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Abstract

The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals. Even though the domain was discovered just 5 years ago, a great deal has been learned regarding its three-dimensional structure and binding specificities. Moreover, a number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell. Interestingly, many of the EH-containing and EH-binding proteins display characteristics of endocytic "accessory" proteins, suggesting that the principal function of the EH network is to regulate various steps in endocytosis. In addition, recent evidence suggests that the EH network might work as an "integrator" of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation.

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... The EH domain mediates a number of important proteinp rotein interactions in endocytosis and molecular tra¤cking [11]. EH domains are about 100 amino acids in length, and bind short peptides in a hydrophobic pocket formed between two K-helices [28,29]. ...
... In support of this result, cDNA segments encoding several novel proteins with multiple NPF motifs [30,31] were isolated from a lambda-cDNA expression library, when the same EH domains were used as probes. The proteins have since been demonstrated to be cellular ligands for Eps15 and intersectin [32^36] and likely interact in a multivalent fashion [11,37]. Thus, analysis of the Fig. 1. ...
... The requirement for a free carboxylate group has limited the use of phage-displayed libraries in de¢ning the speci¢city of PDZ domains, since peptides are typically displayed at the N-terminus of proteins III or VIII of bacteriophage M13. However, it was possible to select peptide ligands from a Intersectin is a protein component of the endocytic machinery, and contains two EH domains, a central region believed to adopt a coiled-coil structure, and ¢ve SH3 domains [11]. A phage-display combinatorial (X 9 ) peptide library was screened for peptide ligands to the N-terminal EH domain of intersectin; the isolated ligands are shown on the left [31]. ...
Article
Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.
... EGF receptor pathway kinase substrate clone 15 (Eps15) has been found in protein complexes with multiple endocytic proteins, including dynamin and the vertebrate homologue of Dap160, intersectin (Santolini et al., 1999;Sengar et al., 1999;Salcini et al., 2001). Originally discovered as an EGF receptor kinase Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development JCB • VOLUME 178 • NUMBER 2 • 2007 310 substrate and a putative oncogene, Eps15 was later implicated in endocytosis and endosomal traffi cking by in vitro studies (Fazioli et al., 1993;Tang and Cai, 1996;Tebar et al., 1996;Wendland et al., 1996;Carbone et al., 1997;Benmerah et al., 1999;Torrisi et al., 1999;Bache et al., 2003). ...
... We then tested the requirement of N-and C-terminal domains of Eps15 in SV endocytosis and synaptic bouton development by neuronally expressing three eps15 cDNA constructs in the eps15-null background: (1) a fulllength eps15 cDNA (Eps15wt), (2) a truncated cDNA encoding only the N-terminal EH domains and coiled coil domains (∆DPF; Fig. S1 A), and (3) a full-length cDNA with point mutations in the EH domains (EHmut; unpublished data). It is expected that the ∆DPF truncation protein will lose the ability to interact with α-adaptin and ubiquitinated proteins, whereas the EHmut protein will only lose the ability to bind Dap160 and other EH binding proteins (Benmerah et al., 1999;Santolini et al., 1999). EHmut is expressed but fails to localize at the NMJ and was not further analyzed (unpublished data). ...
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Epidermal growth factor receptor pathway substrate clone 15 (Eps15) is a protein implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization. Its role is, however, still unclear, because of reasons including limitations of dominant-negative experiments and apparent redundancy with other endocytic proteins. We generated Drosophila eps15-null mutants and show that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis. Consistent with a role in SV endocytosis, Eps15 moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15, and eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 functions in concert with Dap160 during SV endocytosis. Based on these data, we hypothesize that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.
... These processes may be regulated by phosphorylation and dephosphorylation [1,2]. One such module is the EH domain, first identified in Eps15 [3], which binds an NPF (aspargine-proline-phenylalanine) sequence in the target proteins [4]. Among the EH domain-containing proteins are members of the EHD subfamily [4], characterized by three structural modules: an N-terminal domain, harboring nucleotide binding motifs [5,6]; a central coiled-coil domain, involved in oligomerization; and a single C-terminal EH domain. ...
... One such module is the EH domain, first identified in Eps15 [3], which binds an NPF (aspargine-proline-phenylalanine) sequence in the target proteins [4]. Among the EH domain-containing proteins are members of the EHD subfamily [4], characterized by three structural modules: an N-terminal domain, harboring nucleotide binding motifs [5,6]; a central coiled-coil domain, involved in oligomerization; and a single C-terminal EH domain. There are four vertebrate paralogs [7,8], two plant orthologs and one gene in C. elegans [9] and Drosophila [10]. ...
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Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.
... POB1 and Reps1 both contain Eps homology (EH) domains, which are found in many proteins associated with endocytosis (106). RalBP1 can also bind to the µ2 subunit of the AP2 adaptor complex, which is involved in promoting the interaction of AP2 with integral membrane proteins and recruiting them into clathrin-coated pits (107). ...
... RalB function in endocytosis is dependent upon interaction with RalBP1, which interacts with the EGFR substrate POB1, which contains an Eps homology domain (104). Eps homology domains are found in many proteins associated with endocytosis and vesicle sorting, including epsin and Eps15, two proteins that bind to POB1 (106,108). RalBP1 also binds to the µ2 subunit of the AP2 adaptor complex, which is involved in promoting the interaction of AP2 with integral membrane proteins and recruiting them into clathrin-coated pits (107). Given these observations, it has been speculated that RalA and RalB may serve distinct roles in mediating vesicle trafficking, ...
... After internalization into the endosomal compartment, a significant pool of EGF and EGFR escape recycling to the cell surface, and are sorted to a degradation pathway (Sorkin & Waters, 1993). Proteins that contain an EPS15 homology (EH) domain, and proteins that bind to these proteins, are considered to be involved in regulating various steps of endocytosis (Santolini et al., 1999). EGFR pathway substrate 15 (EPS15) (Fazioli et al., 1993) itself has three EH domains. ...
... Third, REPS2 contains one imperfect Eps15 homology (EH) domain (amino acids 16-69) and one consensus EH domain (amino acids 265-366). EH domain proteins are thought to play a role in receptor-mediated endocytosis (Santolini et al., 1999), and for REPS2 it has been demonstrated that the consensus EH domain binds Epsin and Eps15, two proteins that are implicated in endocytosis . In addition, it was shown that deletion mutants of REPS2 can inhibit internalization of EGF and insulin receptors . ...
