InteractiveFly: GeneBrief

Hepatocyte growth factor regulated tyrosine kinase substrate : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. Factors that regulate endosomal sorting of TKRs are largely unknown, however Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) has been identified as one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active Epidermal growth factor receptor (Egfr) and Torso, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling (Lloyd, 2002).

Membrane trafficking events are tightly regulated to ensure proper spatial and temporal delivery of membrane bound cargo. Fusion of intracellular vesicles with their target membrane requires the formation of a highly stable core complex. Regulation of the formation of this complex may modulate vesicle fusion. One proposed regulator of core complex assembly is Hrs; mammalian Hrs binds to the plasma membrane t-SNARE SNAP-25 and inhibits core complex formation in vitro (Bean, 1997; Kwong, 2000). Addition of Hrs to a neuroendocrine cell assay inhibits neurotransmitter release, suggesting that Hrs may regulate Ca²⁺-triggered exocytosis. Notably, Hrs is predominantly localized to early endosomes (Komada, 1997), and Hrs mutant mice have enlarged endosomes (Komada, 1999). Furthermore, Hrs interacts with Eps15, a protein implicated in receptor-mediated endocytosis (Bean, 2000). Thus, Hrs has been proposed to play roles in both exo- and endocytosis (Lloyd, 2002).

Hrs is homologous to yeast Vps27p (vacuolar protein sorting), which regulates protein trafficking from a prevacuolar compartment to the vacuole (Piper, 1995). Vps27p belongs to the Class E subset of VPS proteins, which are implicated in sorting proteins into the vacuole lumen (Odorizzi, 1998). Hrs and Vps27p contain a FYVE domain which binds specifically to phosphatidyl-inositol-3-phosphate (PI3P), and this domain has been demonstrated to localize many proteins to the early endosome. Several FYVE domain-containing proteins have been implicated in endosomal trafficking, including early endosome autoantigen 1 (EEA1), which is essential for early endosome fusion, and Fab1p, which is required for sorting into MVBs. Thus, the FYVE domain may allow proteins to mediate membrane trafficking from or to the endosome through its interaction with PI3P (Lloyd, 2002 and references therein).

In addition to a role in vesicle trafficking, Hrs has been proposed to play different roles in several signal transduction pathways. Hrs binds to Stam, a protein implicated in cytokine signaling (Asao, 1997), and Hrs and Stam both contain VHS (Vps27p, Hrs, Stam) domains present in several proteins implicated in membrane trafficking or signal transduction. Overexpression of Hrs inhibits IL-2-mediated cell growth, suggesting that Hrs may function with STAM to negatively regulate cytokine signaling (Asao, 1997). In contrast to this inhibitory role, Hrs has recently been proposed to play positive roles in both TGF-β and EGFR signaling. Hrs binds to SMAD-2, and hrs mutant mouse embryos exhibit a reduced response to activin and TGF-β (Miura, 2000). Furthermore, overexpression of Hrs in HeLa cells inhibits ligand-induced degradation of EGFR (Chin, 2001), suggesting that Hrs may normally promote EGFR signaling by inhibiting endosome to lysosome trafficking of the receptor (Lloyd, 2002 and references therein).

Thus, although numerous data suggest Hrs may play a role in vesicle trafficking and signal transduction, the precise function of Hrs in these processes is unclear. To further investigate the function of Hrs, the effects of the loss of Hrs was analyzed in Drosophila. The data suggest that Hrs regulates inward budding of endosome membrane and MVB formation. More importantly, hrs mutant animals are unable to degrade active EGFR and Torso TKRs leading to enhanced TKR signaling (Lloyd, 2002).

A mutant allele of hrs that contains only the amino terminal third of the protein behaves genetically as a null mutation, because all phenotypes observed (e.g., enlarged endosomes and increased Egfr tyrosine phosphorylation) were equivalent in hrs/hrs and hrs/Df animals (Lloyd, 2002).

Hrs has been proposed to play a role in at least three vesicle trafficking events: exocytosis, endocytosis, and endosome to lysosome trafficking. Each of these trafficking steps was examined in hrs mutant larvae, and a role for Hrs was found only for endosomal trafficking. Fluid-phase tracer studies and TEM of garland cells reveal a dramatic enlargement of early endosomes, a phenotype that is also seen in mice lacking Hrs (Komada, 1999). Furthermore, TEM analysis of garland cells indicates that the enlarged endosomes result from an inability to invaginate endosomal membrane. A defect in endosomal invagination is consistent with the reduction in MVBs at hrs mutant synapses and with a proposed role for yeast VPS27 (Odorizzi, 1998) in sorting proteins inside the vacuole lumen (Lloyd, 2002).

Although the data demonstrate that Hrs regulates endosomal maturation enroute to lysosomes, several pieces of evidence suggest that Hrs is not essential for endosome to lysosome trafficking. (1) Absence of Hrs does not lead to a generalized increase in expression levels of surface membrane proteins, as would be expected if degradation was blocked. (2) Internalized avidin partially colocalizes with the low-pH indicator dye lysotracker in mutant garland cells at similar time points as in wild-type cells, suggesting that internalized proteins may be delivered to lysosomes. Thus, the data suggest that Hrs specifically functions in endosome invagination, separating membrane and membrane-associated proteins that are destined for lysosomes from those destined for recycling (Lloyd, 2002).

Extracellular signals are communicated to cells with remarkable temporal and spatial resolution. The rapid kinetics of signal amplification and termination are critical to the precision of signal transduction. One mechanism thought to mediate signal downregulation is the internalization and degradation of cell surface receptors. Although internalization of receptors may inhibit ligand binding, many receptors are still active, or in some cases, more active, after internalization. Once inside the early endosome, TKRs may either be recycled back to the surface of the cell or sorted into the multivesicular body (MVB) for degradation in the lysosome (Lloyd, 2002).

It has long been proposed that lysosomal delivery of cell surface receptors is a negative feedback mechanism for downregulation of receptor signaling. However, there is little in vivo evidence for this model, and it remains possible that deactivation of the receptor or downstream components may compensate for a failure to downregulate active receptor. The data suggest that trafficking of TKRs into the MVB plays an important role in signal attenuation. Interestingly, several of the morphological phenotypes observed in hrs mKO embryos are also seen in mutations affecting the torso pathway. For example, posterior cellularization defects are also observed in fs(1)polehole and l(1)polehole/D-raf embryos, and twisted gastrulation phenotypes are also observed in torso embryos (Lloyd, 2002).

Recently, overexpression of Hrs in HeLa cells has been shown to inhibit ligand-mediated degradation of EGFR, suggesting that Hrs may function to prolong EGFR signaling (Chin, 2001). In contrast, the data suggest the opposite function for Hrs, namely that it functions to attenuate TKR signaling by promoting degradation of the tyrosine-phosphorylated, or active, receptor. Interestingly, although active Egfr is upregulated in hrs mutants, total levels of the receptor are decreased, suggesting that Hrs is specifically required for degradation of active receptors. This reduction in total receptor is likely due to a well-characterized negative feedback mechanism whereby Egfr hyperactivation inhibits receptor transcription (Lloyd, 2002).

In summary, the following model is proposed for Hrs function. (1) Endocytosis of activated tyrosine kinase receptors (2) leads to the phosphorylation of Hrs on the early endosome membrane (Urbe, 2000). Phosphorylation may enhance the activity of Hrs, which then (3) leads to localized invagination of endosomal membrane. Ubiquitinated receptors may be sorted into the invagination directly via an interaction with the UIM of Hrs or indirectly through an interaction with Hrs binding proteins SNX1, Clathrin, or Eps15 (Bean, 2000; Chin, 2001; Raiborg, 2001b). Finally, (4) the membrane is pinched off to form a MVB, and (5) the internalized vesicles are trafficked to the lysosome for degradation. This process of MVB formation leads to a reversal of membrane topology such that the cytoplasmic portion of TKRs is now inside the MVB and unable to signal to downstream components. In this model, receptor-mediated activation of Hrs and MVB formation serves a critical role in attenuating tyrosine kinase receptor signaling (Lloyd, 2002).

