Endocytosis and subsequent lysosomal degradation of activated signalling receptors can attenuate signalling. Endocytosis may also promote signalling by targeting receptors to specific compartments. A key step regulating the degradation of receptors is their ubiquitination. Hrs/Vps27p, an endosome-associated, ubiquitin-binding protein, affects sorting and degradation of receptors. Drosophila embryos mutant for hrs show elevated receptor tyrosine kinase (RTK) signalling. Hrs has also been proposed to act as a positive mediator of TGF-beta signalling. Drosophila epithelial cells devoid of Hrs accumulate multiple signalling receptors in an endosomal compartment with high levels of ubiquitinated proteins: these receptors include not only RTKs (EGFR and PVR) but also Notch and receptors for Hedgehog and Dpp. Hrs is not required for Dpp signalling. Instead, loss of Hrs increases Dpp signalling and the level of the type-I receptor Thickveins (Tkv). Finally, most hrs-dependent receptor turnover appears to be ligand independent. Thus, both active and inactive signalling receptors are targeted for degradation in vivo and Hrs is required for their removal (Jékely, 2003).
Monoubiquitination of membrane proteins has an important role in regulating their internalization and sorting to lysosomal degradation. The ubiquitin tag is recognized by proteins containing a ubiquitin interaction motif (UIM), such as epsins, Hse1p/STAM and Eps15. Hrs and its budding yeast homologue, Vps27p, also have a UIM and bind to ubiquitin (Bilodeau, 2002; Lloyd, 2002; Polo, 2002; Raiborg, 2002). The ubiquitin-binding ability of Hrs and Vps27p is required for the efficient sorting of ubiquitinated transferrin receptors in mammalian cells and Fth1p in yeast (Bilodeau, 200; Raiborg, 2002; Jékely, 2003 and references therein).
To determine whether Hrs was generally required for sorting and degradation of ubiquitinated proteins in Drosophila tissues, clones of cells mutant for hrs were generated within an epithelium using somatic recombination. Follicle cells of the ovary and wing imaginal disc cells from third instar larvae were examined. Follicular cells form a simple monolayer epithelium surrounding the germline cells and are large enough to detect subcellular localization of protein. The imaginal disc cells are smaller and form a pseudo-stratified epithelium. The mosaic tissues were stained with an antibody that recognizes mono- and poly-ubiquitinated proteins. Both follicle cells and wing disc cells lacking Hrs show a dramatic accumulation of ubiquitinated proteins. Most of the signal localizes to intracellular structures. In some cases accumulation is observed at the cell cortex. Thus, Hrs is required for the efficient removal of ubiquitinated proteins from the cell (Jékely, 2003).
An enlarged vesicular structure, the 'class E' compartment, has been observed in yeast cells mutant for VPS27 (Piper, 1995). Genetic studies in mice and Drosophila have also shown that cells mutant for hrs have enlarged endosomes (Komada, 1997; Lloyd, 2002), possibly due to impaired membrane invagination and multivesicular body (MVB) formation (Lloyd, 2002). To determine whether ubiquitinated proteins accumulate in the endosomal compartment in hrs mutant cells, GFP-Rab5 or GFP-2xFYVE fusion proteins were expressed in hrs mutant cells. Rab5, a small GTPase regulating endosome fusion, is a marker of early endosomes. FYVE domains bind to phosphatidylinositol-3-phosphate, which is enriched in endosomal membranes, and can also be used to specifically label endosomes. The ubiquitinated protein signal and the GFP-2xFYVE signal show extensive overlap in hrs mutant follicle cells. GFP-Rab5 and ubiquitinated proteins also show significant, although not complete, overlap. These data indicate that nondegraded ubiquitinated proteins accumulate in the endosomal compartment. Additionally, when the GFP-2xFYVE signal in hrs mutant and nonmutant cells was compared, an enlargement of FYVE-positive structures was observed in mutant cells, consistent with an enlargment of the endosomal compartment (Jékely, 2003).
Hrs was already known to affect degradation of receptor tyrosine kinases (RTKs). Indeed the two RTKs analysed in follicle cells, EGFR and PVR (PDGF/VEGF receptor), accumulate within hrs mutant cells, mostly in intracellular structures. These structures are also positive for the ubiquitinated protein signal, indicating that the receptors accumulate in endosomes (Jékely, 2003).
