Gene name - patched
Cytological map position - 44D3-4
Function - hedgehog receptor
Keywords - segment polarity, hedgehog receptor
Symbol - ptc
Genetic map position - 2-59
Classification - transmembrane protein
Cellular location - surface
|Recent literature||Brigui, A., Hofmann, L., Arguelles, C., Sanial, M., Holmgren, R. A. and Plessis, A. (2015). Control of the dynamics and homeostasis of the Drosophila Hedgehog receptor Patched by two C2-WW-HECT-E3 Ubiquitin ligases. Open Biol 5. PubMed ID: 26446620
The conserved Hedgehog (HH) signals control animal development, adult stem cell maintenance and oncogenesis. In Drosophila, the HH co-receptor Patched (PTC) controls both HH gradient formation and signalling. PTC is post-translationally downregulated by HH, which promotes its endocytosis and destabilization, but the mechanisms of PTC trafficking and its importance in the control of PTC remain to be understood. PTC interacts with E3 Ubiquitin (UB)-ligases of the C2-WW-HECT family; two of them-SMURF and NEDD4-are known to regulate its levels. Mutation of the PTC PY motif, which mediates binding of C2-WW-HECT family members, inhibits its internalization but not its autonomous and non-autonomous signalling activities. In addition, the two related UB-C2-WW-HECT ligases NEDD4 and SU(DX) regulate PTC trafficking and finely tune its accumulation through partially redundant but distinct functions. While both NEDD4 and SU(DX) promote PTC endocytosis, only SU(DX) is able to induce its lysosomal targeting and degradation. In conclusion, PTC trafficking and homeostasis are tightly regulated by a family of UB-ligases.
|Gonzalez-Mendez, L., Gradilla, A. C., Sanchez-Hernandez, D., Gonzalez, E., Aguirre-Tamaral, A., Jimenez-Jimenez, C., Guerra, M., Aguilar, G., Andres, G., Falcon-Perez, J. M. and Guerrero, I. (2020). Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc. EMBO J: e103629. PubMed ID: 32311148
Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and it has been proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. This study show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.
Patched is involved in Hedgehog signaling. Hedgehog, secreted by posterior segmental compartments acts on adjacent anterior compartments to induce wingless and decapentaplegic. The influences of patched are repressive, both in early segment development and in imaginal discs. PTC represses wingless in anterior segmental domains and acts to repress decapentaplegic in the anterior compartment of wing imaginal discs. Thus although the targets differ, the same type of effect occurs.
How does Patched function? In addition to patched, the cellular components of of the Hedgehog pathway involved in wingless regulation include, shaggy/zeste white 3, fused, smoothened and the segment polarity transcription factor cubitus interruptus. Evidence has pointed to Patched maintaining an active signal through cyclic AMP-dependent protein kinase A (PKA). Removal of the catalytic subunit of PKA results in the disregulation of wg and the induction of an ectopic morphogenic furrow in the eye (Strutt, 1995). This view is problematic in light of the discovery that high PKA activity cannot counteract the phosphorylation of Fused that depends on HH signaling. On the other hand the phosphorylation of Fused can be inhibited by PTC suggesting that Fused is downstream of PTC. In this model PKA signals are integrated further downstream (Thérond, 1996 and Alcedo, 1996).
Setting aside the finer details of Hedgehog signaling, the question of whether Patched serves as a receptor for Hedgehog is examined. The evidence is rapidly becoming conclusive. In fact Patched is found closely associated with Wingless when double staining techniques are used (Capdevila, 1994a). Patched structure is still in doubt, and it might not even be a distant relative of a seven transmembrane G-protein coupled receptor. Evidence from the mouse points to a 12 pass transmembrane protein more closely related to ion transporters and channels (Goodrich, 1996). Thus Patched structure cannot be related to known protein receptor types.
Nevertheless, genetic and expression evidence point to a role of both Patched and Smoothened in Hedgehog receptor function. In wings bearing large anterior compartment mutant smo clones there appears to be an anterior shift in the distribution of dpp-expressing cells. This shift is interpreted as evidence that the loss of smo activity abolishes the ability of anterior cells to respond to HH and also allows HH to spread abnormally far into the anterior compartment until it reaches, and is transduced by smo+ cells. One mutant of ptc, ptcS2 gives rise to a PTC protein that appears indistinguishable from null alleles when assayed for its ability to repress inappropriate activity of the HH signal transduction pathway. Nevertheless, ptcS2 retains an activity that allows anterior compartment cells to sequester HH. Additionally, up-regulation of ptc by HH, a conserved feature of HH signaling, is required to limit the movement of HH from the posterior into the anterior compartment. Finally, PTC represses the HH signal transduction pathway by blocking the intrinsic activity of SMO. This suggests that PTC is positioned upstream of SMO in the HH signal transduction pathway, either as a factor that regulates SMO-transducting activity in response to HH or as a factor that facilitates the direct modulation of SMO activity by HH (Chen, 1996).
