Patched

Symbol: ptc	Flybase ID: {Flybase_ID}
Synonyms: {Name}	{GadFly}
Function: {Short_Function}	{LocusLink}
Keywords: segment polarity, hedgehog receptor	{Interactive_Fly}

{Summary}

hedgehog receptor
Several observations suggest that the mechanism by which Ptc controls Smo signaling involves vesicular transport. First, extensive structural similarity is shared between Ptc proteins (Ptc1 and Ptc2 in vertebrates) and the Niemann-Pick C1 (NPC1) protein, which includes Libut is not limited to the sterol-sensing domain (SSD). NPC1 functions in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system. NPC1 and Ptc1 colocalized extensively in transfected cells; Ptc1 functions in the receptor-mediated endocytosis of Hh proteins, and Ptc⁺ vesicles observed in Drosophila include multivesicular late endosomes. Second, a diverse group develof steroidal compounds and hydrophobic amines inhibits both Shh signaling, regulated by Ptc1, and vesicular transport of intracellular cholesterol, regulated by NPC1. Third, mutations in the SSD of Drosophila Ptc render the protein incapable of inhibiting Smo. Similar mutations in NPC1 render the protein incapable of regulating endosomal lipid sorting. Fourth, Rab23 a Ras-like GTPase, a component of the vesicular transport machinery, is required for negative regulation of Shh signaling in the vertebrate neural tube. (Incardona, 2002)
"Ptc might traffic Smo to an intracellular compartment where it would be targeted for degradation (Fig. 6). In the case of SCAP, this trafficking is regulated by SSD-dependent sensing of cholesterol levels in the membrane, point mutations in the SSD causing constitutive translocation of SREBP to the Golgi compartment (Hua et al. 1996). Intriguingly, an identical mutation in the SSD of Ptc results in the loss of its Smoinhibiting activity (Martin et al. 2001; Strutt et al. 2001). In contrast to SCAP, however, altering cholesterol levels within the cell by various means has a relatively minor influence on Hh signaling, suggesting that Ptc activity is not similarly modulated by membrane sterol levels (Incardona et al. 2000a). An alternative possibility is that the SSD serves to direct Ptc to a specific membrane microdomain where it inactivates Smo." [Ingham, 2001]
"The activity of NPC1, an SSD-containing protein more closely related to Ptc, raises other possibilities. In the absence of NPC1, unesterified cholesterol accumulates in the late endosome compartment, which, in turn, disrupts sorting between the endosome and trans-Golgi network (Higgins et al. 1999; Kobayashi et al. 1999; Neufeld et al. 1999; Blanchette-Mackie 2000). This has led to the suggestion that NPC1 acts both as a sensor (through its SSD domain) and as a regulator of endosomal cholesterol content, with Ko et al. (2001) suggesting that NPC1 may cause vesicle budding by moving lipid molecules from one leaflet of the organellar membrane to the other. Recently it has been shown that NPC1 has weak similarity to the RND family of prokaryotic permeases and, indeed, has permease activity in both eukaryotic and prokaryotic cells (Davies et al. 2000). In both cases, NPC1 can transport fatty acids across membranes. But although it is thus able to transport lipophilic molecules out of the endosome–lysosome system, it is not clear how this modulates cholesterol levels. Strikingly, Ptc is more closely related to the bacterial AcrBand MexD permeases than it is to NPC1, raising the possibility that it may also function as a permease." [Ingham 2001]
"Although Ptc is clearly a Hh receptor, recent evidence in Drosophila has indicated that Hh can in some circumstances signal to cells by a Ptc-independent mechanism (Ramirez-Weber et al. 2000). This raises the possibility that some other protein may control the activity of Smo in the absence of Ptc, but the identity of such a protein so far remains a mystery. In contrast to the fly, vertebrates have a second Ptc gene, Ptc2, the product of which could in principle also act as a Hedgehog receptor. Indeed, both Ptc1 and Ptc2 bind all mammalian Hedgehog proteins (Carpenter et al. 1998). In many tissues, however, Ptc2 is actually expressed in Hh-secreting cells, suggesting that either Ptc2 acts in an autocrine signaling loop, or has a function distinct from that of Ptc1. Direct binding studies have identified a second Shh-binding protein in vertebrates, Hedgehog-interacting protein (Hip1), a membrane-bound protein (Chuang and McMahon 1999). Hip1 binds all mammalian Hh proteins with an affinity similar to that of Ptc1, but this binding most likely regulates the availability of ligand, thereby attenuating signaling rather than activating a novel pathway (P. Chuang and A. McMahon, in prep.)." [Ingham 2001]
Ptc destabilizes Smo in the absence of Hh (Denef, 2000)
Ptc acts indirectly to regulate Smo activity perhaps by promoting activity of a phosphatase that dephosphorylates Smo in the absence of Hh (Denef, 2000)
In both Drosophila and mammalian cells, the internalization of Ptc or Ptc1 is dynamin-dependent, implying that it is mediated via clathrin-coated pits (Capdevila, 1994; Incardona et al. 2000b). Although the finding that Hh accumulates in lipid rafts (Rietveld, 1999) would be consistent with an alternative mode of internalization, namely, via caveolae, it is not clear from these studies (which involved the fractionation of embryo extracts) whether the lipid raft accumulation represents protein in the sending or the receiving cell. Evidence that caveolae play some role in Ptc behavior comes from the reported association of the vertebrate Ptc1 protein with caveolin in tissue culture cells (Karpen, 2001). However, analysis of this system suggests a role for caveolin in the delivery of Ptc1 to the plasma membrane via lipid rafts rather than the internalization of a Shh/Ptc1 complex (Karpen, 2001).