Endocytic pathway is required for Drosophila Toll innate immune signaling

The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. This study shows that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. It is concluded that endocytosis is required for the activation of the Toll signaling pathway (Huang, 2010).

The Toll signaling pathway is essential for the Drosophila innate immune response to infections by fungi or Gram-positive bacteria. Most of the Toll signaling components were identified through genetic screens for mutants defective in embryonic dorsal-ventral patterning. This study describes Mop, a putative protein tyrosine phosphatase, as a regulator of the Toll pathway. Mop is an endosomal protein that colocalizes with Hrs, a subunit of the ESCRT-0 complex. Knocking down mop by RNAi inhibits Toll pathway activation both in vitro and in vivo. Epistasis studies show that Mop functions upstream of MyD88 and Pelle, at the same level as the Toll receptor. This study shows that Hrs is required for signal-dependent Cactus degradation, and Hrs is present in a complex together with Mop and the Toll receptor. The findings strongly suggest endocytosis plays an essential role in Drosophila Toll signaling (Huang, 2010).

Mop contains two conserved domains, an N-terminal Bro1 domain and a C-terminal PTP domain. Although Mop ortholog has not been identified in the yeast genome, the Bro1 domain itself is evolutionarily conserved from yeast to humans. The yeast Bro1 protein is a component of the ESCRT machinery and is localized to endosomes through the interaction with Snf7, an ESCRT-III subunit. Bro1 recruits the Doa4 deubiquitinating enzyme to endosomes and also functions as a cofactor to activate Doa4, which removes the ubiquitin moiety of ubiquitinated membrane proteins before the cargos invaginate into MVB vesicles. The presence of the Bro1 domain in Mop suggests that Mop is an endosomal protein, which is supported by the data. However, mutant Mop protein lacking the entire Bro1 domain (Mop deltaBro1) still localizes to endosomes. The C-terminal region of the yeast Bro1 protein, outside the Bro1 domain, also contributes to its endosomal location. It is likely that the Mop protein is targeted to endosomes through the nonconserved sequences between two domains and/or the Bro1 domain. This hypothesis is consistent with the finding that HD-PTP, the human Mop homolog, is endosomal in HeLa cells but is distributed throughout the cytoplasm in Drosophila S2 cells. Although Mop deltaBro1 localizes to endosomes, it cannot complement the function of wild-type Mop in Toll pathway activation. The Bro1 domain may target Mop to the specific endosomal domain or recruit other proteins involved in endocytosis or signaling (Huang, 2010).

This study also found that Mop proteins bearing a mutation in the putative phosphatase catalytic motif or missing the phosphatase domain can substitute for the endogenous Mop protein, indicating that the putative phosphatase activity is not required for Toll signaling. Immunoprecipitation experiments show that Hrs, Mop, and Toll are present in the same complex. Mop may act as an adaptor to interact with different proteins to facilitate endocytosis. During the preparation of this work, Mop was reported as an endosomal protein required for EGFR signaling during photoreceptor differentiation in Drosophila eye imaginal disk (Miura, 2008). Genetic evidence suggests that activated Drosophila EGFR is ubiquitylated and sorted through the endocytosis machinery for lysosomal degradation by a mechanism similar to the mammalian EGFR. A mop allele carrying a point mutation in the putative phosphatase catalytic motif functions as well as the wild-type allele in EGFR signaling of eye discs. These observations show that the putative phosphatase activity of Mop is not required for Toll or EGFR signaling. A recent paper demonstrated that the Human Mop homolog, HD-PTP, does not possess enzymatic activity (Huang, 2010).

The Toll signaling pathway has been characterized extensively during Drosophila embryonic development. The Toll protein has been shown to be present at the plasma membrane in the syncytial blastoderm. The majority of MyD88 and Tube also localize to the plasma membrane. However, a significant fraction of these could be detected as punctate structures in syncytial embryos. Mop and Hrs are required for Spätzle-dependent Cactus degradation, and both are essential endosomal proteins. These observations suggest that endocytosis of the Toll receptor is necessary for normal Toll signaling. Expression of chimeric Tube or Pelle proteins fused to the N-terminal 90 amino acid residues of Src activates Toll signaling without ligand binding in Drosophila embryos. The N-terminal region of Src contains a bipartite targeting sequence including the myristylation signal, and the Src protein is known to shuttle between the plasma membrane and endosomes. It is possible that the signal is initiated from endosomes under those experimental conditions (Huang, 2010).

Endocytosis is a dynamic process that regulates various signaling pathways in both a positive and negative manner. Mutations in the Drosophila tumor suppressor gene lethal giant discs (lgd) result in endosomal defects and overactivation of the Notch signaling pathway. In addition to the Toll signaling, endocytic pathway is required for EGFR activation and Wingless signaling. Mammalian TLR4 induces TRAM-TRIF-dependent IRF3 activation from endosomes after initiating the TIRAP-MyD88-dependent NFkappaB signaling at the plasma membrane (Kagan, 2008). The findings that Mop and Hrs are required for Toll signaling suggest that endocytosis has an evolutionarily conserved role in Drosophila Toll and mammalian TLR4 signaling. However, it is interesting to note that endocytosis is required for IRF3, but not NF-kappaB signaling in mammalian cells, but is required for NF-kappaB signaling in Drosophila (Huang, 2010).

REGULATION

Hrs mediates downregulation of multiple signalling receptors in Drosophila

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).

The endocytic control of JAK/STAT signalling in Drosophila

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).

Protein Interactions

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).

Protein Interactions

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).

DEVELOPMENTAL BIOLOGY

Polyclonal antibodies were generated to the full-length (anti-FL-Hrs) and amino-terminal half (aa 1-376, anti-N-Hrs) of the recombinant protein. Western analysis of fly extracts using the anti-FL-Hrs antibody detects a major band of 110 kDa in wild-type animals, whereas no protein is detected in hrs third-instar larvae (L3) or white prepupae (WPP). However, the anti-N-Hrs antibody recognizes a 30 kDa band in mutant animals in addition to the 110 kDa band in wild-type animals, suggesting that a 270 amino acid truncated protein is expressed in mutant animals. Furthermore, the presence of full-length Hrs protein in late stage 17 Df embryos suggests that maternally deposited Hrs protein is very stable and may compensate for the loss of zygotic Hrs in embryonic development. Indeed, embryos produced by mothers homozygous for hrs in their germline cells (maternal knockout or mKO) lack full-length Hrs protein and die early in embryogenesis. Thus, the effects of loss of Hrs function may be analyzed in zygotic mutant third-instar larvae/early pupae or in germline mutant embryos (Lloyd, 2002).

Analysis of the protein expression of Hrs suggests that Hrs is ubiquitously expressed. To determine the subcellular localization of Hrs, expression was examined in garland cells and muscle cells of third-instar larvae. Anti-Hrs labels vesicles enriched in perinuclear regions of muscle cells, whereas labeled vesicles are predominantly in the periphery of garland cells. These staining patterns are specific; they are not seen in hrs mutant cells. Interestingly, overexpression of hrs leads to an enlargement or accumulation of Hrs-positive vesicles and a reduction in cell size (Lloyd, 2002).

Both Hrs antibodies also label type I synaptic boutons of the neuromuscular junction (NMJ). There is some colocalization of Hrs with the synaptic vesicle (SV) marker Synaptotagmin, but most staining appears to be outside SV-rich regions. However, electrophysiological analysis of wild-type and mutant neuromuscular junctions suggests that Hrs does not play an important role in regulating synaptic vesicle exocytosis. Furthermore, Hrs is not enriched in the synapse-rich neuropil of the larval brain, even when overexpressed in neurons using the elav-GAL4 driver (Lloyd, 2002),

Hrs promotes ubiquitination and mediates endosomal trafficking of smoothened in Drosophila hedgehog signaling

In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. This study demonstrated that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. Hrs promotes Smo ubiquitination; deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. The N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, it was found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, these data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation (Fan, 2013).