Surprisingly, a vertebrate homolog of Smoothened (vSMO), shows no direct interaction with mouse Sonic hedgehog (SHH), and Sonic hedgehog binds mPtc, a murine homolog of Patched, with high affinity. For example, epitope-tagged SHH as well as IgG-Sonic HH, both hybrid proteins containing the N terminal region of Sonic HH attach covalently to second proteins, bind to cells expressing mPTC, and no change in the affinity between SHH and PTC is observed in the presence of vSMO. mPTC can be co-immunoprecipitated with epitope-tagged SHH. Nevertheless, the three proteins can form a physical complex. Thus in the end, it appears that Patched, and not SMO is the protein that directly interacts with SHH. Patched and Smoothened function as receptor and signal transducing proteins respectively. Smoothened and Patched associate with each other on the membrane. In the absence of ligand, PTC inhibits signaling of Smoothened, while in the presence of ligand, PTC inhibition of SMO signaling is released and SMO signaling becomes activated (Stone, 1996).
Patched sends signals during the development of segmentation and development of the eye imaginal disc. hedgehog is required to overcome the repressive effects of Patched, allowing for the synthesis of DPP and Wingless. DPP is required to form a segmental boundary; both hedgehog and patched are required for differentiation and maintenance of segment compartment identity.
Interestingly, hedgehog interaction with patched does not always result in the synthesis of Wingless. The anterior-posterior boundary of the wing is drawn by dpp, which is required for differentiation of both compartments, but wingless is not induced in the anterior compartment. It functions instead on the proximal-distal axis. The same holds true for leg development.
The discovery of a hedgehog homolog in the mouse (sonic hedgehog) adds a new dimension to the debate over the function and regulation of patched. sonic hedgehog appears to regulate mouse patched. The argument is made, from the perspective of mouse biology, that the same holds true in the fly (Goodrich, 1996), but only when the whole hh-ptc pathway is understood in terms of the regulation of the genes involved can the nature of the interactions be fully understood.
The negative effects of patched point to a pervasive and significant aspect of developmental biology: inductive forces often counteract established repressive forces. There is a compelling logic to this view: if development traveled a straight and narrow path, each gene having only positive effects (that is, no negative effects) in the induction of the next gene along a pathway, there would be no order and no restraint. But contrary to this supposition, development is an orderly, restrained process. Control systems prevent cells from achieving unrestrained growth. The Notch pathway of lateral inhibition is just one example from among many. Only through the enforcement of negative regulation can a highly ordered biological system come into being.
The negative effects of patched are particularly relevant to the expression of dpp. In the wing disc, dpp is expressed in only a narrow band of cells between the anterior and posterior compartments. Patched not only inhibits wingless, but also dpp. Likewise dpp is inhibited in the posterior compartment by engrailed and invected (Sancola, 1995). Hedgehog, only able to act at short range, counteracts the repression of dpp by patched in just a single band of anterior compartment cells, those closest to the the posterior compartment, thus forming a dpp expressing boundary between the two compartments.
The multipass membrane-spanning proteins Patched and Smoothened have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc. Activation of the intracellular signaling pathway occurs when Hh binds to Ptc, an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is thus far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems. To test this model, the Drosophila Smo protein has been overexpressed in vivo and it has been found that increasing the levels of Smo protein per se is not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos reveals distinct patterns of Smo distribution, depending on which region of the protein is detected by the antibody. These findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner (Ingham, 2000).