Knot/Collier
- 71B Gal4/UAS-ptc eliminated A/P stripe of col Fig 3D) (Johnson, 2000)
- 71B Gal4/UAS-ptc1130X expanded the A/P stripe and repressed the stripe near the D/V border (Fig 3F)(Johnson, 2000)
Hedgehog
- Overexpression of hh in embryos with a HS-hh construct wg is ectopically expressed anterior to each normal wg domain, but more anterior cells don't express wg. ptc, however, is expressed in all cells except engrailed expressing cells. (Ingham, 1993)
Effect on engrailed
- en is expressed in ptc mutants in wing imaginal discs (Fig 2B), but not in ptc ci double mutants (Fig 2C) (Methot, 2001)
Effect on patched itself
- in ptc^S2 (an allele that generates a signaling-inactive Ptc protein that can be visulized with an antibody against Ptc: Chen and Struhl, 1996) mutant cells ptc was strongly upregulated. Clones double mutant for ci and ptc were incapable of upregulating Ptc^S2 protein (Methot, 2001)
Ptc and Fu in the germarium undergo changes in expression that are coincident with Sxl (Vied, 2001)
In smo clones (no Hh signaling) dpp-lacZ, Ptc and anterior En expression is inhibited, suggesting that they are direct targets for regulation by Hh signaling (Strigini, 1997)
smo, fu, and ci all act downstream of ptc to activate wg
free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity thereby affecting the level of pathway activity (Taipale, 2002)
In fused mutant wing discs Col and Ptc expreesion (protein and transcript) is absence approximately six rows of cells on either side of the DV boundary (Fig 1) (Glise, 2002) and the stripe is broadened due to increased range of Hh in fused mutants (Fig 1) (Glise, 2002)
In contrast to Pka-C1 clones, clones of cells lacking Ptc activated Col in all cells within the clones except for cells along the prospective wing margin (Fig 3D) (Glise, 2002)
Ectopically expressed N^intra (a dominat active form) produces a cell-autonomous down-regulation of both Col and Ptc (Fig 6C and 6D). By contrast, expression of En was not affected by Nintra expression in either the posterior compartment or the anterior compartment in response to Hh (Fig. 6E), consistent with the fact that en is normally activated in the prospective wing margin (Glise, 2002)

Binds Shh w/ high affinity, 2 extracellular loops are required
in vitro: Shh can bind to the large extracellular domains of Ptc1 when expressed in tissue culture cells or Xenopus oocytes (Marigo et al. 1996a; Stone, 1996; Fuse et al. 1999)
Ptc and Hh colocalize to intracellular vesicles in Hh-responding cells both in the Drosophila embryo and imaginal disc (Bellaiche et al. 1998; Burke, 1999; Martin et al. 2001; Strutt et al. 2001), suggesting that on binding to Ptc, the Hh–Ptc complex is internalized by responding cells.complex is internalized by responding cells.
- Support: In mammalian tissue culture soluble recombinant Shh protein can be internalized by cells transfected with the vertebrate Ptc1 gene (Incardona et al. 2000b).
in vitro assays indicate that neither cholesterol nor palmitoyl modification increases the affinity of Hh proteins for Ptc, although they do increase the specific activity of the protein (Pepinsky et al. 1998).
ShhN internalized by Ptc1 accumulated to much higher levels with leupeptin treatment (Incardona, 2002), consistent with observations on endogenous Shh in embryonic neural tissue (Incardona, 2000)

Mutant clones lacking both en and invected ectopically express dpp-lacZ and ptc in the posterior compartment where dpp activity ordinarily is repressed (Sanicola, 1995)
Hh binding causes removal of Ptc from surface (Denef, 2000)
ptc-lacZ construct only defines the stripe of strongest ptc expression (00049)
In KNRK cells: vertebrate Ptc1's half-life is short, with loss of an immunofluorescent signal and protein levels within 4-6 hr of cycloheximide treatment (Incardona, 2002)
- ShhN treatment reduced Ptc1 levels even further (Incardona, 2002)
- Cellular uptake of the lysosomal protease inhibitor leupeptin extended Ptc1 half-life both in the absence and presence of ligand (Incardona, 2002)