Article
Like normal prostate cells, prostate cancer cells are dependent on androgens for growth and survival, and prostate cancer can be treated by androgen ablation therapy. However, after a period of time some of the prostate cancer cells no longer respond to androgen ablation and survive the therapy. This transition of androgen-dependent prostate cancer (ADPC) to androgen-independent prostate cancer (AIPC) is critical, since no effective therapy is available for the androgen-independent stage of the disease. The molecular mechanisms that underlie the transition are largely unknown. REPS2, the protein that is studied in this PhD thesis project, might be involved in a molecular mechanism that contributes to AIPC development, since REPS2 mRNA is downregulated in AIPC compared to ADPC. With specific antibodies it was shown that the REPS2 protein level in AIPC is decreased compared to ADPC. Transient overexpression of REPS2 in prostate cancer cell lines induced apoptosis within 48 h, which indicates that REPS2 may play a role in the life-death balance of the cell. To elucidate cellular functions of REPS2, proteins were identified that bind REPS2. A large fragment of the NF-κB subunit p65 (RELA) was found to bind REPS2. This protein p65 is inactive in ADPC but active in AIPC, and might cause cell survival through inhibition of apoptotic cell death. Two other protein sequences that were found to bind REPS2 represent parts of TRAF4 and STAT6. Interestingly, these two proteins, like p65, are implicated in control of some aspects of the NF-κB pathway. Taken together, the results point to a putative inhibitory effect of REPS2 on NF-kB signalling and prostate cancer cell survival.
... EH domains that have been identified in several endocytic accessory proteins including ITSN, eps15 and eps15R confer binding to the tripeptide sequence asparagine-prolinephenylalanine (NPF motif) [25]. EH domain-based networks have been suggested to function as signal integrators in diverse cellular processes including exo/endo-cytic vesicle cycling [26]. EH domain-NPF interactions were originally thought to be of low affinity and relatively promiscuous. ...
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During neurotransmitter release, SVs (synaptic vesicles) fuse at the active zone and are recovered predominantly via clathrin-mediated endocytosis at the presynaptic compartment surrounding the site of release, referred to as the periactive zone. Exo- and endo-cytosis in synapses are tightly temporarily and spatially coupled to sustain synaptic transmission. The molecular mechanisms linking these two cellular events, which take place in separate compartments of the nerve terminal, remain largely enigmatic. Several lines of evidence indicate that multiple factors may be involved in exocytic-endocytic coupling including SV integral membrane proteins, SV membrane lipids and the membrane-associated actin cytoskeleton. A number of recent studies also indicate that multimodular adaptor proteins shuttling between the active and periactive zones aid the dynamic assembly of macromolecular protein complexes that execute the exo- and endo-cytic limbs of the SV cycle. Here, we discuss recent evidence implicating the multidomain scaffolding and adaptor protein ITSN1 (intersectin 1) as a central regulator of SV cycling.
... Irs4p consists of 638 amino acids and contains a predicted epidermal growth factor substrate 15 homology (EH) domain. EH domains are highly conserved eukaryotic protein-binding regions that target the amino acid motif Asn-Pro-Phe (NPF) and form the framework of the EH network, an extensive protein interaction network that coordinates pathways regulating cell wall biogenesis and other cellular processes (de Beer et al., 1998; Confalonieri & Di Fiore, 2002; Salcini et al., 1997; Santolini et al., 1999; Tang et al., 2000). C. albicans Irs4p is the sole homologue of the duplicated Saccharomyces cerevisiae proteins Irs4p and Tax4p, which were recently shown to bind and activate Inp51p, a phosphatidylinositol-(4,5)-bisphosphate [PI(4) (5)P 2 ] 5- phosphatase (Morales-Johansson et al., 2004). ...
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We previously identified Candida albicans Irs4p as an epidermal growth factor substrate 15 homology (EH) domain-containing protein that is reactive with antibodies in the sera of patients with candidiasis and contributes to cell wall integrity, hyphal formation and virulence. In this study, we use a yeast two-hybrid method and co-immunoprecipitation to show that Irs4p physically interacts with the phosphatase Inp51p. Disruption of the Inp51p Asn-Pro-Phe (NPF) motif eliminates the interaction, suggesting that this motif is targeted by Irs4p. Both inp51 and irs4 null mutants exhibit significantly increased levels of phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)] without changes in levels of other phosphoinositides. Like the irs4 mutant, the inp51 mutant demonstrates increased susceptibility to cell wall-active agents, impaired hyphal formation and abnormal chitin distribution along hyphal walls during growth within solid agar. Moreover, the inp51 and irs4 mutants overactivate the cell wall integrity pathway as measured by Mkc1p phosphorylation. As anticipated, mortality due to disseminated candidiasis is significantly attenuated among mice infected with the inp51 mutant, and tissue burdens and inflammation within the kidneys are reduced. Hyphal formation and chitin distribution in vivo are also impaired, consistent with observations of embedded growth in vitro. All phenotypes exhibited by the inp51 and irs4 mutants are rescued by complementation with the respective genes. In conclusion, our findings suggest that Irs4p binds and activates Inp51p to negatively regulate PI(4,5)P(2) levels and the cell integrity pathway, and that PI(4,5)P(2) homeostasis is important for coordinating cell wall integrity, hyphal growth and virulence under conditions of cell wall stress.
... The EH network is implicated in the control of endocytosis and actin cytoskeleton organization (reviewed in Santolini et al., 1999). Our results indicate additional levels of involvement, i.e., modulation of nucleocytoplasmic shuttling and possibly of protein degradation. ...
Article
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The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.
... They display impaired organization of the actin cytoskeleton, and deeper plasma membrane invaginations than the control wild type cells [182]. Pan1p interacts with several proteins that are necessary for endocytosis: End3p in yeast [10], and yAP180 (the yeast homologue of the clathrin assembly polypeptide AP180), the epsin homologues Ent1p and Ent2p and the synaptojanin homologue, Slj1p in both yeast and mammals [142,181,183] and therefore has been proposed to function as a multivalent adapter required for endocytosis [181]. The observation of a genetic interaction between PAN1 and RSP5 [192] was the first indication of a possible role of Rsp5p in endocytosis beyond its role in ubiquitination of endocytosed proteins. ...
Article
In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca2+-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na+ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins.