The PI3K class III complex promotes axon pruning by downregulating a Ptc-derived signal via endosome-lysosomal degradation

Developmental axon pruning is essential for wiring the mature nervous system, but its regulation remains poorly understood. This study shows that the endosomal-lysosomal pathway regulates developmental pruning of Drosophila mushroom body γ neurons. The UV radiation resistance-associated gene (Uvrag) functions together with all core components of the phosphatidylinositol 3-kinase class III (PI3K-cIII) complex to promote pruning via the endocytic pathway. By studying several PI(3)P binding proteins, this study found that Hrs, a subunit of the ESCRT-0 complex, required for multivesicular body (MVB) maturation, is essential for normal pruning progression. Thus, the existence of an inhibitory signal that needs to be downregulated is hypothesized. Finally, the data suggest that the Hedgehog receptor, Patched, is the source of this inhibitory signal likely functioning in a Smo-independent manner. Taken together, this in vivo study demonstrates that the PI3K-cIII complex is essential for downregulating Patched via the endosomal-lysosomal pathway to execute axon pruning (Issman-Zecharya, 2014).

EFFECTS OF MUTATION

To determine if Hrs functions in endocytosis, trafficking of internalized tracers was investigated in third-instar larval garland cells, large cells with a high rate of fluid-phase endocytosis. Wild-type garland cells show strong labeling of peripheral vesicles after a 5 min incubation with avidin-Cy3, indicating rapid internalization of dye into endosomes. Mutant cells are much larger than wild-type cells but show strong labeling of peripheral vesicles, suggesting that dye internalization is not significantly impaired. However, many labeled vesicles (endosomes) in mutant cells are much larger than those observed in wild-type cells. Furthermore, analysis of lysosomal markers suggest that while lysosomes are reduced in size in hrs mutant cells, smaller endosomes are capable of delivering avidin to low pH compartments at a rate similar to wild-type cells (Lloyd, 2002).

Next, hrs mutant endosomes were analyzed using transmission electron microscopy (TEM). Garland cells were incubated with HRP for 5 min, fixed, and sectioned for TEM. In wild-type garland cells, HRP labels the lumen and internal membrane of peripherally located endosomes. In hrs mutant larvae, mutant endosomes are dramatically enlarged, but do not show significant HRP labeling, despite their ability to internalize avidin dye. Rather, in mutant cells, HRP labels a vast tubulo-vesicular network at the periphery of mutant cells, which is not seen in wild-type cells. Finally, endosomes in wild-type garland cells undergo invagination of their limiting membrane to eventually collapse upon themselves. In hrs garland cells, there is a strong reduction in the relative number of invaginated endosomes (5-fold, P = 0.008) and collapsed endosomes (10-fold, P = 0.003). These data suggest that endosomes are enlarged in hrs mutant cells due to an inability of endosomes to invaginate their limiting membrane (Lloyd, 2002).

Inward budding of endosome membrane is believed to be the first step of multivesicular body (MVB) formation. To determine if MVB formation is impaired in hrs mutant animals, electron microscopy was performed at the NMJ. In wild-type synapses, large, classical MVBs are occasionally observed, and they are believed to be endosomal intermediates containing synaptic vesicle proteins destined to be delivered to somatic lysosomes. Much more frequently, though, 60-120 nm vesicles were observe that also appear to contain small internal vesicles. Remarkably, in hrs mutant synapses, there is a 5-fold reduction in the number of these small MVBs. These data suggest that Hrs regulate formation of MVBs at the synapse (Lloyd, 2002).

One proposed function of multivesicular bodies is to partition transmembrane proteins and lipids destined for delivery to the lysosome from those destined to be recycled back to the surface of the cell. While most cell surface proteins are recycled from endosomes, activated TKRs such as the EGFR are trafficked inside MVBs for degradation in the lysosome. Because Hrs is phosphorylated in response to TKR activation (Komada, 1995), the possibility that Hrs regulates TKR degradation and signaling was investigated in Drosophila (Lloyd, 2002).

The first TKR pathway analyzed was the Torso pathway, which functions very early in development to pattern the embryonic termini. The Torso receptor is present throughout the surface of the early embryo, but it binds ligand only at the poles. Ligand binding of the Torso receptor triggers the ras/MAPK cascade leading to the phosphorylation of ERK/MAPK. A diphospho-ERK (dpMAPK)-specific antibody has been used to investigate TKR signaling in the early embryo. In wild-type embryos, terminal dpMAPK staining initiates approximately 2 hr after egg laying (AEL), peaks about 30 min later, and then rapidly terminates during the initiation of gastrulation. In cellular blastoderm hrs maternal knockout (mKO) embryos, dpMAPK staining is enhanced and spatially broadened when compared to wild-type embryos. Furthermore, dpMAPK expression remains elevated at the anterior and posterior termini during early gastrulation, when compared to wild-type. Hence, Torso signaling is both spatially broadened and temporally prolonged (Lloyd, 2002).

MAPK activation induces the expression of the terminal gap genes huckebein (hkb) and tailless (tll), and the pattern of expression of these genes can be used as a quantitative readout for Torso receptor activation. In mutant embryos, both anterior and posterior domains of hkb are significantly expanded by 23% and 50%, respectively. Similarly, the anterior stripe of tll is moved posteriorly by 50%, and the posterior stripe is expanded by 25%. Thus, these data confirm that Torso signaling is enhanced and broadened both spatially and temporally in mutant embryos and demonstrate that Hrs negatively regulates signaling of the Torso TKR pathway (Lloyd, 2002).

Next, whether an increase in Torso signaling was due to the failure of receptor degradation was investigated. Total levels of the Torso receptor in early (0-4 hr) embryos are similar between wild-type and hrs mKO embryos. However, a 56 kDa band corresponding to the cytoplasmic domain of Torso (500 aa) is prevalent in mutant animals but barely detectable in wild-type embryos. These data suggest that a failure to degrade the active cytoplasmic portion of the receptor may underlie the increased Torso signaling observed in early embryos. To further examine degradation of the Torso receptor, the kinetics of receptor protein expression were examined in wild-type and mutant embryos. In wild-type embryos, Torso protein expression is maximal just prior to receptor activation (1-2 hr AEL) and is rapidly downregulated such that it is barely detectable by 5 hr AEL. In hrs mKO embryos, in contrast, Torso protein levels fail to decline until 9-10 hr AEL, at which time zygotic expression of hrs is abundant. These data demonstrate that hrs is essential for Torso TKR degradation (Lloyd, 2002).

During early gastrulation, hrs mKO embryos display numerous morphological defects. Some mKO embryos fail to complete posterior terminal cellularization, and their yolk appears to be extruded posteriorly during gastrulation. During germ band elongation, most hrs mKO embryos undergo highly abnormal development and exhibit aberrant folding or twisting. The vast majority of mutant embryos arrest morphological development prior to germband retraction, and few secrete cuticle. These phenotypes suggest that hrs may be required to regulate multiple early embryonic signaling pathways (Lloyd, 2002).

Next, the effects of loss of Hrs function on the Egfr pathway were examined. Egfr signaling is required for a wide variety of cell fate decisions throughout Drosophila development. Secretion of the Egfr ligand, Spitz, in the ventral midline leads to graded activation of Egfr and the ras/MAPK cascade in the ventral ectoderm. dpMAPK staining is present in the ventral neuroectoderm and dorsal cephalic regions of the gastrulating embryo, and this staining pattern requires Egfr activation. dpMAPK staining in these regions is expanded in hrs mKO embryos, demonstrating that Egfr signaling is enhanced in the absence of Hrs (Lloyd, 2002).

Next to be examined was whether enhanced MAPK signaling in the ventral ectoderm results in an expansion of ventral fate. The transcription factor ventral nervous system defective (vnd) is a primary target of Egfr signaling in the ventral ectoderm, and genetic manipulations leading to enhanced Egfr signaling lead to an expansion of cells expressing vnd. In hrs mKO embryos, there is an expansion of the number of cells expressing Vnd from 1-2 in wild-type to 3-4 in the mutant. Furthermore, Fas III expression, known to be a downstream target of Egfr activation, is upregulated in hrs mKO embryos. Thus, a dorsal expansion of cells expressing dpMAPK and Vnd and an upregulation of Fas III expression suggest that Egfr signaling is enhanced in hrs mKO embryos (Lloyd, 2002).

