of UAS expression plasmids together with an actin-Gal4 construct. Cells were lysed, and protein extracts were immunoprecipitated and immunoblotted. Since coexpression of the two halves of Ptc provides function almost as well as the intact protein, the two halves must assemble and associate noncovalently. Indeed, Ptc-N and Ptc-C are coexpressed in S2 cells, both halves are immunoprecipitated using a monoclonal antibody to the Ptc C terminus (Figure 6B). This association is specific since little or none of either Ptc half coimmunoprecipitated with an unrelated polytopic membrane protein, Drosophila Frizzled 1 (Fz1; Vinson et al., 1989).

Reconstitution of Ptc Activity from Two Separate Polypeptides Our study shows that while neither half of Ptc is functional when expressed alone, the protein’s activity is almost completely reconstituted when both polypeptides are produced in vivo. In spite of the similar topology predicted for the two halves of Ptc, neither half of the protein can form an active multimer. However, the functional reconstitution accomplished by producing both halves indicates that each half can assemble properly in the membrane and associate through a noncovalent linkage. This association is strong enough to permit coimmunoprecipitation of both halves using an antibody that recognizes Ptc-C. Our experiments were guided by protein assembly studies done with transporter family members including lactose permease (Bibi and Kaback, 1990; Wrubel et al., 1990) and the yeast a-factor transporter (Berkower and Michaelis, 1991). In these studies, activity was restored by producing both halves of the protein but not by producing either alone. CFTR is a notable exception, where production of the amino terminal half alone forms a regulated chloride channel while the carboxyl terminal half has no activity (Sheppard et al., 1994). These experiments suggest that even if the halves of Ptc and transporter family members arose through duplication, the

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functions of each half have become specialized and dependent on other parts of the protein. Separation of Ptc Activities Studies by Chen and Struhl (1996) indicate that the induction of endogenous ptc transcription in response to Hh is a critical feedback mechanism that sequesters Hh and restricts its range of signaling. They found that the ptcS2 allele could sequester Hh but not inhibit signaling (Chen and Struhl, 1996). The molecular defect of ptcS2 has not been identified. We have found that Ptc-N⫹C⌬, which lacks the final 156 amino acids, has similar properties. Neither Ptc-N⫹C⌬ nor Ptc1130X can properly regulate Hh signaling, as is indicated by their failure to rescue ptc mutant embryos (Table 1). However, Ptc-N⫹C⌬ causes dominant phenotypes in the adult wing like that of full-length Ptc. A likely explanation is that Ptc-N⫹C⌬ acts as a “ligand sink” to bind and sequester Hh, enabling endogenous Ptc to repress target genes. This idea is bolstered by our finding that production of either Ptc-N⫹C⌬ or Ptc1130X in the posterior compartment, where no endogenous Ptc is present, also cause similar defects in the wing. These wing defects probably arise from the sequestration of Hh in the posterior compartment, which prevents its movement across the compartment boundary. The C terminus of Ptc appears dispensable for ligand binding, although it may have subtle effects on Hh affinity. Studies on mouse Ptc1 show that truncation of the final 140 residues of the 270 amino acid C terminus does not alter the protein’s affinity for Sonic hedgehog (Shh) (Fuse et al., 1999). The putative extracellular loops of Ptc are required for Shh binding since removal of either loop from chick Ptc eliminates binding (Marigo et al., 1996). We find that removal of the second extracellular loop of fly Ptc results in a stable protein that has no function (data not shown). The loops may directly interact with ligand, or removal of the loops may cause improper assembly or localization of the protein. Mechanism of Ptc1130X Action The C-terminal truncation has different effects on Ptc activity depending on whether the protein is expressed as two half proteins or as a single polypeptide. Both Ptc-N⫹C⌬ and Ptc1130X can sequester Hh, but only Ptc1130X has the dominant-negative activity of blocking Ptc inhibition of signaling. Perhaps the dominant-negative activity requires a conformational change transmitted between two domains of Ptc. This might not happen when the Ptc C-terminal truncation is produced as two polypeptides. The dominant-negative properties of Ptc1130X may activate Hh signaling in several ways. Our studies rule out a mechanism by which Ptc1130X facilitates Hh movement further into the anterior compartment, since Ptc1130X stimulates signaling in the absence of functional Hh. Hence, Ptc1130X must interfere with components downstream of Hh. Two possible proteins are Ptc and Smo. Ptc1130X might associate with Ptc to block its function or proper localization within the cell. This model suggests that Ptc normally forms a multimer in its active state. Alternatively, Ptc1130X might associate nonproductively with Smo or another signaling component and thereby shield such a component from interaction with endogenous Ptc. Our biochemical studies in S2 cells indicate that small amounts of Ptc1130X associate