... However, they both contain an Eps15 homology (EH) domain involved in protein-protein interactions, and also polyproline stretches and a coiled-coil domain located in the same order. POB1 interacts directly with Eps15 and Epsin (for reviews see [17], [18]) and Reps1 probably does so as well [19]. Eps15 and Epsin, and their yeast orthologs Pan1 and End3, are essential components of the endocytotic machinery. ...
Article
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The FGF/Ras/Ral/RLIP pathway is required for the gastrulation process during the early development of vertebrates. The Ral Interacting Protein (RLIP also known as RalBP1) interacts with GTP-bound Ral proteins. RLIP/RalBP1 is a modular protein capable of participating in many cellular functions. To investigate the role of RLIP in early development, a two-hybrid screening using a library of maternal cDNAs of the amphibian Xenopus laevis was performed. Xreps1 was isolated as a partner of RLIP/RalBP1 and its function was studied. The mutual interacting domains of Xreps1 and Xenopus RLIP (XRLIP) were identified. Xreps1 expressed in vivo, or synthesized in vitro, interacts with in vitro expressed XRLIP. Interestingly, targeting of Xreps1 or the Xreps1-binding domain of XRLIP (XRLIP(469-636)) to the plasma membrane through their fusion to the CAAX sequence induces a hyperpigmentation phenotype of the embryo. This hyperpigmented phenotype induced by XRLIP(469-636)-CAAX can be rescued by co-expression of a deletion mutant of Xreps1 restricted to the RLIP-binding domain (Xreps1(RLIP-BD)) but not by co-expression of a cDNA coding for a longer form of Xreps1. We demonstrate here that RLIP/RalBP1, an effector of Ral involved in receptor-mediated endocytosis and in the regulation of actin dynamics during embryonic development, also interacts with Reps1. Although these two proteins are present early during embryonic development, they are active only at the end of gastrulation. Our results suggest that the interaction between RLIP and Reps1 is negatively controlled during the cleavage stage of development, which is characterized by rapid mitosis. Later in development, Reps1 is required for the normal function of the ectodermic cell, and its targeting into the plasma membrane affects the stability of the ectoderm.
... One such module is the EH (Eps15 homology) domain first identified in Eps15 [15,16]. The EH domain structure generally consists of two EF-hands and a helix-loop-helix structure that binds calcium (or a pseudo EFhand), connected by an anti-parallel beta-sheet [17,18,19]. Many EH-containing proteins were identified in different species, among them EHD1-4 (EH domain containing proteins), Eps15 and Intersectin 1-2 [20,21,22,23]. ...
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Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain.
... The presence of the EH domains suggested that ITSN has a role in endocytosis. In support of this, multiple studies demonstrated that the EH domains directly interact with components of the endocytic machinery such as Epsin 1 and 2, Rev interacting protein (Rip/RAB/Hrb), SCAMP1, and Stonin2 [2,[9][10][11]. Also, ITSN localizes to clathrin-coated vesicles, and regulates clathrin-dependent endocytosis [7,12]. Further, ITSN plays a conserved role in endocytosis and vesicle recycling in multiple species (reviewed in [13]). ...
Article
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Intersectins (ITSNs) represent a family of multi-domain adaptor proteins that regulate endocytosis and cell signaling. ITSN genes are highly conserved and present in all metazoan genomes examined thus far. Lower eukaryotes have only one ITSN gene, whereas higher eukaryotes have two ITSN genes. ITSN was first identified as an endocytic scaffold protein, and numerous studies reveal a conserved role for ITSN in endocytosis. Subsequently, ITSNs were found to regulate multiple signaling pathways including receptor tyrosine kinases (RTKs), GTPases, and phosphatidylinositol 3-kinase Class 2beta (PI3KC2β). ITSN has also been implicated in diseases such as Down Syndrome (DS), Alzheimer Disease (AD), and other neurodegenerative disorders. This review summarizes the evolutionary conservation of ITSN, the latest research on the role of ITSN in endocytosis, the emerging roles of ITSN in regulating cell signaling pathways, and the involvement of ITSN in human diseases such as DS, AD, and cancer.
... 10 Proteins fated for degradation are directed to lysosomes while those destined for reuse are recycled back either to the plasma membrane or to the Golgi apparatus. 11 The EH domain is well represented among proteins involved in vesicular transport across the endolysosomal system, 12 which prompted efforts in linking EHD functions to endocytic recycling. Indeed, several of the EHD interacting partners appear to be effectors of Rab proteins. ...
Article
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Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins (DSPs). Among them is the evolutionarily conserved group of ATP‐binding Eps15‐homology domain‐containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T‐tubules in skeletal muscles and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles. This article is protected by copyright. All rights reserved.
... One such module is the EH (Eps15 homology) domain first identified in Eps15 [13], [14]. The EH domain structure generally consists of two EF-hands and a helix-loop-helix structure that binds calcium (or a pseudo EF-hand), connected by an anti-parallel beta-sheet [15]–[17]. Thirty-five EH-containing proteins were identified so far in different species, with 11 proteins identified in human, among them EHD1-4 (EH domain containing proteins), Eps15 and Intersectin 1–2 [18]. ...
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Endocytosis has been suggested to be crucial for the induction of plant immunity in several cases. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and LeEix2. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD2 that are required for its inhibitory activity on endocytosis remained unknown. In this work we demonstrate that the coiled-coil domain of EHD2 is crucial for the ability of EHD2 to inhibit endocytosis in plants, as mutant EHD2 forms lacking the coiled-coil lost the ability to inhibit endocytosis and signaling of LeEix2. The coiled-coil was also required for binding of EHD2 to the LeEix2 receptor. It is therefore probable that binding of EHD2 to the LeEix2 receptor is required for inhibition of LeEix2 internalization. We also show herein that the P-loop of EHD2 is important for EHD2 to function properly. The EH domain of AtEHD2 does not appear to be involved in inhibition of endocytosis. Moreover, AtEHD2 influences actin organization and may exert its inhibitory effect on endocytosis through actin re-distribution. The coiled-coil domain of EHD2 functions in inhibition of endocytosis, while the EH domain does not appear to be involved in inhibition of endocytosis.
... Analysis of the CpMuc4 sequence and the C. hominis Muc4 polymorphic variants (accession #s FJ184998 to FJ185010) with the Eukaryotic Linear Motif (ELM) program [39] identified the presence of NPF and/or GPF motifs in all the sequences. These motifs interact with EH-domain containing proteins that are involved in endocytic processes [40][41][42][43]. This observation suggests that nCpMuc4 may play a role in the initiation of host cell responses that result in parasite engulfment, and encourages further investigation into the identity of the host cell receptor. ...