To analyse the expression of the endogenous Smo protein, an antibody was raised against the membrane-proximal portion of the putative intracellular carboxy-terminal tail of Smo (anti-SmoC antibody) and this was used to stain wild-type and transgenic embryos. In contrast to the ubiquitous distribution of the SMO mRNA, immunohistochemical staining of wild-type embryos with the anti-SmoC antibody reveals a modulated distribution of the protein. Smo accumulates in a series of sequentially repeating stripes, each of which is about one-half a segment in width and spans the parasegmental boundary, the site of Hh activity. Staining of h-Gal4;UAS-smo embryos with the anti-SmoC antibody reveals a significant increase in the levels of Smo protein in the GAL4-expressing segments; strikingly, however, this staining is also restricted to cells flanking the parasegmental boundary. To determine the precise location of these Smo-positive cells, embryos were probed simultaneously with the anti-SmoC antibody and monoclonal antibodies for Engrailed (En), a marker of Hh-expressing cells, and Wg, a marker of Hh-responding cells. Double staining with the anti-En antibody reveals that Smo accumulates in and around cells expressing En; double staining with the anti-Wg antibody shows that Smo also accumulates in cells expressing Wg (Ingham, 2000).
The accumulation of Smo in and around cells secreting Hh protein, strongly suggests that the translation and/or stability of Smo is promoted by Hh activity. To test this possibility, Smo distribution was analyzed in embryos in which Hh is ectopically expressed under the control of the Kr promoter. Such embryos display a ubiquitous expression of Smo between parasegments 5 and 9, precisely the region in which ectopic Hh expression is driven by the Kr-Gal4 driver. Since Hh acts by inhibiting Ptc activity, the effects of Hh on Smo would be expected to be mediated by Ptc. In embryos homozygous for a ptc loss-of-function mutation, the modulated pattern of staining typical of the wild type is lost, indicating that Smo protein accumulates uniformly in the absence of Ptc activity (Ingham, 2000).
The simplest interpretation of these data is that Ptc functions to block the translation or promote the degradation of Smo. And, since the spatial distribution of Smo is unaltered in h-Gal4;UAS-smo transgenic embryos, it would follow that Ptc activity can suppress accumulation of Smo protein independent of the levels at which the gene is transcribed. This effect of Hh/Ptc-mediated signaling on Smo accumulation provides a simple explanation for the lack of an effect of ectopic smo expression, namely that the exogenous protein never accumulates outside the normal domain of Smo activity (Ingham, 2000).
Surprisingly, however, when h-Gal4;UAS-smo embryos were probed with the anti-FLAG antibody, a strikingly different pattern of exogenous protein accumulation is observed. In contrast to the narrow stripes detected by the anti-SmoC antibody, staining is seen throughout each h-Gal4 expression domain. This indicates that the SMO mRNA is translated in all cells in which it is transcribed. It follows that the Ptc-dependent staining pattern revealed by the anti-SmoC antibody reflects a post-translational modification of the Smo protein. One possibility is that Ptc could promote the cleavage of Smo, yielding a relatively stable but functionally inert truncated form of the protein in cells not exposed to Hh. In this connection, it is interesting to note that the SREPB cleavage activating protein (SCAP), with which Ptc shares some homology, acts to promote the cleavage of SREBP by chaperoning the latter from the endoplasmic reticulum to the Golgi. Alternatively, however, it could be that Ptc induces a modification of the Smo protein that masks the epitope recognised by the anti-SmoC antibody; binding of Hh to Ptc would suppress this modification, activating the protein and making it accessible to the antibody. To discriminate between these two possibilities, a second tagged form of Smo was generated, in this case inserting a hemagglutinin (HA) tag at the end of the carboxy-terminal tail. When embryos expressing this construct under h-Gal4 control were stained with an anti-HA antibody, a similar broad distribution of the tagged protein as that revealed by the anti-FLAG antibody is seen. This argues against the cleavage model, but instead suggests that the carboxy-terminal tail undergoes a Ptc-dependent modification. Since the anti-SmoC antibody was raised against an unmodified bacterially expressed protein, it seems most likely that this modification results in a conformational change that exposes epitopes recognised by the antibody. In this regard, it is notable that a putative dominant gain-of-function mutation in the human Smo protein is predicted to change the conformation of the equivalent region of the carboxy-terminal tail against which the anti-SmoC antibody is directed (Ingham, 2000).
Further studies indicate that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumor suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule (Taipale, 2002).
Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. This study investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localizes to cilia and inhibites Smoothened (Smo) by preventing its accumulation within cilia. When Shh binds to Ptc1, Ptc1 leaves the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function (Rohatgi, 2007).