To test whether the requirement for Hrs is limited to RTKs, other types of signalling receptors were analysed. The Hedgehog receptor Patched and the Hedgehog signal transducer Smoothened are multi- and seven-pass transmembrane proteins, respectively. Thickveins (Tkv) is a type-I serine-threonine kinase receptor for the TGF-beta family ligand Dpp. Notch is a single-pass transmembrane protein that undergoes specific proteolytic cleavage upon activation. Notably, hrs mutant follicle cells show a marked accumulation of each of these receptors. As for RTKs, most of the receptor molecules accumulate intracellularly and show significant colocalization with the ubiquitinated protein signal. Thus, Hrs has a general role in regulating the sorting and degradation of diverse classes of signalling receptors. The homotypic adhesion molecule DE-cadherin was not affected visibly in hrs mutant cells. The latter observation is in agreement with previous observations that nonsignalling transmembrane proteins are not upregulated in hrs mutant animals (Lloyd, 2002). Either the trafficking of these proteins is independent of Hrs function or they have a low turnover rate in the examined tissues (Jékely, 2003).
The high degree of overlap between the signal for each of the receptors and the signal for ubiquitinated proteins means that the receptors accumulate in roughly the same endosomal compartment. This, together with the increase in receptor levels in hrs mutant cells, suggests that these receptors are degraded through the same Hrs-dependent pathway. Ubiquitination of the inhibitory Smad7 by the E3 ubiquitin ligase Smurf2 has been shown to target the Smad7-TGF-beta receptor complex for lysosomal degradation. In follicle cells, a similar complex may be sorted for degradation in an Hrs-dependent manner. It has been argued that the turnover of Hedgehog receptors is strongly regulated and may be critical for signalling, but a role of ubiquitination in this event has not been reported. The observation that Patched and Smoothened accumulate in compartments highly enriched in ubiquitinated proteins in hrs mutant cells suggests that trafficking of Patched and Smoothened is also regulated by ubiquitination (Jékely, 2003).
When analysing hrs mutant clones, an increase of ubiquitinated proteins at the cell cortex was occasionally noticed in addition to the intracellular accumulation. Some cortical accumulation could also be observed directly for the signalling receptors, in particular for Tkv. This accumulation could be due to inefficient endocytosis from the plasma membrane or increased recycling of endocytosed proteins. Hrs does not appear to be required directly for endocytosis (Lloyd, 2002), but downstream defects may 'clog up' the endocytosis machinery. Hrs can also affect receptor recycling. Overexpression of Hrs in tissue culture cells increases the retention of ubiquitinated transferrin receptors (Raiborg, 2002). The strong intracellular accumulation of receptors in hrs mutant cells could therefore either be due to defective sorting towards lysosomal degradation or due to defective post-endocytic retention, a concomitant general increase in the steady-state levels of the receptors at the plasma membrane, and therefore in endosomes. The first explanation is favored because often no increased surface levels of receptors or ubiquitinated proteins were detected even when strong intracellular accumulation was evident. Therefore, in hrs mutant cells, receptors seem to be retained intracellularly, rather than recycled. Hrs is most likely not the only factor responsible for the post-endocytic retention of receptors. Redundancy in sorting to the vacuole has been reported for the yeast alpha-factor receptor Ste3p. In this case, Vps27p and Hse1 have overlapping roles to sort Ste3p to the vacuolar lumen (Bilodeau, 2002; Jékely, 2003).
Hrs has been suggested to play a critical positive role in TGF-beta signal transduction in mice by stimulating the recruitment of Smad2 to the receptor. Whether Hrs is required for TGF-beta/Dpp signalling in Drosophila and thus might serve a conserved role in this pathway was tested. In the egg chamber, Dpp is expressed in the anterior follicle cells and contributes to the patterning of the follicular epithelium. The receptor Tkv is expressed uniformly in the epithelium. Active signalling downstream of the receptor can be monitored by the presence of the phosphorylated form of MAD (P-MAD) in the nucleus (anti-P-MAD). In wild-type stage 10 egg chambers, P-MAD was detected in the Dpp-producing anterior follicle cells and 1-2 rows of follicle cells immediately adjacent to the source. In hrs mutant follicle cells close to the Dpp ligand source, MAD phosphorylation and nuclear translocation still occurs efficiently. Thus, Hrs is not required for Dpp signalling in this context (Jékely, 2003).