Article
Cryptosporidium spp. are intracellular apicomplexan parasites that cause outbreaks of waterborne diarrheal disease worldwide. Previous studies had identified a C. parvum sporozoite antigen, CpMuc4, that appeared to be involved in attachment and invasion of the parasite into intestinal epithelial cells. CpMuc4 is predicted to be O- and N-glycosylated and the antigen exhibits an apparent molecular weight 10kDa larger than the antigen expressed in E. coli, indicative of post-translational modifications. However, lectin blotting and enzymatic and chemical deglycosylation did not identify any glycans on the native antigen. Expression of CpMuc4 in T. gondii produced a recombinant protein of a similar molecular weight to the native antigen. Both purified native CpMuc4 and T. gondii recombinant CpMuc4, but not CpMuc4 expressed in E. coli, bind to fixed Caco-2A cells in a dose dependent and saturable manner, suggesting that this antigen bears epitopes that bind to a host cell receptor, and that the T. gondii recombinant CpMuc4 functionally mimics the native antigen. Binding of native CpMuc4 to Caco2A cells could not be inhibited with excess CpMuc4 peptide, or an excess of E. coli recombinant CpMuc4. These data suggest that CpMuc4 interacts directly with a host cell receptor and that post-translational modifications are necessary for the antigen to bind to the host cell receptor. T. gondii recombinant CpMuc4 may mimic the native antigen well enough to serve as a useful tool for identifying the host cell receptor and determining the role of native CpMuc4 in host cell invasion.
... RalBP1, through a conserved Ral-binding domain in its C-terminal, interacts with the activated form of RalA and RalB [38,41] and may also form a complex with the tyrosine-phosphorylated proteins, Reps1 (RalBP1-associated Eps homology (EH) domain protein 1) and Reps2/POB1 (partner of RalBP1) [42,43]. Both of these proteins contain EH domains which exist in proteins involved in endocytosis [44]. Reps1 binds to Rab11-FIP2 via its EH domains [45], while POB1 binds to either Epsin or Eps15 to make a complex with AP2clathrin [46]. ...
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The Ral (Ras-Like) signaling pathway plays an important role in the biology of cells. A plethora of effects is regulated by this signaling pathway and its prooncogenic effectors. Our team has demonstrated the overactivation of the RalA signaling pathway in a number of human malignancies including cancers of the liver, ovary, lung, brain, and malignant peripheral nerve sheath tumors. Additionally, we have shown that the activation of RalA in cancer stem cells is higher in comparison with differentiated cancer cells. In this article, we review the role of Ral signaling in health and disease with a focus on the role of this multifunctional protein in the generation of therapies for cancer. An improved understanding of this pathway can lead to development of a novel class of anticancer therapies that functions on the basis of intervention with RalA or its downstream effectors.
... An Asn-Pro-Phe (NPF) motif acts as the ligand for the EH domain (Salcini et al. 1997). EH-containing and EH-binding proteins establish a complex network of interactions within the cell that regulates internalization and/or trafficking processes (Santolini et al. 1999). Numb is an EH-interacting protein that binds to Eps15, and to the related protein Eps15R, through a NPF motif contained in its COOH-terminal region. ...
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Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.
... The Eps15 homology domain (EH domain), a highly conserved region comprising about 100 residues, exists in various organisms ranging from yeast to mammals (Santolini et al., 1999;Miliaras and Wendland, 2004). Most of the EH domains are generally present in the N-terminus of Eps15 and Eps15related proteins that are involved in internalization events (Fazioli et al., 1993;Benmerah et al., 1995;Wong et al., 1995;Carbone et al., 1997;Delft et al., 1997;Polo et al., 2003). ...
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Objective To provide essential information for peptide inhibitor design, the interactions of Eps15 homology domain of Eps15 homology domain-containing protein 1 (EHD1 EH domain) with three peptides containing NPF (asparagine-proline-phenylalanine), DPF (aspartic acid-proline-phenylalanine), and GPF (glycine-proline-phenylalanine) motifs were deciphered at the atomic level. The binding affinities and the underlying structure basis were investigated. Methods Molecular dynamics (MD) simulations were performed on EHD1 EH domain/peptide complexes for 60 ns using the GROMACS package. The binding free energies were calculated and decomposed by molecular mechanics/ generalized Born surface area (MM/GBSA) method using the AMBER package. The alanine scanning was performed to evaluate the binding hot spot residues using FoldX software. Results The different binding affinities for the three peptides were affected dominantly by van der Waals interactions. Intermolecular hydrogen bonds provide the structural basis of contributions of van der Waals interactions of the flanking residues to the binding. Conclusions van der Waals interactions should be the main consideration when we design peptide inhibitors of EHD1 EH domain with high affinities. The ability to form intermolecular hydrogen bonds with protein residues can be used as the factor for choosing the flanking residues.
... Among the latter, a prominent function is exerted by NUMB that, through its PTB domain, can dock directly to the NVYY motif of E-cadherin, and also bind p120CTN through its proline-rich region (107,108) (Figures 2B,C). Through these interactions, NUMB may serve as a docking site for the recruitment of endocytic adaptors, including AP-2 and EPS15 that ultimately fine tune the rate of E-cadherin internalization (22,(114)(115)(116)(117)(118)(119). Within this framework, NUMB may act on E-cadherin endocytosis in two ways: (1) bridging together p120CTN and endocytic adaptors, thereby promoting the internalization of the entire E-cadherin/p120CTN complex; (2) binding directly to the NVYY motif in E-cadherin, which is usually masked by p120CTN, and facilitating the internalization of p120CTN-unbound E-cadherin, ultimately opposing p120CTN-mediated suppression of endocytosis (107,108). ...
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EH domains are protein-protein interaction domains that function in vesicular trafficking and endocytosis. Here, we report the NMR spectral assignments of the high-affinity complex between the second EH domain of Eps15 and a stonin 2 peptide--providing the basis for the characterization of a two-site binding mode.