2 large extracellular loops and 12 transmembrane domains

Ptc is more apical along the lateral membrane (Denef, 2000)
Once internalized, both Ptc and Shh appear to be targeted to the lysosome, at least in some cell types (Mastronardi et al. 2000).
In KNRK cells: vertebrate Ptc1, in the absence of Smo, undergoes constitutive internalization and transport to late endosomes/lysosomes (Incardona, 2002)
In KNRK cells: treatment with Leupeptin alone dramatically increased Ptc1⁺ late endosomes/lysosomes marked by lysosome-associated membrane glycoprotein (Incardona, 2002)
In the absenceof leupeptin, the small amount of internalized ShhN detected was localized to LBPA late endosomes, and, in the presence of leupeptin, ShhN and Ptc1 accumulated highly in LAMP-1⁺ vesicles. Double labeling for Ptc1 and LBPA was precluded by a requirement for Triton X-100 permeabilization to expose Ptc1HA immunoreactivity, which extracts LBPA. Concanamycin A treatment resulted in the accumulation of Ptc1 and ShhN in transferrin early endosomes, and ShhN did not colocalize with LBPA. Ptc1⁺ early endosomes appeared with similar kinetics and degree with or without ligand, indicating that Ptc1 reaches endosomes from the cell surface rather than by another route. (Incardona, 2002)
Ptc up-regulation (protein and trascript) is not observed in the central row of cells that correspond to the prospective wing margin (Figs. 1J and 1K). This region of refractory to Hh signaling exists along the entire DV boundary. See (Glise, 2002).

{Modifications}

Share structural homology with Disp in the form of a sterol-sensing domain
Ptc proteins (Ptc1 and Ptc2 in vertebrates) and the Niemann-Pick C1 (NPC1) protein, which includes but is not limited to the sterol-sensing domain
- NPC1 functions in the sorting and recycling of cholesterol and glycosphingolipids in the late endosomal/lysosomal system
Ptc has homology to bacterial proton-driven transmembrane molecular transporters

Embryo
- ptc^IIW only have two isolated denticle rows per segment (Fig 6F) (Methot, 2001)
very few ptc clones survive in the region between L1 and L2 as opposed to ci clones (Table 1: Methot, 2001; Philllips et al., 1990)
Mutant leads to derepression of wg
anterior ptc mutant clones cells minimize contact with neighboring ptc+ cells, causing mutant clones to be round with smooth borders (Fig 2 & 3) (Methot, 2001)
- ptc ci double mutants cells mix well with surrounding cells (Fig 2 & 3) (Methot, 2001)
transcription of wg becomes independent of hh in the absence of ptc
mutations in the sterol-sensing domain render it unable to repress Smo, but do not affect its binding of Hh (Martin, V. 2001, and Strutt, H. 2001)
other proteins w/ sterol-sensing domains are thought to control vesicle transport
Open-brain (opb) [Rab23] is involved in negative regulation of Shh signaling (Eggenschwiler 2001, rev Jeong, J. 2001)
ptccon is a Trp236->Arg mutation (region that binds Shh) and is a dominant gain-of-function allele. (Mullor, J.R., 2000)
ptccon reduces the sensitivity of Ptc to perceive [Hh]
Behaves like fu mutants and larvae lethality is rescued by Su(fu)LP (b/c there is more free Ci)
there is a slight cos2 phenotype
Over expressing smo or hh rescues
Clones crossed boundary into P especially with reduced Hh present
In ptc^con/ptc⁺ wing discs, anterior en expression is lost (dpp and ptc exp is unaffected)
In ptccon homozygotes: died at 3rd larval instar, imaginal discs smaller, no up-regulation of ptc at A-P border, high levels of Ci were uniformally detected
ptccon embryos display no phenotype
mutation of the SSD does not appear to compromise either binding or internalization of Hh (Martin et al. 2001; Strutt et al. 2001).
Mutations of hPtc or hSmo that trigger ligand-independent activity of the Hh signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma (Bale, 2001; Wechsler-Reya, 2001; Taipale, 2001)
Ptc has homology to bacterial proton-driven transmembrane molecular transporters; and Ptc function is altered when residues that are conserved in and required for function of these bacterial transporters is mutated. These results suggest that the Ptc functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule (Taipale, 2002)
clones of cells lacking Ptc activated Col in all cells within the clones except for cells along the prospective wing margin (Fig 3D) (Glise, 2002)
Ptc function can be rescued by two separate polypeptides (ptc-N: aa 1-676 and ptc-C: aa 676-1286), however individualy they have no effect (Johnson, 2000)
Deletion of the Ptc-C terminus (1130-1286) compromises target gene repression but not Hh sequestration (Johnson, 2000)
71B Gal4 / UAS-ptc1130X (aa 1130-1286 del) increased the venation and size of the anterior compartment while leaving the posterior region unaffected (Fig 2D). Clones expressing ptc-1130X have increased Ci levels and dpp expression, and this appears to be Hh independent (Johnson, 2000)

en-Gal4/UAS-ptc flies had a partially fused veins 3 & 4 and a narrowing of the 3-4 intervein (Johnson, 1995)
en-Gal4/UAS-ptc-N+C or UAS-ptc-N+CΔ (Johnson, 1995)

{Reagents}