To determine if enhanced Egfr signaling is due to increased receptor activity, tyrosine phosphorylation of the receptor was examined by immunoprecipitating Egfr from pupal lysates and Western blotting with a phosphotyrosine-specific antibody. Levels of tyrosine-phosphorylated receptor are increased 2- to 3-fold in hrs/hrs and hrs/Df animals when compared to wild-type (+/+) or heterozygous (hrs/+) animals. When these same membranes are stripped and reprobed with anti-Egfr antibody, total levels of the receptor are found to be decreased approximately 2-fold in hrs/hrs and hrs/Df mutant animals. Together, these data indicate that the relative levels of the active form of Egfr are increased approximately 5-fold in hrs mutant animals. Finally, levels of other ubiquitously expressed surface transmembrane proteins examined at this stage are unchanged or slightly decreased in mutant animals, suggesting that there is not a general defect in turnover of plasma membrane proteins (Lloyd, 2002).

A mutation in dVps28 reveals a link between a subunit of the endosomal sorting complex required for transport-I complex and the actin cytoskeleton in Drosophila

Proteins that constitute the endosomal sorting complex required for transport (ESCRT) are necessary for the sorting of proteins into multivesicular bodies (MVBs) and the budding of several enveloped viruses, including HIV-1. The first of these complexes, ESCRT-I, consists of three proteins: Vps28p, Vps37p, and Vps23p or Tsg101 (see Drosophila Tsg 101) in mammals. A mutation was characterized in the Drosophila homolog of vps28. The dVps28 gene is essential: homozygous mutants die at the transition from the first to second instar. Removal of maternally contributed dVps28 causes early embryonic lethality. In such embryos lacking dVps28, several processes that require the actin cytoskeleton are perturbed, including axial migration of nuclei, formation of transient furrows during cortical divisions in syncytial embryos, and the subsequent cellularization. Defects in actin cytoskeleton organization also become apparent during sperm individualization in dVps28 mutant testis. Because dVps28 mutant cells contained MVBs, these defects are unlikely to be a secondary consequence of disrupted MVB formation and suggest an interaction between the actin cytoskeleton and endosomal membranes in Drosophila embryos earlier than previously appreciated (Sevrioukov, 2005).

In the Drosophila genome, a single gene, CG12770, exhibits significant homology to the yeast and mammalian Vps28 proteins. The cDNA GH04443 is derived from this locus and encodes a predicted protein of 210 amino acids that is 62% and 35% identical to its human (hVPS28) and yeast (ScVPS28) counterparts, respectively. There are no similarities to other protein sequence motifs in the database. An antibody raised against dVps28 recognizes a protein of the expected size that is widely expressed during Drosophila development and also in cultured Drosophila S2 cells. In S2 cells as well as in cells of the eye disc and in isolated spermatocysts, dVps28 protein was uniformly distributed throughout the cytosol with no obvious enrichment in any organelle (Sevrioukov, 2005).

To test whether the homology of Vps28 proteins extends to their biochemical activity, its binding to Vps23p/Tsg101 was examined. The Drosophila homolog dTsg101 is encoded by cDNA GH09529. Because antibodies are not yet available against endogenous dTsg101 protein, epitope-tagged versions of dTsg101 and dVps28 were coexpressed to test their interaction in S2 cells. Immunoprecipitation of dVps28 from S2 cells resulted in the coprecipitation of expressed HA-dTsg101, which was increased after coexpression of Myc-dVps28. These results indicated that, like its yeast and mammalian orthologs, dVps28 binds specifically to dTsg101 (Sevrioukov, 2005).

Consistent with ESCRT's conserved function from yeast to mammalian cells, loss of dVps28 function causes morphological changes in MVBs and developmental defects in the compound eye that indicate a subtle misregulation of cell signaling molecules. However, for the ligands and receptors, no significant changes were detected in their cell surface levels or their delivery to lysosomes in dVps28 cells, indicating that any changes were too subtle to be detected by the immunofluorescence methods used (Sevrioukov, 2005).

One possible explanation for this observation is that some Vps28 functions are partially fulfilled by another protein. It is not likely that this hypothetical protein is similar to Vps28p in sequence because no another Vps28-like molecule in the completed genome sequences of Drosophila melanogaster. Another possibility is that the dVps28^l(2)k16503 allele does not completely inactivate dVps28 function. Although this possibility cannot be ruled out, it is unlikely for two reasons: (1) the dVps28^l(2)k16503 allele is a strong mutation that causes lethality early in development at the transition from first to second instar, much earlier than a null mutation in the hrs gene, which regulates the sorting of receptors into MVBs and (2) the lethal phase and the loss of dVps28 protein were indistinguishable between larvae homozygous for dVps28^l(2)k16503 and those that were hemizygous over a deficiency of the region. This indicates that the dVps28^l(2)k16503 allele removes most if not all of dVps28 function (Sevrioukov, 2005).

Another possibility for the relatively mild phenotypes is the perdurance of dVps28 protein. This is suggested by the dramatic phenotypes after removal of the maternal contribution in mKO dVps28 embryos: many remained unfertilized and the remaining embryos were arrested in their development before reaching the cellular blastoderm. It is interesting to compare this phenotype to that of embryos lacking any maternal Hrs contribution. Hrs binds to mono-ubiquitinated membrane proteins, whose sorting into MVBs is thought to be mediated by Hrs binding to ESCRT-1. Consistent with this notion, mKO hrs embryos exhibit a reduced down-regulation of cell surface receptors and a resulting enhanced activation of the MAP kinase pathway. Importantly, these defects were observed after cellularization had been completed and during gastrulation. The finding that mKO dVps28 embryos exhibit major defects before completion of cellularization indicates functions of dVps28 in addition to the down-regulation of membrane proteins mediated by the interaction of ESCRT-I with Hrs (Sevrioukov, 2005).

Surprisingly, the earliest defect found in fertilized eggs was an uneven distribution of nuclei. In Drosophila, the first 13 nuclear divisions occur without accompanying cell divisions. The first five of these syncytial divisions occur close to the center of the embryo, with nuclei subsequently spreading out along the anterior-posterior axis. This process, referred to as axial expansion, depends on the function of the actin cytoskeleton. In mKO dVps28 embryos, this process often is disorganized resulting in an uneven distribution of nuclei (Sevrioukov, 2005).

Other dVps28 phenotypes emerged during cellularization. After axial expansion, the nuclei move to the embryo's cortex where the last four of the syncytial nuclear divisions occur. The resulting nuclei are surrounded by invaginating membranes in a process that requires the actin-myosin network. At this stage, two defects were evident in embryos lacking dVps28. Many of the cells that formed were droplet shaped instead of the usual cuboidal shape. The actin cytoskeleton is required for normal cellularization and interfering with is function, by cytochalasin, or altering the activity of the Rho or Cdc42 GTPases, interferes with normal cellularization. Furthermore pole cells, usually the first cells to form, were absent in most embryos. The lack of pole cells has previously been observed in embryos in which axial expansion is perturbed upon interference with the actin cytoskeleton (Sevrioukov, 2005).

All of these early phenotypes are consistent with a role of dVps28 in directly or indirectly organizing the actin cytoskeleton. A role for endosomes in actin remodeling during cellularization has previously been established in embryos mutant for nuf or rab11. The small GTPase Rab11 localizes to recycling endosomes, and Nuf is a homolog of Arfo2 that directly binds Rab11 and acts in recycling endosomes. Mutants eliminating either of these proteins cause gaps in which actin fails to be recruited to the furrows during cortical nuclear divisions, similar to the observations in mKO dVps28 embryos. In all three of these mutants, the actin defects may be a consequence of the failure to recruit Discontinuous actin hexagon (Dah: a membrane-associated protein that localizes to invaginating furrows in syncytial blastoderm embryos and during cellularization) to invaginating furrows. Dah has significant similarity to dystrobrevin and dystrophin. Dystrophin plays a critical role in anchoring the actin cytoskeleton to membranes, and consistent with a similar function for Dah, embryos lacking maternally contributed Dah fail to properly assemble the actin cytoskeleton at furrows (Sevrioukov, 2005 and references therein).