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Eps15 homology (EH) domains are protein interaction modules that recognize Asn-Pro-Phe (NPF) motifs in their biological ligands to mediate critical events during endocytosis and signal transduction. To elucidate the structural basis of the EH-NPF interaction, the solution structures of two EH-NPF complexes were solved using NMR spectroscopy. The first complex contains a peptide representing the Hrb C-terminal NPFL motif; the second contains a peptide in which an Arg residue substitutes the C-terminal Leu. The NPF residues are almost completely embedded in a hydrophobic pocket on the EH domain surface and the backbone of NPFX adopts a conformation reminiscent of the Asx-Pro type I beta-turn motif. The residue directly following NPF is crucial for recognition and is required to complete the beta-turn. Five amino acids on the EH surface mediate specific recognition of this residue through hydrophobic and electrostatic contacts. The complexes explain the selectivity of the second EH domain of Eps15 for NPF over DPF motifs and reveal a critical aromatic interaction that provides a conserved anchor for the recognition of FW, WW, SWG and HTF ligands by other EH domains.
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Keywords:proteome;phage-display;combinatorial peptides;protein-protein interactions
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Eps15 homology (EH)-domain containing proteins are regulators of endocytic membrane trafficking. EH-domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH-domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH-domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues.
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Plasma membrane translocation of specific cytosolic proteins plays an important role in cell signaling pathways. We have recently shown that prohibitin (PHB) , a protein present in the plasma membranes of various cell types, interacts with Eps 15 homology domain protein 2 (EHD2), a lipid raft protein. However, the mechanism involved in membrane translocation of PHB is not known.We report that PHB undergoes palmitoylation at cysteine 69 (Cys69), and that this palmitoylation is required for PHB's membrane translocation. Furthermore, we demonstrate that membrane translocation of PHB facilitates tyrosine phosphorylation and its interaction with EHD2. Thus, the palmitoylation and membrane translocation of PHB and its interaction with EHD2 may play a role in cell signaling.
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The protein Numb does not live up to its name. This passive-sounding protein is anything but spent. Originally identified as a cell-fate determinant in Drosophila development, Numb received a good deal of attention as an inhibitor of the Notch receptor signaling pathway. It turns out, however, that Numb does a lot more than simply regulate Notch. It has been implicated in a variety of biochemical pathways connected with signaling (it regulates Notch-, Hedgehog- and TP53-activated pathways), endocytosis (it is involved in cargo internalization and recycling), determination of polarity (it interacts with the PAR complex, and regulates adherens and tight junctions), and ubiquitination (it exploits this mechanism to regulate protein function and stability). This complex biochemical network lies at the heart of Numb's involvement in diverse cellular phenotypes, including cell fate developmental decisions, maintenance of stem cell compartments, regulation of cell polarity and adhesion, and migration. Considering its multifaceted role in cellular homeostasis, it is not surprising that Numb has been implicated in cancer as a tumor suppressor. Our major goal here is to explain the cancer-related role of Numb based on our understanding of its role in cell physiology. We will attempt to do this by reviewing the present knowledge of Numb at the biochemical and functional level, and by integrating its apparently heterogeneous functions into a unifying scenario, based on our recently proposed concept of the "endocytic matrix". Finally, we will discuss the role of Numb in the maintenance of the normal stem cell compartment, as a starting point to interpret the tumor suppressor function of Numb in the context of the cancer stem cell hypothesis.
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The endocytic pathway is involved in activation and inhibition of cellular signaling. Thus, defining the regulatory mechanisms that link endocytosis and cellular signaling is of interest. An emerging link between these processes is a family of proteins called intersectins (ITSNs). These multidomain proteins serve as scaffolds in the assembly of endocytic vesicles and also regulate components of various signaling pathways, including kinases, guanosine triphosphatases, and ubiquitin ligases. This review summarizes research on the role of ITSNs in regulating both endocytic and signal transduction pathways, discusses the link between ITSNs and human disease, and highlights future directions in the study of ITSNs.
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Traumatic spinal cord injury is a common and severe complication after an accident. As we all know that neurite outgrowth of neurons is difficult after a spinal cord injury. Endosome system is associated with cargoes transportation and contributes in promoting the neuronal capability for neurite outgrowth. EH domain‐containing protein 1 (EHD1) transports proteins through the endosome system, especially in the recycling endosomes and regulating the neurite outgrowth. In mammalian cells, the involvement of the ubiquitin‐proteasome system in endosomal sorting has been well established. Two RING fingers and a DRIL (double RING finger‐linked) 1 (Triad1) plays an important role in membrane trafficking and its mutant results in the wrong accumulation of receptors in endosomes and plasma membrane. In this current study, we reasonably integrated the results of the above research and investigated the regulating function of Triad1 to EHD1 following the spinal cord injury. We characterized the upregulated expression and distribution of Triad1 and EHD1 in the neurons after SCI and declared the interaction between Triad1 with EHD1 both in vitro and in vivo. Triad1 regulated the interaction between itself and the full‐length or EH domain of EHD1, which influenced the neurite outgrowth of PC12 cells. Our data delineate a novel interaction between Triad1 and EHD1 that may contribute to the regulation of neurite outgrowth for neurons after the spinal cord injury.
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Clathrin-mediated endocytosis involves a coordinated series of molecular events regulated by interactions among a variety of proteins and lipids through specific domains. One such domain is the Eps15 homology (EH) domain, a highly conserved protein-protein interaction domain present in a number of proteins distributed from yeast to mammals. Several lines of evidence suggest that the yeast EH domain-containing proteins Pan1p, End3p, and Ede1p play important roles during endocytosis. Although genetic and cell-biological studies of these proteins suggested a role for the EH domains in clathrin-mediated endocytosis, it was unclear how they regulate clathrin coat assembly. To explore the role of the EH domain in yeast endocytosis, we mutated those of Pan1p, End3p, or Ede1p, respectively, and examined the effects of single, double, or triple mutation on clathrin coat assembly. We found that mutations of the EH domain caused a defect of cargo internalization and a delay of clathrin coat assembly but had no effect on assembly of the actin patch. We also demonstrated functional redundancy among the EH domains of Pan1p, End3p, and Ede1p for endocytosis. Of interest, the dynamics of several endocytic proteins were differentially affected by various EH domain mutations, suggesting functional diversity of each EH domain.