The P-MAD expression domain is expanded to 3-4 rows of follicle cells if the epithelium is mutant for hrs. The P-MAD signal is graded, indicating that signalling is still dependent on the Dpp ligand gradient. Apparently, the hrs mutant follicle cells have increased sensitivity to Dpp. Since a higher level of Tkv protein is known to sensitize cells to low levels of Dpp, the expansion of the P-MAD domain can most simply be explained by the increased amount of Tkv at the surface of hrs mutant cells (Jékely, 2003).
The effect of Hrs on Dpp target gene activation was determined. The wing disc was used for this analysis since Dpp signalling and target gene activation are well characterized. Dpp is expressed in the middle of the wing disc [at the anterior-posterior (A-P) boundary] and forms a morphogen gradient. Spalt, a target of Dpp signalling, is expressed in a characteristic band at both sides of the A-P boundary. hrs mutant patches within the endogenous Splat domain show a slight increase in Spalt levels. When hrs clones are located at the edge of the Spalt domain, a modest expansion of the expression is observed. Thus, Hrs is not required for Dpp target gene activation in the wing disc. Instead, Hrs has a slightly negative effect on the pathway. As for the P-MAD signal in follicle cells, the Spalt signal is still graded in hrs mutant clones and no ectopic Spalt expression was observed in hrs mutant cells far from the Dpp source. This indicates that Spalt activation is still dependent on endogenous Dpp. The effects of hrs appear to be cell autonomous and positive in all parts of the Spalt expression domain, suggesting that hrs mutant wing disc cells are simply more sensitive to Dpp (Jékely, 2003).
Hrs mutant mouse embryonic cells show dramatically decreased responses to TGF-beta stimulation (Miura, 2000). Hrs is not required for Dpp signalling in Drosophila wing disc cells and ovarian follicle cells. The difference between these results may reflect an acquired aspect of TGF-beta pathway regulation in mammals or a specific regulation in mouse embryonic stem cells. However, it is clear that Hrs does not play a conserved general role in this otherwise quite conserved signalling pathway (Jékely, 2003).
To analyse the importance of ligand stimulation for Hrs-dependent downregulation of receptors, Tkv accumulation in hrs mutant cells was compared close to, and far from, the endogenous Dpp source. Follicle cells, which allow a clear detection of intracellular as well as cortical Tkv staining, were examined. There could be other TGF-beta-related ligands in the ovary, but the P-MAD staining indicates that the only significant source of signal stimulating this pathway comes from the anterior. Interestingly, high levels of Tkv accumulation were observed even in those hrs mutant follicle cells that were farthest from the Dpp source, experiencing no or very little Dpp ligand and signalling. Tkv accumulation was apparently uniform in all hrs mutant follicle cells, that is, cells closest and farthest from the Dpp source accumulated similar amounts of the receptor. These observations indicate that the bulk of Hrs-dependent downregulation of Tkv is constitutive in these cells, independent of ligand. This does not rule out the possibility that ligand-induced endocytosis also occurs. In the follicular epithelium, the spread of Dpp may be limited to a few cell diameters by high levels of receptor. It is possible that only a small fraction of the receptor molecules bind Dpp ligand. In this case, given the high rate of constitutive receptor turnover, stimulation would not affect visibly the bulk of receptor trafficking (Jékely, 2003).
Domeless (Dome) is an IL-6-related cytokine receptor that activates a conserved JAK/STAT signalling pathway during Drosophila development. Despite good knowledge of the signal transduction pathway in several models, the role of receptor endocytosis in JAK/STAT activation remains poorly understood. Using both in vivo genetic analysis and cell culture assays, it was shown that ligand binding of Unpaired 1 (Upd1) induces clathrin-dependent endocytosis of receptor-ligand complexes and their subsequent trafficking through the endosomal compartment towards the lysosome. Surprisingly, blocking trafficking in distinct endosomal compartments using mutants affecting either Clathrin heavy chain, rab5, Hrs or deep orange led to an inhibition of the JAK/STAT pathway, whereas this pathway was unchanged when rab11 was affected. This suggests that internalization and trafficking are both required for JAK/STAT activity. The requirement for clathrin-dependent endocytosis to activate JAK/STAT signalling suggests a model in which the signalling 'on' state relies not only on ligand binding to the receptor at the cell surface, but also on the recruitment of the complex into endocytic vesicles on their way to lysozomes. Selective activation of the pool of receptors marked for degradation thus provides a way to tightly control JAK/STAT activity (Devergne, 2007).