A subset of the defects in rab11 mutant embryos has been linked to a requirement for trafficking through the recycling endosome during cellularization. Consistent with this notion, defects in rab11 and nuf embryos only become apparent during cortical divisions. In mKO dVps28 embryos, by contrast, defects were detected in the distribution of nuclei, long before cellularization initiates. This indicates a function of dVps28 in actin remodeling independent of recycling to the cell surface and independent of Rab11 and Nuf. This is consistent with the finding that in mKO dVps28 embryos, recycling endosomes are not affected, as judged by Rab11 and Nuf localization, suggesting that dVps28 is required for furrow localization of Dah down-stream or in parallel to Rab11 and Nuf's function in recycling endosomes. Such a model is difficult to reconcile with the canonical function of Vps28 as an ESCRT-1 subunit involved in targeting proteins into the interior vesicles of late endosomes (Sevrioukov, 2005 and references therein).

It is unlikely that Dah mediates all effects of dVps28 on the actin cytoskeleton. No defects are observed in embryos lacking Dah before cycle 10, long after defects have become apparent in mKO dVps28 embryos. Furthermore, Dah is not required during spermatogenesis, another developmental context in which effects of dVps28 on the organization of the actin cytoskeleton were observed. Spermatogenesis in dVps28 mutant testis progresses until bundles of 64 syncytial spermatids are formed. These spermatids are separated by a process called individualization that requires complex membrane rearrangements. At the site of these rearrangements, syncytial membrane, cytoplasm, and vesicles accumulate in the cystic bulge. In wild-type testis, an actin-dependent process drives the cystic bulge away from the 64 spermatid nuclei toward the distal end. The loss of synchrony in the movement of the cystic bulge is the first detectable defect during spermatogenesis in dVps28 mutant testis. Because dah mutants have no phenotype in males, these results indicate an independent connection between dVps28 function and the actin cytoskeleton (Sevrioukov, 2005 and references therein).

Actin acts at many stages in the endocytic pathway. In yeast, the initial internalization step requires actin polymerization, and in mammalian cells, early endosomes move on the tip of actin tails. Additionally, late endocytic organelles require the actin cytoskeleton for fusion in yeast and mammalian cells. Furthermore, a screen of the yeast genome for mutations interfering with protein sorting to the vacuole identified several regulators of the actin cytoskeleton (Sevrioukov, 2005 and references therein).

Importantly, several of these mutants identified on the basis of a vacuolar sorting phenotype also exhibited defects in the organization of the actin cytoskeleton. For example, aberrant actin patches and a reduction of actin cables were observed in yeast lacking Vps36p, one of the subunits of the ESCRT-II complex. It will be interesting to see whether a similar functional connection between the actin cytoskeleton and the ESCRT-I complex may underlie the enigmatic phenotypes of Tsg101 mutations in mice that cause cell cycle arrest and early embryonic lethality (Sevrioukov, 2005 and references therein).

Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing

During animal development, Wnt/Wingless (Wg) signaling is required for the patterning of multiple tissues. While insufficient signal transduction is detrimental to normal development, ectopic activation of the pathway can be just as devastating. Thus, numerous controls exist to precisely regulate Wg signaling levels. Endocytic trafficking of pathway components has recently been proposed as one such control mechanism. This study characterizes the vesicular trafficking of Wg and its receptors, Arrow and DFrizzled-2 (DFz2), and investigates whether trafficking is important to regulate Wg signaling during dorsoventral patterning of the larval wing. A role for Arrow and DFz2 in Wg internalization has been demonstrated. Subsequently, Wg, Arrow and DFz2 are trafficked through the endocytic pathway to the lysosome, where they are degraded in a hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)-dependent manner. Surprisingly, Wg signaling is not attenuated by lysosomal targeting in the wing disc. Rather, it is suggested that signaling is dampened intracellularly at an earlier trafficking step. This is in contrast to patterning of the embryonic epidermis, where lysosomal targeting is required to restrict the range of Wg signaling. Thus, signal modulation by endocytic routing will depend on the tissue to be patterned and the goals during that patterning event (Rives, 2006).

During patterning and growth of the wing imaginal disc, cells along the D/V axis interpret positional information and, hence, their fate, from the concentration of Wg ligand. The graded distribution of Wg, with high levels near the source at the D/V boundary and low levels toward the edges of the wing pouch, is therefore crucial for normal wing development. Lysosomal targeting of Wg and its receptors has been proposed as a mechanism for shaping the Wg gradient and attenuating signal transduction. To address this model, both trafficking to the lysosome and lysosome function was interfered with using genetic and pharmacological means (Rives, 2006).

In Drosophila, the hrs loss of function allele is a valuable tool for interrupting vesicular traffic to the lysosome. Hrs functions in late endosome invagination, a process that separates endocytic cargo to be recycled from cargo destined for the lysosome. Trafficking of the EGFR and Torso RTKs into the late endosome/MVB is an important step in signal attenuation; hrs mutant embryos experience elevated tyrosine kinase signaling due to the persistence of active receptors. Likewise, in the wing disc and the ovarian follicle cell, Hrs is required for downregulation of Tkv levels and dampening of the Dpp signal. Thus, Hrs activity is required to attenuate multiple developmental signals (Rives, 2006).

The fact that RTK and Dpp signaling levels are elevated in hrs mutant cells implies that active receptor complexes continue to signal inside the cell from an endocytic compartment. Although receptor internalization may turn off signaling by preventing ligand-receptor interaction, it is clear that many receptors remain active on endosomal membranes. For instance, activated EGFR can be detected in association with downstream signaling effectors on early endosomes, suggesting that signaling persists after endocytosis. This study reports dramatic intracellular accumulation of Wg, Arrow, and DFz2 in hrs or deep orange (dor) mutant wing discs; dor encodes a yeast VPS homologue required for delivery of vesicular cargo to lysosomes. Similar observations have been made for Wg and for Wg and Arrow. Given this dramatic intracellular accumulation of ligand, receptors, and a signal transducer, Wg signaling levels are expected to be elevated in hrs mutant cells. However, based on antibody stains for three Wg targets, no altered Wg signaling was detected in mutant cells. This was true for large null mutant clones, induced early in development, as well as in discs from larvae bearing a null hrs allele. The attenuation of Wg signaling, therefore, appears to be regulated differently from the attenuation of RTK and Dpp signaling (Rives, 2006).

The data suggest that Wg signaling is attenuated prior to Hrs-mediated lysosomal targeting of the receptor complex. In this case, removal of hrs prevents receptor and ligand degradation but has no bearing on signal output. Following endocytosis, internalized receptor-ligand complexes may be deactivated by physical dissociation in the increasingly acidic environment as they move through the endocytic compartment or by targeting to the lysosome for degradation. A model is favored in which the active Wg receptor complex is attenuated by dissociation earlier in the endocytic pathway; perhaps, this complex is more sensitive to pH levels in early endosomes, whereas, for example, a Dpp receptor complex is only uncoupled at the lower pH of later endosomal compartments (Rives, 2006).

Alternatively, there may be residual lysosomal degradation in hrs cells sufficient to effectively terminate signaling despite the accumulation of Wg, Arrow, and DFz2. It is not certain that Hrs is obligatory in targeting endocytic cargo to the lysosome. Internalized avidin, an endocytic tracer, still localizes to a low pH compartment in hrs mutant garland cells, suggesting that some trafficking to the lysosome continues in the absence of Hrs. Perhaps, this residual trafficking is sufficient to dampen Wg signaling levels but not RTK or Dpp levels (Rives, 2006).

In contrast to the genetic removal of hrs, treatment of wing discs with the lysosomal protease inhibitors chloroquine or NH₄Cl leads to expansion of SOPs, a Wg gain-of-function phenotype. While this result agrees with the previous finding that chloroquine-treated embryos generate excess smooth cuticle, indicative of enhanced Wg signaling, it is surprising that disruption of lysosome function can affect signaling. Once internalized, receptors are sorted into inner MVB vesicles, they are presumably sequestered from intracellular effectors and thereby deactivated. If mild bases, such as chloroquine, solely affect lysosomal protease function, a step subsequent to MVB sorting, this should not affect Wg signaling output in embryos or in imaginal discs. As all endocytic compartments maintain an acidic environment that is crucial to their function, it is unlikely that alkalizing agents solely inhibit the lysosome. In a caution to their use, pharmacological reagents such as chloroquine and NH₄Cl almost certainly disturb earlier pH-dependent trafficking steps as well, resulting in the accumulation of active receptor complexes. It is hypothesized that chloroquine- and NH₄Cl-mediated alkalization prevents the dissociation of Wg from its receptor(s), thereby resulting in prolonged signaling (Rives, 2006).