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Changes in synaptic efficacy underlying learning and memory processes are assumed to be associated with alterations of the protein composition of synapses. Here, we performed a quantitative proteomic screen to monitor changes in the synaptic proteome of four brain areas (auditory cortex, frontal cortex, hippocampus striatum) during auditory learning. Mice were trained in a shuttle box GO/NO-GO paradigm to discriminate between rising and falling frequency modulated tones to avoid mild electric foot shock. Control-treated mice received corresponding numbers of either the tones or the foot shocks. Six hours and 24 h later, the composition of a fraction enriched in synaptic cytomatrix-associated proteins was compared to that obtained from naïve mice by quantitative mass spectrometry. In the synaptic protein fraction obtained from trained mice, the average percentage (±SEM) of downregulated proteins (59.9 ± 0.5%) exceeded that of upregulated proteins (23.5 ± 0.8%) in the brain regions studied. This effect was significantly smaller in foot shock (42.7 ± 0.6% down, 40.7 ± 1.0% up) and tone controls (43.9 ± 1.0% down, 39.7 ± 0.9% up). These data suggest that learning processes initially induce removal and/or degradation of proteins from presynaptic and postsynaptic cytoskeletal matrices before these structures can acquire a new, postlearning organisation. In silico analysis points to a general role of insulin-like signalling in this process.
Chapter
Synapses locally recycle vesicles to ensure the maintenance of neuronal communication critical for normal brain function. While several modes of vesicle regeneration are thought to exist, clathrin-mediated endocytosis (CME) is the best studied mechanism of synaptic vesicle recycling. During CME, the membrane bends, invaginates, and forms a new vesicle that is pinched from the synaptic membrane. As this is not easily achieved, numerous proteins and lipids have been identified that orchestrate vesicle formation from the presynaptic membrane and by integrating structural, biochemical, and in vivo loss of function studies in various organisms a molecular description of the process is starting to emerge. KeywordsClathrin-mediated endocytosis-synaptic vesicle recycling-adaptors-AP2-AP180-actin-amphiphysin-auxilin-calcineurin-cdk-5-clathrin heavy chain-clathrin light chain-dap160-intersectin-dynamin-endophilin-eps15-epsin-hsc70-phosphatidylinositol kinase-talin-stonin-syndapin-synaptojanin and synaptotagmin
Chapter
The Eps15 homology (EH) domain is a 100-residue protein-interaction module that consists of a pair of helix-loop-helix EF hand motifs. The second EF hand typically binds calcium tightly, providing structural stability. The EH domain is either found singly or in a series of two or three tandem repeats in a variety of eukaryotic proteins that are involved in endocytosis, vesicle transport, and signal transduction. Its function is to mediate interactions with Asn-Pro-Phe (NPF) sequences or related motifs that are also often repeated in ligand proteins. The structures, calcium coordination, and peptide binding modes of five monomeric EH domains have recently been elucidated, providing insights into their mechanisms of action.
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Protein interaction modules coordinate the connections within and the activity of intracellular signaling networks. The Eps15 Homology (EH) module, a protein-protein interaction domain that is a key feature of the EH-network, was originally identified in a few proteins involved in endocytosis and vesicle trafficking, and has subsequently also been implicated in actin reorganization, nuclear shuttling, and DNA repair. Here we report an extensive characterization of the physical connections and of the functional wirings of the EH-network in the nematode. Our data show that one of the major physiological roles of the EH-network is in neurotransmission. In addition, we found that the proteins of the network intersect, and possibly coordinate, a number of "territories" of cellular activity including endocytosis/recycling/vesicle transport, actin dynamics, general metabolism and signal transduction, ubiquitination/degradation of proteins, DNA replication/repair, and miRNA biogenesis and processing.
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The AP1 complex is one of a family of heterotetrameric clathrin-adaptor complexes involved in vesicular trafficking between the Golgi and endosomes. The complex has two large subunits, γ and β1, which can be divided into trunk, hinge, and appendage domains. The 1.8 Å resolution structure of the γ appendage is presented. The binding site for the known γ appendage ligand γ-synergin is mapped through creation of point mutations designed on the basis of the structure. We also show that Eps15, a protein believed to be involved in vesicle formation at the plasma membrane, is also a ligand of γ appendage and binds to the same site as γ-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex.
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Ligand binding to the EGF receptor initiates both the activation of mitogenic signal transduction pathways plus trafficking events that relocalize the receptor on the cell surface and within intracellular compartments. The trafficking compartments include caveolae, clathrin-coated pits, and various endosome populations prior to receptor degradation in lysosomes. Evidence is presented that distinct signaling pathways are initiated from these different compartments. These include the Ras/MAP kinase cascade and the PLC-dependent hydrolysis of PI-4,5 P2. Multiple tyrosine kinase substrates that facilitate EGF receptor trafficking between these various compartments, as well as the participation of phosphoinositides and Ras-like G proteins in the trafficking pathway are also described. BioEssays 22:697–707, 2000.
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The major process that regulates the amplitude and kinetics of signal transduction by tyrosine kinase receptors is endocytic removal of active ligand-receptor complexes from the cell surface, and their subsequent sorting to degradation or to recycling. Using the ErbB family of receptor tyrosine kinases we exemplify the diversity of the down regulation process, and concentrate on two sorting steps whose molecular details are emerging. These are the Eps15-mediated sorting to clathrin-coated regions of the plasma membrane and the c-Cbl-mediated targeting of receptors to lysosomal degradation. Like in yeast cells, sorting involves not only protein phosphorylation but also conjugation of ubiquitin molecules. The involvement of other molecules is reviewed and recent observations that challenge the negative regulatory role of endocytosis are described. Finally, we discuss the relevance of receptor down regulation to cancer therapy.
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Neuronal cell fate decisions are directed in Drosophila by NUMB, a signaling adapter protein with two protein–protein interaction domains: a phosphotyrosine-binding domain and a proline-rich region (PRR) that functions as an SH3-binding domain. Here we show that there are at least four human NUMB isoforms and that these serve two distinct developmental functions in the neuronal lineage: differentiation (but not proliferation) is promoted by human NUMB protein isoforms with a type I (short) PRR. In contrast, proliferation (but not differentiation) is directed by isoforms that have a type II (long) PRR. The two types of PRR may promote distinct intracellular signaling pathways downstream of the NOTCH receptor during mammalian neurogenesis.
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In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for \underline{E}ps15 \underline{h}omology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows ≈60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for \underline{E}ps15-\underline{r}elated). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.
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Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.
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We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX 1–2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.
