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).
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).
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).
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).
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).
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 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 (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).
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).
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).
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 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).
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