Consistent with the excess SOP specification in chloroquine- and NH₄Cl-treated discs, RNAi knockdown of Rab5 in cultured cells causes an increase in Wg-dependent reporter activation. These findings suggest that Wg signaling is normally attenuated at a trafficking step after internalization from the plasma membrane, but prior to Hrs-mediated lysosomal targeting. Such findings should be interpreted cautiously, however, as S2 cells are reported to be macrophage-like, and, thus, any effects on signaling output in these cells might not compare to that in wing disc cells in vivo. Nevertheless, attempts were made to define more precisely the trafficking step involved by treating cultured S2 cells with Shi dsRNA. So far the results have been ambiguous, since two trials demonstrated increased reporter activation while two other trials exhibited no such increase. Unfortunately, due to the compromised viability of endocytosis-defective cells in the wing disc, the DRab5 or Shi cell culture results could not be varified in vivo. However, in agreement with the data, a recent report shows enhanced Wg signaling, as evidenced by accumulation of the signal transducer Armadillo, in cells expressing a temperature-sensitive dominant negative variant of Shi. The viability issue was circumvented by transiently expressing dominant negative Shi with a 3-h upshift to the non-permissive temperature. Interestingly, no change was observed in Wg target gene expression under these conditions, suggesting that cell viability becomes compromised before such changes can occur (Rives, 2006).

While no evidence was found that lysosomal targeting modulates Wg signal output in the developing wing, it is clear that Wg, Arrow, and DFz2 are trafficked to the lysosome by Hrs. Hrs contains a conserved ubiquitin-interacting motif (UIM) and binds ubiquitin in vitro, suggesting that it regulates MVB sorting via direct interaction with ubiquitinated receptors. Monoubiquitination of cell surface receptors is emerging as an important signal for internalization and lysosomal sorting. It will be of interest to determine whether Arrow, Fz, and DFz2 undergo signaling-dependent monoubiquitination, and whether this has a consequence for Wg signaling output (Rives, 2006).

Signaling ligands are commonly internalized by receptor-mediated endocytosis, during which a ligand–receptor complex accumulates in coated pits on the plasma membrane and enters the cell in clathrin-coated vesicles. In the embryonic epidermis, endocytosis of Wg is thought to be receptor-mediated; expression of DFz2-GPI, which presumably lacks an endocytic signal, binds Wg but does not cause internalization. A similar model is predicted in the wing imaginal disc, where expression of DFz2-GPI stabilizes Wg to a greater extent than full length DFz2, most likely due to an inability to internalize Wg. Consistent with these views, it was found that extracellular Wg accumulates on the surfaces of arrow and fz⁻dfz2⁻ mutant cells. This striking accumulation cannot be explained by ectopic wg gene expression and likely results from impaired Wg internalization. In support of this conclusion, Wg and Arrow can colocalize in endosomes. It was still possible to detect residual Wg internalization into arrow mutant cells and fz⁻dfz2⁻ mutant cells. Yet, given the striking excess of extracellular Wg on receptor-deficient cells, a large increase was expected in the number of intracellular Wg puncta if Wg is internalized at a normal rate. This was not observed and led to a suggestion that Arrow, Fz, and DFz2 function as endocytic receptors for Wg. Since Fz does not contain an obvious endocytic signal, it is presumed that Arrow and DFz2 play more prominent roles. The residual intracellular Wg in receptor-deficient cells might be explained by a functional redundancy of Arrow and DFz2 in ligand internalization, in which case an absolute defect could only be observed by producing arrow^-dfz2⁻ doubly mutant cells. While this manuscript was in preparation, Piddini (2005) also reported that both DFz2 and Arrow contribute to Wg trafficking and degradation. A model was proposed in which DFz2 is important for Wg binding and internalization, while Arrow targets the Wg-DFz2 complex for degradation in the lysosome (Rives, 2006).

Contrary to hypothesis, recent evidence suggests that the accumulation of extracellular Wg on arrow and fz⁻dfz2⁻ mutant clones is due to upregulation of the glypican Dally-like protein (Dlp) (Han, 2005). That study also observed an increase in the level of extracellular Wg on arrow and fz⁻dfz2⁻ mutant clones. However, Wg accumulation was reduced if the mutant cells were compromised for the ability to make HSPGs by additional removal of sulfateless (sfl), an enzyme required for heparan sulfate biosynthesis, or brother of tout-velu (botv), a heparan sulfate copolymerase required for HSPG biosynthesis. This suggests that some of the build-up of extracellular Wg is due to trapping by excess HSPGs, rather than to a defect in endocytic trafficking (Rives, 2006).

In the process of evaluating endocytosis-defective cells for changes in Wg signaling levels, cells were frequently observed undergoing apoptosis. This is not surprising, since endocytosis is an important means for the cell to acquire macromolecules essential for viability as well as to gauge the growth needs of the tissue in which it resides. The results are troubling, though, given the widespread use of shi^ts, DRab5^DN and Shi^DN in the Drosophila community. Thus, it is necessary to monitor cell viability and assay for expression of control genes when using these reagents in order to draw accurate conclusions about signaling levels (Rives, 2006).

One notable question that was not addressed experimentally is whether endocytosis of Arrow or DFz2 is induced by Wg stimulation or proceeds continuously, independent of ligand. Some evidence for Wg-induced endocytosis of DFz2 has recently been presented (Piddini, 2005). Signal-induced endocytosis is well established, especially for RTK signaling, and plays an important role in controlling signal duration. Constitutive endocytosis and recycling provide a more general means of regulating receptor concentration at the cell surface but may also be used to downregulate signaling by clearing activated receptors, as suggested for the Tkv receptor in the developing wing. Future investigation of this issue will provide insight into the regulation of Wg signaling by endocytosis (Rives, 2006).

EVOLUTIONARY HOMOLOGS

Vps27p, a yeast homolog of Hrs, is a sorting receptor for ubiquitinated membrane proteins destined for degradation

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it was shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. A complex of Vps27p and Hse1p that localizes to endosomal compartments is described that is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is to act as a sorting receptor for ubiquitinated membrane proteins destined for degradation (Bilodeau, 2002).

Down-regulation (degradation) of cell surface proteins within the lysosomal lumen depends on the function of the multivesicular body (MVB) sorting pathway. To function, this pathway requires the class E vacuolar protein sorting (Vps) proteins. Of the class E Vps proteins, both the ESCRT-I complex (composed of the class E proteins Vps23, 28, and 37) and Vps27 (mammalian hepatocyte receptor tyrosine kinase substrate, Hrs) have been shown to interact with ubiquitin, a signal for entry into the MVB pathway. Activation of the MVB sorting reaction is dictated largely through interactions between Vps27 and the endosomally enriched lipid species phosphatidylinositol 3-phosphate via the FYVE domain (Fab1, YGL023, Vps27, and EEA1) of Vps27. ESCRT-I then physically binds to Vps27 on endosomal membranes via a domain within the COOH terminus of Vps27. A peptide sequence in this domain, PTVP, is involved in the function of Vps27 in the MVB pathway, the efficient endosomal recruitment of ESCRT-I, and is related to a motif in HIV-1 Gag protein that is capable of interacting with Tsg101 (see Drosophila Tumor suppressor protein 101), the mammalian homologue of Vps23. It is proposed that compartmental specificity for the MVB sorting reaction is the result of interactions of Vps27 with phosphatidylinositol 3-phosphate and ubiquitin. Vps27 subsequently recruits/activates ESCRT-I on endosomes, thereby facilitating sorting of ubiquitinated MVB cargoes (Katzmann, 2003).

Structural studies of Hrs

Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate [PtdIns(3)P]-binding FYVE domain and two coiled-coil domains. The roles of these domains in the subcellular localisation of Hrs were investigated. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 os abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes is Rab5 independent. The VHS-domain is nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, which has been shown to bind to SNAP-25, are required for targeting of Hrs to early endosomes. A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutation (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs. Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an additional domain. It is speculated that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes (Raiborg, 2001b).