In Drosophila, Ptc inhibits the movement of Smo to the plasma membrane. Binding of Hh causes the internalization of Ptc from the plasma membrane to vesicles, allowing Smo to translocate to the plasma membrane and activate downstream signaling. The discovery that protein components of primary cilia are required for Hh signaling suggested that subcellular localization has an important role in mammalian Hh signaling. Primary cilia are cell surface projections found on most vertebrate cells that function as sensory 'antennae' for signals. Several components of the Hh pathway, including Smo and the Gli proteins, accumulate in primary cilia, and Smo is enriched in cilia upon stimulation with Shh (Rohatgi, 2007).
The dynamic subcellular localization of Ptc1 and Smo in mammalian cells was studied with the use of novel antibodies to the two proteins. These antibodies allowed detection of endogenous Ptc1 and Smo in cultured mouse fibroblasts (NIH 3T3 cells) and mouse embryonic fibroblasts (MEFs), two Hh-responsive cell types. Hh signaling was activated in NIH 3T3 cells by treatment with either Shh or SAG (Shh-agonist), a small molecule that directly binds and activates Smo. Because ptc1 is itself a transcriptional target of Hh signaling, increases in Ptc1 protein levels can serve as a metric for pathway activation. Ptc1 protein levels began to rise by 4 hours and continued to increase until 24 hours after addition of Shh. After stimulation of cells with Shh or SAG, endogenous Smo was enriched in primary cilia. The mean fluorescence intensity of Smo in cilia began to increase as early as 1 hour after stimulation of cells with Shh or SAG. This likely represented relocalization from a cytoplasmic pool, because the total amount of Smo protein did not increase at this time point (Rohatgi, 2007).
To determine whether Ptc1 regulates the localization of Smo, Smo localization was examined in MEFs from ptc1-/- mice. These cells showed constitutive activation of Hh target gene transcription. Consistent with a role of Ptc1 in regulating Smo trafficking, Smo was constitutively localized to primary cilia in these cells even in the absence of Shh or SAG. Reintroduction of Ptc1 into these cells by means of a retrovirus suppressed Hh-pathway activity and prevented Smo accumulation in primary cilia. Thus, the regulation of Smo localization by Ptc1 is conserved from flies to mammals (Rohatgi, 2007).
To understand how Ptc1 may regulate entry of Smo into the cilium, the localization of Ptc1 was examined in MEFs and mouse embryos. Endogenous Ptc1 was present in small amounts in MEFs, near the limit of detection by immunofluorescence. Therefore the amounts of Ptc1 protein was increased by stimulating cells with SAG. Under these conditions, Ptc1 was highly enriched in primary cilia. The ciliary localization of Ptc1 was confirmed in three additional ways. First, Ptc1 fused to yellow fluorescent protein (Ptc1-YFP) was found around the base and in the shaft of cilia in unstimulated ptc1-/- cells infected with a retrovirus encoding Ptc1-YFP. Second, Ptc1-YFP overproduced in ptc1-/- cells by transfection showed clear ciliary localization in both live and fixed cells. Third and most important, endogenous Ptc1 was found in the cilia of mouse embryo mesoderm cells responsive to Shh (Rohatgi, 2007).
Ptc1 staining in cross sections of embryonic day 9.5 (E9.5) embryos was detected in cells of the ventral neural tube, notochord, splanchnic mesoderm, and paraxial mesoderm, precisely the regions where Hh signaling is known to be active and Shh target genes such as ptc1 are highly expressed. Focus was placed on mesoderm cells because they are likely the cells that gave rise to the MEFs that were analyzed in culture. Endogenous Ptc1 showed asymmetric localization to a domain surrounding the base of the cilium and in particles along the shaft of the cilium. This localization pattern around the base and in a particulate pattern along the shaft of the cilium is similar to that seen in cultured fibroblasts. In embryo cells, there was more variability in the amount of Ptc1 in the shaft of cilia, a finding likely related to differences in the amount of Shh signal received by cells in the complex milieu of embryonic tissue. The concentration of Ptc1 at the base of primary cilia suggests a mechanism for how it may inhibit Smo activation. Transport of proteins in and out of primary cilia is thought to be regulated at their base, and Ptc1 could function at this location to inhibit a protein-trafficking step critical for Smo activation (Rohatgi, 2007).