Using genetic analysis this study shows that several regulators of the endocytic pathway are required for normal JAK/STAT signalling in vivo. The membrane-bound Dome receptors undergo ligand-dependent internalization in clathrin-coated vesicles, which are then targeted to the sorting endosome via Rab5. The function of Hrs is required for JAK/STAT activation and to direct most of the active receptors to the MVBs, targeting them to the lysosome for degradation (Devergne, 2007).
One important question is to know whether the trafficking of ligand-bound receptors has any effect on signalling. This question was addressed by looking at Stat nuclear localization, which represents a robust readout to assess JAK/STAT activity, and at pnt-lacZ expression (Devergne, 2007).
The effect of Hrs is opposite on the JAK/STAT pathway compared with its effect on other pathways. Indeed, in the egg chamber, Hrs plays a positive role on JAK/STAT activity, whereas it has been shown to downregulate the EGFR, Notch and TGF-β pathways in the same tissue. Interestingly, HRS has been shown to interact with STAM in the same mono-ubiquitylated recognition complex (Lohi, 2001). STAM is a known JAK/STAT activator (Pandey, 2000), suggesting that HRS could control STAT signalling through its interaction with STAM. So, Hrs could play two crucial roles: first, allowing the sequestration and the sorting of the receptor to the lysosome and, second, activating the ligand-receptor complex in collaboration with STAM (Devergne, 2007).
The data challenge the simple view whereby binding of the ligand to the receptor at the membrane would be sufficient to activate the pathway. Indeed, it was found that equally essential is the need of clathrin for the activation of JAK/STAT signalling. Thus, activation can occur only when the ligand-receptor complex is assembled into clathrin-coated vesicles. In this view, activation would proceed in two steps, requiring both binding of the ligand and interaction with clathrin. The role of clathrin could be to concentrate/cluster receptors and/or bring them together with other signal transducers in the endosomal compartment. This finding is in agreement with a recent work showing that, in mammals, clathrin is required to transduce JAK/STAT signals through the IFNα-receptor, but not the IFNγ-receptor, suggesting a conserved function for clathrin in JAK/STAT signalling (Marchetti, 2006). Interestingly, like in mammals, JAK/STAT signalling in Drosophila might be controlled in a cell-type-specific manner by Chc-dependent endocytosis. Indeed, in Drosophila eyes, Vps25 and TSG101 mutations lead to Upd upregulation and JAK/STAT activation in a Notch-dependent manner (Devergne, 2007).
What is the significance of clathrin function and, more generally, of the requirement for internalization, in JAK/STAT signalling? It has been shown for several signalling pathways that internalization brings together membrane receptors and intracellular pathway components in the endosomal compartment, which thus serves as a platform for signalling. The fact that Dome internalization and activation are coupled to degradation has important consequences. Making signalling complexes only active in the endosomal compartment is a powerful mechanism to control the number of active complexes in the cell. Their targeting to the lysosome allows the control of their lifetime as active receptors, providing a temporal -- hence quantitative -- control on signalling (Devergne, 2007).
Activation of JAK/STAT follows an off/on/off model in which two conditions are required for correct JAK/STAT activation: (1) formation of a ligand-receptor complex (as proposed in the classical model), followed by (2) the internalization of the complex via Chc-containing budding vesicles. The sole formation of the ligand-receptor interaction would lead to an inactive complex (off). However, interaction with Chc and subsequent internalization activate the complex (on), thus ensuring that only the complexes targeted for degradation are activated. Arrival of the complex in the MVB/lysosome turns it into the off state (Devergne, 2007).