Hrs is part of a multivalent complex which includes STAM and Eps15, that sorts ubiquitinated proteins into the multivesicular body pathway

The signal-transducing adaptor molecule STAM is involved in cytokine-mediated intracellular signal transduction. A 110-kDa phosphotyrosine protein inducible by stimulation with interleukin 2 (IL-2) has been cloned. The 110-kDa molecule was found to be a human counterpart of mouse Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and to be associated with STAM. Tyrosine phosphorylation of Hrs is induced rapidly after stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor as well as hepatocyte growth factor. The mutual association sites of Hrs and STAM include highly conserved coiled-coil sequences, suggesting that their association is mediated by the coiled-coil structures. Exogenous introduction of the wild-type Hrs significantly suppresses DNA synthesis upon stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor, while the Hrs mutant deleted of the STAM-binding site loses such suppressive ability. These results suggest that Hrs counteracts the STAM function, which is critical for cell growth signaling mediated by the cytokines (Asao, 1997).

Hrs-2, via interactions with SNAP-25, plays a regulatory role on the exocytic machinery. Hrs-2 physically interacts with Eps15, a protein required for receptor-mediated endocytosis. The Hrs-2/Eps15 interaction is calcium dependent, inhibited by SNAP-25 and alpha-adaptin, and results in the inhibition of receptor-mediated endocytosis. Immunoelectron microscopy reveals Hrs-2 localization on the limiting membrane of multivesicular bodies, organelles in the endosomal pathway. These data show that Hrs-2 regulates endocytosis and delineates a biochemical pathway (Hrs-2-Eps15-AP2) in which Hrs-2 functions. These data also suggest that Hrs-2 acts to provide communication between endo- and exo-cytic processes (Bean, 2000).

STAM1 and STAM2, which have been identified as regulators of receptor signaling and trafficking, interact directly with Hrs, which mediates the endocytic sorting of ubiquitinated membrane proteins. The STAM proteins interact with the same coiled-coil domain that is involved in the targeting of Hrs to endosomes. STAM1 and STAM2, as well as an endocytic regulator protein, Eps15, can be co-immunoprecipitated with Hrs both from membrane and cytosolic fractions. Recombinant Hrs, STAM1/STAM2, and Eps15 form a ternary complex. Overexpression of Hrs causes a strong recruitment of STAM2 to endosome membranes. Moreover, STAM2, like Hrs and Eps15, binds ubiquitin, and Hrs, STAM2, and Eps15 colocalize with ubiquitinated proteins in clathrin-containing endosomal microdomains. The localization of Hrs, STAM2, Eps15, and clathrin to endosome membranes is controlled by the AAA ATPase mVps4, which has been implicated in multivesicular body formation. Depletion of cellular Hrs by small interfering RNA results in a strongly reduced recruitment of STAM2 to endosome membranes and an impaired degradation of endocytosed epidermal growth factor receptors. It is proposed that Hrs, Eps15, and STAM proteins function in a multivalent complex that sorts ubiquitinated proteins into the multivesicular body pathway (Bache, 2003).

Members of the STAM family of proteins, STAM1 and STAM2, are associated with Hrs through their coiled-coil regions. Both Hrs and STAM bind ubiquitin and are involved in endosomal sorting of ubiquitinated cargo proteins for trafficking to the lysosome. This study examines the biological significance of STAM binding to Hrs. Endogenous STAM1 and STAM2 are mostly localized on the early endosome, suggesting that they are resident endosomal proteins. A STAM2 mutant that lacks the coiled-coil region and does not bind Hrs, in contrast, mislocalizes to the cytoplasm. Deletion of a region located N-terminal to the coiled-coil region and conserved among STAM proteins also severely affects Hrs binding and the endosomal localization of STAM2, suggesting that this region is also involved in these activities. Depletion of endogenous Hrs by RNA interference similarly causes the mislocalization of exogenously expressed STAM2 to the cytoplasm. These results indicate that STAM is localized to the early endosome by binding to Hrs on the target membrane. In addition, the expression level of endogenous STAM proteins is drastically reduced in Hrs-depleted cells, suggesting that STAM is stabilized by binding to Hrs. Finally, STAM2 mutants lacking the Hrs-binding activity are defective in causing the enlargement of early endosomes, accumulating ubiquitinated proteins on this aberrant organelle, and inhibiting the degradation of ligand-activated epidermal growth factor receptors, suggesting that the association with Hrs is a prerequisite for STAM function (Mizuno, 2004).

Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. Three mammalian Vps (vacuolar protein sorting) proteins have been identified, Hrs and signal transducing adaptor molecules (STAMs) 1 and 2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif. Through formation of a complex, they are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, an Hrs-defective fibroblastoid cell line [hrs^-/-] was established; embryos with this genotype died in utero. In the hrs^-/- cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restores STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) fail to express the STAMs. This regulated control of STAM expression by Hrs is independent of transcription. Interestingly, STAM1 degradation is mediated by proteasomes and is partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs^-/- cells not only leads to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restores STAM1 levels in early endosomes and eliminates the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins (Kobayashi, 2005).

TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation

Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (tsg101) recently was found to be centrally involved in this process. TSG101 interacts with HRS, an early endosomal protein, and disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevents delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. These results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes (Lu, 2003).

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the 'late domain') binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. This study reports that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs; residues 222-777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222-777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding (Pornillos, 2003).

Hrs and the trafficking of epidermal growth factor

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p; however, the role of Hrs in lysosomal trafficking is unclear. Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form an approximately 550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using deletion analysis, the binding domains of Hrs and SNX1 that mediate their association have been mapped. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1 (Chin, 2001).

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs (an early endosomal protein) in this sorting process, Hrs was overexpressed in HeLa cells and the intracellular trafficking of epidermal growth factor receptor (EGFR) was examined in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibits the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. In contrast, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain does not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs (Morino, 2004).

Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. This study reports that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors (Yan, 2005).

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates multiple signal-transduction pathways as well as trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Although there is growing awareness that endocytic transport can play a direct role in signal specification, relatively little is known about the molecular mechanisms underlying this link. This study shows that human Sprouty 2 (hSpry2), a protein that has been implicated in the negative regulation of receptor tyrosine kinase (RTK) signaling, interferes with the trafficking of activated EGFR specifically at the step of progression from early to late endosomes. This effect is mediated by the binding of hSpry2 to the endocytic regulatory protein, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), and leads to a block in intracellular signal propagation. These observations suggest that EGFR signaling is controlled by a novel mechanism involving trafficking-dependent alterations in receptor compartmentalization (Kim, 2007).

Hrs recruits clathrin to early endosomes

Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Hrs co-localizes with clathrin, and the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes is observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin causes the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs does not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran are retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes (Raiborg, 2001a).

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Hrs is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy Hrs was shown to localize to flat clathrin lattices on early endosomes. WHrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. It is concluded that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface (Raiborg, 2002).

Huntingtin-associated protein 1 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate and functions in endosomal trafficking

Huntingtin-associated protein 1 (HAP1) is a novel protein of unknown function with a higher binding affinity for the mutant form of Huntington's disease protein, huntingtin. HAP1 interacts with Hrs, a mammalian homologue of yeast vacuolar protein sorting protein Vps27p involved in the endosome-to-lysosome trafficking. This novel interaction was identified in a yeast two-hybrid screen using full-length Hrs as bait, and confirmed by in vitro binding assays and co-immunoprecipitation experiments. Deletion analysis reveals that the association of HAP1 with Hrs is mediated via a coiled-coil interaction between the central coiled-coil domains of both proteins. Immunofluorescence and subcellular fractionation studies show that HAP1 co-localizes with Hrs on early endosomes. Like Hrs, overexpression of HAP1 causes the formation of enlarged early endosomes, and inhibits the degradation of internalized epidermal growth factor receptors. Whereas overexpression of HAP1 affects neither constitutive nor ligand-induced receptor-mediated endocytosis, it potently blocks the trafficking of endocytosed epidermal growth factor receptors from early endosomes to late endosomes. These findings implicate the involvement of HAP1 in the regulation of vesicular trafficking from early endosomes to the late endocytic compartments (Li, 2002).