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We previously reported a new type of signal-transducing adaptor molecule, STAM, which was shown to be involved in cytokine-mediated intracellular signal transduction. In this study, we molecularly cloned a 110-kDa phosphotyrosine protein inducible by stimulation with interleukin 2 (IL-2). 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 suppressed DNA synthesis upon stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor, while the Hrs mutant deleted of the STAM-binding site lost such suppressive ability. These results suggest that Hrs counteracts the STAM function which is critical for cell growth signaling mediated by the cytokines.
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The role of Eps15 in clathrin-mediated endocytosis is supported by two observations. First, it interacts specifically and constitutively with the plasma membrane adaptor AP-2. Second, its NH terminus shows significant homology to the NH terminus of yeast End3p, necessary for endocytosis of α-factor. To gain further insight into the role of Eps15-AP-2 association, we have now delineated their sites of interactions. AP-2 binds to a domain of 72 amino acids (767-739) present in the COOH terminus of Eps15. This domain contains 4 of the 15 DPF repeats characteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Eps15r protein. Precipitation of proteolytic fragments of AP-2 with Eps15-derived fusion proteins containing the binding site for AP-2 showed that Eps15 binds specifically to a 40-kDa fragment corresponding to the ear of α-adaptin, a result confirmed by precipitation of Eps15 by α-adaptin-derived fusion proteins. Our data indicate that this specific part of AP-2 binds to a cellular component and provide the tools for investigating the function(s) of the association between AP-2 and Eps15.
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We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.
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A genetic screen for factors required for endocytosis in the budding yeast Saccharomyces cerevisiae previously identified PAN1. Pan1p is a homologue of the mammalian protein eps15, which has been implicated in endocytosis by virtue of its association with the plasma membrane clathrin adaptor complex AP-2. Pan1p contains two eps15 homology (EH) domains, a protein-protein interaction motif also present in other proteins that function in membrane trafficking. To address the role of Pan1p and EH domains in endocytosis, a yeast two-hybrid screen was performed using the EH domain-containing region of Pan1p. This screen identified yAP180A, one of two yeast homologues of a class of clathrin assembly proteins (AP180) that exhibit in vitro clathrin cage assembly activity. In vitro binding studies using GST fusion proteins and yeast extracts defined distinct binding sites on yAP180A for Pan1p and clathrin. yAP180 proteins and Pan1p, like actin, localize to peripheral patches along the plasma membrane. Mammalian synaptojanin, a phosphatidylinositol polyphosphate-5-phosphatase, also has been implicated in endocytosis recently, and three synaptojanin-like genes have been identified in yeast. We observed genetic interactions between the yeast SJL1 gene and PAN1, which suggest a role for phosphoinositide metabolites in Pan1p function. Together with other studies, these findings suggest that Pan1p coordinates regulatory interactions between proteins required for both endocytosis and actin-cytoskeleton organization; these proteins include the yAP180 proteins, clathrin, the ubiquitin-protein ligase Rsp5p, End3p, and synaptojanin. We suggest that Pan1p (and by extension eps15) serves as a multivalent adaptor around which dynamic interactions between structural and regulatory components of the endocytic pathway converge.
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The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein–protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal.
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The F1-20 protein is a novel neuronal-specific, synapse-associated protein that is expressed nonuniformly in mouse brain. Expression of the F1-20 protein is developmentally regulated in a pattern coincident with active synaptogenesis and synaptic maturation. Here we report the cloning of the cDNA sequence for the F1-20 protein. We found two distinct isoforms of F1-20 cDNA that differed by the presence of 15 additional nucleotides, which does not interrupt the open reading frame. RNase protection analysis and PCR amplification of mouse brain RNA revealed that both isoforms are present in cellular RNA. It is likely that the two F1-20 mRNA isoforms are derived from RNA splicing events utilizing alternative 3' acceptor sites. Analysis of the deduced amino acid sequence for the complete open reading frame revealed that the predominant F1-20 mRNA encodes an 896 amino acid polypeptide with a molecular weight of 91,319 Da. The deduced amino acid sequence does not contain a signal sequence, or any extensive hydrophobic regions. The deduced amino acid sequence does contain a number of consensus sequences for protein kinases. Searches of the protein and nucleic acid sequence data bases revealed that the F1-20 protein has not been previously characterized at the primary structure level, although a weak similarity was found between rabbit calpastatin and the C-terminal portion of the F1-20 protein. We then determined biochemically that the F1-20 protein is a substrate for Ca(2+)-dependent proteolysis, which is specifically inhibited by calpain inhibitors in vitro. This indicates that the F1-20 protein is a substrate for neuronal calpain. We observed that treatment of a synaptosomal lysate with alkaline phosphatase led to an increase in the electrophoretic mobility of the F1-20 protein, as well as to an increase in the sharpness of the electrophoretic band. This indicates that the F1-20 protein is phosphorylated in vivo.
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A molecular description of the nerve terminal will be required to understand synaptic function fully. The goals of this study were to contribute toward such a description by characterizing a novel synapse-specific protein. A monoclonal antibody library was screened for antibodies to synaptic proteins. The antibodies were then used to isolate cDNA clones by expression screening. Here we report a detailed characterization of the protein reactive with monoclonal antibody F1-20. Immunohistochemical and biochemical analyses revealed that the F1-20 protein is synapse associated. Western blot analyses revealed that the F1-20 protein is a brain-specific polypeptide with an apparent molecular weight on SDS-PAGE of 190,000 Da. Northern blot analyses indicated that probes generated from an F1-20 cDNA clone hybridize to a single brain-specific mRNA of approximately 4.8 kilobases. In situ hybridization experiments demonstrated that F1-20 mRNA expression is neuronal specific. Northern and Western blot analyses indicated that F1-20 mRNA levels increase abruptly at postnatal day 4 and protein levels increase abruptly at postnatal day 7. This corresponds to a period of active synaptogenesis and synaptic maturation in the mouse CNS. We characterized the neuroanatomical distribution of the F1-20 protein by immunohistochemistry, and of the F1-20 mRNA by in situ hybridization. We found that the F1-20 mRNA and protein are expressed nonuniformly in brain. Variation in the expression of the F1-20 protein is complex and reveals patterns also exhibited by probes directed against other synapse-associated molecules. The highest levels of F1-20 protein are found in the cortically organized regions of the brain. The highest levels of F1-20 mRNA are found in long-distance projection neurons. There is also variation in the expression of F1-20 mRNA between different classes of large output neuron, as well as extensive variation in the expression of F1-20 mRNA between different nuclear groups.