nonspecifically with overexpressed Ptc, Smo, or Fz1. The apparent lack of significant interactions between Ptc and Smo may indicate that their association during signaling is transient or that other proteins are involved. The dominant-negative activity of Ptc1130X suggests a different mechanism by which PTCH1 mutations could cause tumors. PTCH1 acts as a tumor suppressor gene, with skin tumors arising from inactivating mutations in both PTCH1 alleles (Gailani et al., 1992., 1996). However, in mice some medulloblastomas arise by haploinsufficiency of ptc (Zurawel, 2000; Wetmore et al., 2000). Our results indicate that dominant-negative mutations in PTCH1, in addition to loss of function mutations, could cause abnormal activation of Hh signaling. Indeed, several PTCH1 mutations map to positions corresponding to the C terminus of Ptc1130X (Johnson and Scott, 1997). Experimental Procedures Vector Construction and Germline Transformation Deletions of Drosophila ptc were derived from a full-length cDNA, ptc S9 (Schuske et al., 1994) as follows: 1130X, a stop codon and XbaI site, were introduced by PCR after codon 1130. Loop 2 (⌬738– 939), ptc was digested with BstEII and BsmI, blunted with T4 polymerase, and ligated together. Ptc-N (676X), a XhoI/FspI fragment of ptc, was cloned into the XhoI/blunted XbaI sites of pUAST (Brand and Perrimon, 1993). Ptc-C⌬ (⌬9–676, 1130X), a FspI fragment was ligated into a MluI/NruI digested and blunted form of ptc S9. Ptc-C (⌬9–676), a XbaI site was introduced after the stop codon of fulllength ptc by PCR, the PCR product was digested with EheI/XbaI and cloned into EheI/XbaI digested Ptc-C⌬. All fragments except Ptc-N were digested with XhoI/XbaI and cloned into the XhoI/XbaI sites of pUAST. All PCR-derived coding sequence and in-frame fusions were sequenced to ensure the correct sequence. The pUAST-derived vectors were injected into yw embryos and transgenic Drosophila lines were established (Spradling and Rubin, 1982). Fly Stocks Flies used were da Gal4 (Wodarz et al., 1995), 71B Gal4 (Brand and Perrimon, 1993), en Gal4 (Porter et al., 1996), UAS ptc B1 (Johnson et al., 1995), hhMrt (Felsenfeld and Kennison, 1995), hhts2, hhac, ptcB98, and ptcW109. Crosses w; ptcW109/wg lacZ CyO; da Gal4 ⫻ w; ptcB98/wg lacZ CyO; UAS ptc or UAS ptc⌬; w; UAS ptc1130X; hhts2/TM6Tb ⫻ w; 71B Gal4; hhAC/TM6Tb; and yw, actin-cd2-Gal4; UAS lacZ; HSFLP MKRS Sb/ TM6Tb x UAS ptc1130X. Preparation of Wings and Cuticles Crosses were incubated at 25⬚C or 29⬚C to modulate the severity of the wing phenotypes. Wings were dissected from adults and mounted as described (Basler and Struhl, 1994). For the complementation studies, embryos were collected and aged on agar plates at 29⬚ C for at least 24 hr and cuticles prepared as described (Schuske et al., 1994). Antibody and RNA In Situ Staining of Imaginal Discs For the FLP-mediated expression of UAS ptc1130X in clones, embryos were raised at room temperature until the first and second instar, heat shocked at 37⬚C between 30–60 min, and raised at 29⬚C until third instar. Wing imaginal discs were dissected from wandering third instar larvae and stained with rat anti-Ci monoclonal (gift of R. Holmgren) and rabbit anti-␤ gal antibodies as described (Johnson et al., 1995). RNA in situ hybridizations were performed using riboprobes to col and dpp as described (Johnson et al., 1995). Preparation of Ptc Antibodies Ptc Trp E fusion constructs were made by fusing the coding sequence for amino acid 229–397 (loop) and amino acid 1194 to 1286