Neurofibromatosis 2 (NF2) tumor suppressor schwannomin and its interacting protein HRS regulate STAT signaling

Mutations in the neurofibromatosis 2 (NF2) gene, with the resultant loss of expression of the NF2 tumor suppressor schwannomin, are among the most common causes of benign human brain tumors, including schwannomas and meningiomas. HRS strongly interacts with schwannomin. HRS is a powerful regulator of receptor tyrosine kinase trafficking to the degradation pathway and HRS also binds STAM. Both of these actions by HRS potentially inhibit STAT activation. Therefore, it was hypothesized that schwannomin inhibits STAT activation through interaction with HRS. Both schwannomin and HRS inhibit Stat3 activation and schwannomin suppresses Stat3 activation mediated by IGF-I treatment in the human schwannoma cell line STS26T. Schwannomin inhibits Stat3 and Stat5 phosphorylation in the rat schwannoma cell line RT4. Schwannomin with the pathogenic missense mutation Q538P fails to bind HRS and does not inhibit Stat5 phosphorylation. These data are consistent with the hypothesis that schwannomin requires HRS interaction to be fully functionally active and to inhibit STAT activation (Scoles, 2002).

Mutation of Hrs

Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, mice carrying a null mutation of the gene were generated. Hrs homozygous mutant embryos develop with their ventral region outside of the yolk sac, have two independent bilateral heart tubes (cardia bifida), lack a foregut, and die around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis is present in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs is disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking (Komada, 1999).

Endosomal localization and receptor dynamics determine tyrosine phosphorylation of Hrs

Hrs is a prominent substrate for activated tyrosine kinase receptors that has been proposed to play a role in endosomal membrane trafficking. The protein contains a FYVE domain, which specifically binds to the lipid phosphatidylinositol (PI) 3-phosphate (PI 3-P). This interaction is required both for correct localization of the protein to endosomes that only partially coincides with early endosomal autoantigen 1 and for efficient tyrosine phosphorylation of the protein in response to epidermal growth factor stimulation. Treatment with wortmannin reveals that Hrs phosphorylation also requires PI 3-kinase activity, which is necessary to generate the PI 3-P required for localization. Both hypertonic media and expression of a dominant-negative form of dynamin (K44A) were used to inhibit endocytosis -- under such conditions, receptor stimulation fails to elicit phosphorylation of Hrs. These results provide a clear example of the coupling of a signal transduction pathway to endocytosis, from which it is proposed that activated receptor (or associated factor) must be delivered to the appropriate endocytic compartment in order for Hrs phosphorylation to occur (Urbe, 2000).

Hrs plays an essential role in a recycling mechanism mediating functional resensitization of cell signaling

Hrs is well known to terminate cell signaling by sorting activated receptors to the MVB/lysosomal pathway. A distinct role of Hrs has been identified in promoting rapid recycling of endocytosed signaling receptors to the plasma membrane. This function of Hrs is specific for receptors that recycle in a sequence-directed manner, in contrast to default recycling by bulk membrane flow, and is distinguishable in several ways from previously identified membrane-trafficking functions of Hrs/Vps27p. In particular, Hrs function in sequence-directed recycling does not require other mammalian Class E gene products involved in MVB/lysosomal sorting, nor is receptor ubiquitination required. Mutational studies suggest that the VHS domain of Hrs plays an important role in sequence-directed recycling. Disrupting Hrs-dependent recycling prevented functional resensitization of the beta(2)-adrenergic receptor, converting the temporal profile of cell signaling by this prototypic G protein-coupled receptor from sustained to transient. These studies identify a novel function of Hrs in a cargo-specific recycling mechanism, which is critical to controlling functional activity of the largest known family of signaling receptors (Hanyaloglu, 2005).

Monoubiquitinylation regulates endosomal localization of Lst2, a negative regulator of EGF receptor signaling

Genetic screens performed in worms identified major regulators of the epidermal growth factor receptor (EGFR) pathway, including the ubiquitin ligase Cbl/SLI-1. This study focused on the less-characterized Lst2 protein and confirmed suppression of MAPK signals. Unexpectedly, human Lst2, a monoubiquitinylated phosphoprotein, does not localize to endosomes, despite an intrinsic phosphoinositol-binding FYVE domain. By constructing an ubiquitinylation-defective mutant and an ubiquitin fusion, it is concluded that endosomal localization of Lst2, along with an ability to divert incoming EGFR molecules to degradation in lysosomes, is regulated by ubiquitinylation/deubiquitinylation cycles. Consistent with bifurcating roles, Lst2 physically binds Trim3/BERP, which interacts with Hrs and a complex that biases cargo recycling. These results establish an ubiquitin-based endosomal switch of receptor sorting, functionally equivalent to the mechanism inactivating Hrs via monoubiquitinylation (Mosesson, 2009).

To ensure fidelity of signaling outcomes, activated receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), are subjected to signal desensitizing mechanisms, primarily entailing accelerated endocytosis and degradation in lysosomes. Activated receptors concentrate over clathrin-coated pits at the plasma membrane, which invaginate to form coated vesicles. Vesicles then uncoat prior to fusing with tubulo-vesicular organelles, denoted early endosomes (EEs). Receptor cargos subsequently undergo sorting, either for recycling back to the plasma membrane or for lysosomal destruction at late endosomal compartments termed multivesicular bodies (MVBs). RTKs often undergo ubiquitinylation through recruitment of Cbl family ubiquitin ligases. Mono- and oligoubiquitins in the context of RTKs drive progression of cargos via endosomes, toward lysosomes. Active sorting in endosomes is attributed to various ubiquitin-binding domains (UBDs) carried by multiple adaptors (Mosesson, 2009 and references therein).

Small GTPases of the Rab subfamily are also attributed pivotal functions; enrichment of specific Rabs in different endosomal compartments permits local activation of a particular complement of effectors, which execute requisite endocytic tasks. Activated Rab5, which operates at the cell surface/EE interface, stimulates production of phosphatidylinositol 3-phosphate (PI3P) in EE membranes, thereby nucleating complexes of effector proteins like EEA1, which harbors a PI3P-binding domain called FYVE. In addition to EEA1, ~30 other proteins share the double zinc finger FYVE motif. Notably, PI3P binding by specific FYVE domain proteins is highly regulated both in cis and in trans. For example, a FYVE-containing amino-terminal fragment of Hrs failed to localize to EEs. This may be attributed to ancillary interactions involving the flanking coiled-coil domain, or dimerization of FYVE domains. Similarly, the FYVE domain of EEA1 may not suffice for endosomal localization. Instead, endosomal localization of EEA1 is thought to be complemented in living cells by p38-induced phosphorylation. Thus, FYVE-mediated localization of a variety of endocytic adaptors to EEs is a multifocal regulatory node that impacts on both endocytosis and signaling. This study extends the complexity to regulation by monoubiquitinylation (Mosesson, 2009).

This study describes a mammalian FYVE domain protein, termed hLst2, whose endosomal localization is regulated by monoubiquitinylation. Consistent with its primitive ortholog in C. elegans, which functions as a negative regulator of the worm's EGFR (Yoo et al., 2004), cellular depletion of human Lst2 augments EGF-induced signaling. Like many endosomal adaptors, hLst2 undergoes constitutive monoubiquitinylation, but despite the intrinsic FYVE domain, it displays primarily nonendosomal localization. By identifying a specific lysine acceptor, monoubiquitinylation was discovered as a means to prevent FYVE domain-dependent association of hLst2 with PI3P-enriched endosomes. In line with a unique ubiquitin/PI3P switch, a nonubiquitinylated hLst2 localizes to EEs, promotes degradative sorting of activated EGFRs, and reduces signaling.

REFERENCES

Search PubMed for articles about Drosophila Hepatocyte growth factor regulated tyrosine kinase substrate

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Bache, K. G., Raiborg, C., Mehlum, A. and Stenmark, H. (2003). STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes. J. Biol. Chem. 278(14): 12513-21. 12551915

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date revised: 22 November 2022

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