Shh could inactivate Ptc1 by binding to it at the cilium and inducing its internalization, degradation, or movement to other regions of the plasma membrane. To determine whether Ptc1 at the cilium can bind to Shh, a fluorescently labeled version of the N-terminal signaling fragment of Shh (ShhN-A594) was produced. Minute amounts of ShhN-A594, one-hundredth of those required to activate signaling, were added to live ptc1-/- cells transfected with Ptc1-YFP and a marker for cilia, inversin fused to cyan fluorescent protein (inversin-CFP). Live cells were used because the interaction between Shh and Ptc1 does not survive fixation. ShhN-A594 concentrated at cilia containing Ptc1-YFP and colocalized with puncta of Ptc1-YFP. Ptc1-/- cells expressing inversin-CFP alone did not bind ShhN-A594, and an excess of unlabeled ShhN prevented binding of ShhN-A594 (Rohatgi, 2007).
It was next asked whether the interaction of Shh with Ptc1 influences the localization of Ptc1. Ptc1 was concentrated at cilia after treatment of cells with SAG alone but not after treatment with Shh or a combination of Shh and SAG. This suggested that Shh binding might trigger the removal of the Ptc1-Shh complex from the cilium, or that new Ptc1 produced in response to Shh was not localized in the cilium. To distinguish these possibilities, the production of large amounts of Ptc1 was induced in the cilia of NIH 3T3 cells with SAG treatment and then the cells were switched to control medium or medium containing Shh. Ptc1 levels in the cilium remained stable in the control, but Shh treatment caused a time-dependent disappearance of Ptc1 from the primary cilium. The loss of Ptc1 from cilia was not associated with a decrease in total Ptc1 protein levels and thus implied movement of Ptc1 from cilia to another location in the cell. This delocalization was only evident with the endogenous protein and not upon examination of transfected Ptc1-YFP, a far more abundant protein (Rohatgi, 2007).
Ptc1 and Smo localization were examined in the same experiment. Because the localization changes for Ptc1 and Smo described above were each seen in >80% of the cilia visualized, the levels of Ptc1 and Smo in cilia were inversely correlated. The reciprocal time courses of Ptc1 disappearance and Smo appearance at cilia after Shh addition support a model in which Shh triggers the removal of Ptc1 from the cilium, allowing Smo to enter and activate signaling. Consistent with this idea, cells of the ventral neural tube and floor plate, which receive large amounts of Shh, showed high levels of Smo and low levels of Ptc1 in cilia. The movement of Ptc1 and Smo at the cilium is analogous to the situation in Drosophila, where pathway activation is associated with Smo movement to the plasma membrane and movement of Ptc away (Rohatgi, 2007).
Ptc1 may regulate Smo localization through a small molecule. Because Smo translocation to the primary cilium appears to be a critical step in its activation, a regulatory small molecule would be predicted to control this step. Naturally occurring oxysterols are good candidates for endogenous small molecules that regulate Smo function. Cellular sterol concentrations are important determinants of a cell's responsiveness to Shh, and oxysterols can activate Hh signaling. When NIH 3T3 cells were treated with activating concentrations of the oxysterol 20alpha-hydroxycholesterol, Smo rapidly translocated to the primary cilium with kinetics that were identical to those seen in cells treated with SAG or Shh. Treatment with 7alpha-hydroxycholesterol, an oxysterol that does not activate the Hh pathway, did not induce translocation of Smo. This result provides a specific molecular mechanism -- Smo translocation to cilia -- to explain how oxysterols regulate Hh signaling (Rohatgi, 2007).
Cells treated with 20alpha-hydroxycholesterol also retained Ptc1 in cilia in a pattern similar to that seen in cells treated with SAG. Thus, oxysterols appear to function not like Shh, by causing the removal of Ptc1 from cilia, but at a more downstream step to make Smo insensitive to the inhibitory effects of Ptc1. However, oxysterols function differently from SAG because they likely do not directly bind to Smo (Rohatgi, 2007).
These results suggest that Ptc1 localization to primary cilia inhibits the Hh pathway by excluding Smo and also allows cilia to function as chemosensors for the detection of extracellular Shh. Binding of Shh to Ptc1 at primary cilia is coupled to pathway activation by the reciprocal movement of Ptc1 out of the cilia and Smo into the cilia, a process that may be mediated by oxysterols. Elucidating the molecular machinery that controls Ptc1 and Smo trafficking at primary cilia will likely provide new targets for modulation of this important pathway (Rohatgi, 2007).
The ptc gene encodes a protein with seven putative transmembrane alpha helices (Hooper, 1989). This structure suggests that Patched is homologous to G-protein coupled receptors, although no real sequence homology is apparent.
date revised: 25 April 2001
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