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(C-terminal) of Ptc in-frame following Trp E in pATH vectors (Koerner et al., 1991). Fusion proteins were expressed in bacteria, purified from inclusion bodies, injected into rats (both fusions), and polyclonal antiserum recovered. The C-terminal fusion protein was also injected into mice to make monoclonal antibodies. Several hybridoma cell lines were recovered that reacted to Ptc by immunoblot and one of these, 47H8 (IgG1␬ isotype), was used to immunoprecipitate Ptc. Cell Transfection and Coimmunoprecipitation Studies Drosophila S2 cells were grown at 25⬚C and transfected with combinations of the following vectors: full-length and deleted forms of UAS ptc, UAS smoCFP, UAS fz1myc (gift from E. Ruifson and R. Nusse), UAS GFP-T65 (gift from B. Dickson), and pA5C Gal4 (gift from Y. Hiromi). Cells (4 ⫻ 106) were plated in 6 cm dishes 1 day before transfection. For each treatment, 2 ␮g of pA5C Gal4 and 8 ␮g UAS constructs were introduced into cells by CaPO4, the media changed following about 12 hr, and the cells harvested 48–72 hr after transfection. Cells were resuspended, washed twice in PBS, and counted. The cells were kept at 4⬚C for the rest of the procedure. Cells were lysed at 6 ⫻ 106 cells/ml in lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris [pH 8], and Complete protease inhibitors [Roche] for 1 hr while rocking. Extracts were pelleted at 10,000 ⫻ g for 10 min, the supernatant was incubated with 0.5% Pansorbin cells (Calbiochem) for 1 hr, and pelleted at 10,000 ⫻ g for 10 min. Cleared extract from ⵑ4 ⫻ 106 cells in 0.75–1 ml lysis buffer was incubated with 1 ␮g mouse monoclonal antibody (all IgG1␬ isotype) against either HA (16B12, Convance), myc (9E10, Sigma), GFP (Roche), or Ptc (47H8) for 1 hr. 45 ␮l of 50% purified protein G agarose (Roche) was added to each immunoprecipitate and incubated either 4 hr or overnight by rocking. Immunoprecipitates were washed 10 min each with 1 ml of lysis buffer (twice), lysis buffer with 0.1% NP-40 and 0.5 M NaCl (twice), and lysis buffer with 0.1% NP-40 (once). Samples were suspended in Laemmli buffer, separated on 10% acrylamide gels, and transferred to nitrocellulose at 100V for 1 hr. Samples were immunoblotted with antisera and visualized using HRP-conjugated secondary antibodies and ECL (Amersham). Acknowledgments The authors thank M. Fish for outstanding technical assistance, L. Zheng for UAS CFP smo, E. Bailey for mapping assistance, K. Champion for in situ hybridization assistance, D. Shaw for isotyping, R. Holmgren for Ci antibody, M. Ruppert for myc antibody, D. Casso and T. Kornberg for UAS GFP and A5C Gal4 vectors, C-H. Wu, E. Rulifson, and R. Nusse for S2 cells and UAS fz1myc, A. Vincent for col cDNA, J. Hooper for ptc alleles, and P. Beachy for en Gal4 and UAS hh stocks. We are grateful to E. Bailey, K. Kozopas, J. Horabin, M. Ruppert, and V. Bankaitis for helpful discussions and criticisms of the manuscript. This work was supported by a Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship (DRG 1218), a Walter and Idun Berry Postdoctoral Fellowship, and a National Institutes of Health grant 1R01HD37505-01 (to R. L. J.). M. P. S. is an Investigator of the Howard Hughes Medical Institute. Received January 28, 2000; revised July 25, 2000. References Alcedo, J., Ayzenzon, M., Von Ohlen, T., Noll, M., and Hooper, J.E. (1996). The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal. Cell 86, 221–232. Aza-Blanc, P., Ramirez-Weber, F.A., Laget, M.P., Schwartz, C., and Kornberg, T.B. (1997). Proteolysis that is inhibited by hedgehog targets Cubitus interruptus protein to the nucleus and converts it to a repressor. Cell 89, 1043–1053. Basler, K., and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368, 208–214.

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