To study the interaction between Mom/Domeless and Unpaired, their cDNAs were subcloned into epitope-tagged mammalian expression vectors. 293T cells were cotransfected with V5-tagged Upd and HA-tagged Mom and expression was detected by immunofluorescence and Western blot with specific mouse monoclonal anti-tag antibodies. To examine the direct binding of Upd to Mom, 293T cells were transfected with Upd-V5. The ligand was released to the medium by treating the cells with heparin. Subsequently, the concentrated conditioned medium was applied to 293T cells nontransfected and transfected with HA-Mom and with a truncated form containing the N-terminal domain, Mom-N. Of note, given that there are no specific antibodies available for Mom, and that both anti-HA and anti-V5 antibodies have the same animal origin, (which prohibits double staining), an indirect approach was used to ascertain evidence of their presence in transfected cells. Since it is known that cells transfected with two DNAs will incorporate both at the same time, 293T cells were transfected with HA-Mom along with Stat92E. Therefore, cells stained with rabbit anti-STAT antibody should be those also expressing Mom. As expected, V5 staining was detected in cells containing Stat92E and transfected with Mom or Mom-N. These data show that Upd can be detected in 293T cells only when Mom is present, which indicates a physical interaction between these two molecules (Chen, 2002).
The gp130-subfamily of receptors has no intrinsic tyrosine kinase domain, but is constitutively associated with tyrosine kinase JAKs. A tyrosine residue fitting a YXXQ consensus motif at the C terminus of the receptors provides a binding site for STAT. A C-terminal YXXQ sequence was found in Domeless/Mom, suggesting that Mom may bind Stat92E (Chen, 2002).
The physical interaction between Mom and Stat92E was directly assessedin cotransfection experiments. Cell lysates were prepared from S2 cell lines expressing V5-epitope-tagged Mom, Hop, and Stat92E with either Upd-V5 or vector alone. The lysates were immunoprecipitated using anti-Stat92E antibodies and then probed using anti-V5 antibodies. Mom coimmunoprecipitates with Stat92E only in the Upd-V5 and mom-V5 transfected cells. These data suggest that Stat92E binds to the activated Mom receptor (Chen, 2002).
In the mammalian system, JAK proteins are bound to monomeric cytokine receptors through the membrane-proximal domain. Signaling is triggered when cytokine binding induces receptor dimerization. This brings the receptor-associated JAK kinases into apposition, enabling them to transphosphorylate each other. The JAK kinases, now activated, phosphorylate a distal tyrosine on the receptor. This receptor phosphotyrosyl residue is subsequently recognized by the SH2 domain of the STAT proteins, drawing them into the receptor complex, where they are activated through phosphorylation on the tyrosine residue by JAKs (Chen, 2002).
To show Mom-dependent activation of the Hop/Stat92E pathway, the tyrosine phosphorylation of Mom, Hop, and Stat92E was examined. S2 cells were co-infected with V5-epitope-tagged Mom, Hop, and Stat92E with either Upd-V5 or vector alone. Anti-Stat92E immunoprecipitates were prepared and tested for reactivity with the anti-phosphotyrosine antibody 4G10. Whereas Upd, Mom, Hop, and Stat92E proteins are detectable in the transfected samples, increased tyrosine phosphorylation of Mom, Hop, and Stat92E is detected in immunoprecipitates prepared from Upd-V5- and Mom-V5-transfected cells. These data are consistent with the hypothesis that Mom is a receptor of Upd that activates the Hop/Stat92E signal transduction pathway (Chen, 2002).
Recent evidence suggests that ubiquitination of endosomal TKRs may be a signal for trafficking to the lysosome rather than recycling to the surface. However, factors that bind ubiquitinated TKRs and sort them into multivesicular bodies (MVBs) are unknown. Recently, a 20 amino acid ubiquitin-interacting motif (UIM) conserved in family members of the proteosome subunit 5A (S5A) has been found in a large number of proteins, including several proteins implicated in endocytic trafficking. The UIM present in Hrs is highly conserved among all species examined, so it was determined if Hrs interacts with ubiquitin using GST pull-down assays. GST-ubiquitin but not GST readily pulls down the full-length Hrs protein from pupal extract. This interaction is direct, because GST-ubiquitin also binds purified recombinant N-Hrs (aa 1-376) protein containing the UIM. These data demonstrate that Hrs binds ubiquitin and suggest that Hrs may regulate endosomal sorting of TKRs via a direct interaction of Hrs with ubiquitinated receptors (Lloyd, 2002).
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