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Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.
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We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.
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Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.
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Selective enrichment of clathrin-coated membranes by anticlathrin immunoadsorption was used to examine the internalization of receptor-ligand complexes through coated pits. Using Staphylococcus aureus-anticlathrin antibody and [35S]methionine-labeled KB cells, the kinetics of association of the epidermal growth factor (EGF-R) and transferrin receptors (TF-R) with coated membranes were directly examined. The accumulation of EGF-R in coated pits at the cell surface was dependent upon EGF binding. EGF-R then passed sequentially through a compartment which did not react with anticlathrin antibody and a second clathrin-coated compartment. The EGF-R was degraded in lysosomes with a half-life of approximately 41-55 min. The tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, appears to mimic the action of EGF in inducing EGF-R accumulation in coated pits at the cell surface and receptor internalization. In contrast to the results with EGF-R, the TF-R was found in clathrin-coated membranes in the presence or absence of TF, and the concentration of TF-R in clathrin-coated membranes did not significantly change with time. The method presented should be of great utility for examining the biochemical changes that occur during the receptor-mediated endocytosis and sorting of ligands and receptors.
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This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.
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A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.
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Ligand-activated epidermal growth factor receptors (EGFRs) associate with coated pit adaptor proteins (AP2) in vivo, implying a mechanism for receptor retention in coated pits during internalization. Using an in vitro binding assay, we localized the adaptor binding determinant to residues 970-991 of EGFRs and confirmed specificity by competition with a synthetic peptide corresponding to this sequence. A mutant EGFR lacking this AP2 binding determinant did not associate with AP2 in vivo but demonstrated internalization and down-regulation kinetics indistinguishable from its wild-type counterpart. Immunocytochemistry confirmed ligand-induced internalization of the mutant EGFR. These data suggest that endocytic determinants are distinct from AP2 binding determinants and that processes other than association with AP2 regulate endocytosis of EGFRs.
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Tyrosine-based signals within the cytoplasmic domain of integral membrane proteins mediate clathrin-dependent protein sorting in the endocytic and secretory pathways. A yeast two-hybrid system was used to identify proteins that bind to tyrosine-based signals. The medium chains (mu 1 and mu 2) of two clathrin-associated protein complexes (AP-1 and AP-2, respectively) specifically interacted with tyrosine-based signals of several integral membrane proteins. The interaction was confirmed by in vitro binding assays. Thus, it is likely that the medium chains serve as signal-binding components of the clathrin-dependent sorting machinery.
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Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.
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HIV-1 Rev is the prototype of a class of retroviral regulatory proteins that induce the sequence-specific nuclear export of target RNAs. This function requires the Rev activation domain, which is believed to bind an essential cellular cofactor. We report the identification of a novel human gene product that binds to not only the HIV-1 Rev activation domain in vitro and in vivo but also to functionally equivalent domains in other Rev and Rex proteins. The Rev/Rex activation domain-binding (Rab) protein occupies a binding site on HIV-1 Rev that precisely matches that predicted by genetic analysis. Rab binds the Rev activation domain when Rev is assembled onto its RNA target and can significantly enhance Rev activity when overexpressed. We conclude that Rab is the predicted activation domain-specific cofactor for the Rev/Rex class of RNA export factors.
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Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.
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We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.
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Newly synthesized vacuolar hydrolases such as carboxypeptidase Y (CPY) are sorted from the secretory pathway in the late-Golgi compartment and reach the vacuole after a distinct set of membrane-trafficking steps. Endocytosed proteins are also delivered to the vacuole. It has been proposed that these pathways converge at a "prevacuolar" step before delivery to the vacuole. One group of genes has been described that appears to control both of these pathways. Cells carrying mutations in any one of the class E VPS (vacuolar protein sorting) genes accumulate vacuolar, Golgi, and endocytosed proteins in a novel compartment adjacent to the vacuole termed the "class E" compartment, which may represent an exaggerated version of the physiological prevacuolar compartment. We have characterized one of the class E VPS genes, VPS27, in detail to address this question. Using a temperature-sensitive allele of VPS27, we find that upon rapid inactivation of Vps27p function, the Golgi protein Vps10p (the CPY-sorting receptor) and endocytosed Ste3p rapidly accumulate in a class E compartment. Upon restoration of Vps27p function, the Vps10p that had accumulated in the class E compartment could return to the Golgi apparatus and restore correct sorting of CPY. Likewise, Ste3p that had accumulated in the class E compartment en route to the vacuole could progress to the vacuole upon restoration of Vps27p function indicating that the class E compartment can act as a functional intermediate. Because both recycling Golgi proteins and endocytosed proteins rapidly accumulate in a class E compartment upon inactivation of Vps27p, we propose that Vps27p controls membrane traffic through the prevacuolar/endosomal compartment in wild-type cells.
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The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a-factor receptor, Ste3p. Mutation of this sequence prevented pheromone-stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.
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A complete understanding of the molecular mechanisms of endocytosis requires the discovery and characterization of the protein machinery that mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bulk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in conjunction with FACS to enrich for yeast mutants that exhibit internalization defects. Detailed characterization of two of these mutants, dim1-1 and dim2-1, revealed defects in the endocytic pathway. Like other yeast endocytosis mutants, the temperature-sensitive dim mutant were unable to endocytose FM4-64 or radiolabeled alpha-factor as efficiently as wild-type cells. In addition, double mutants with either dim1-delta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products function in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. Pan1p shares homology with the mammalian clathrin adaptor-associated protein, eps15. Both proteins contain multiple EH (eps15 homology) domains, a motif proposed to mediate protein-protein interactions. Phalloidin labeling of filamentous actin revealed profound defects in the actin cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscent of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur were visualized in the mutants by electron microscopy using cationized ferritin as a marker for the endocytic pathway. This new screening strategy demonstrates a role for She4p and Pan1p in endocytosis, and provides a new general method for the identification of additional endocytosis mutants.
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The acid unfolding of deoxymyoglobin (deoxyMb) from the native (N) form to the unfolded (U) form proceeds through at least two spectroscopically distinct heme intermediates. The 426-nm absorbing heme intermediate (I′-form) occurs in the pH ∼ 3.5–4.5 range. In the I′-form, the iron-proximal histidine bond is broken; however, the heme is five-coordinate due to binding of a water molecule. The I′-form was first observed in pH-jump (neutral to acid conditions) experiments, where it was characterized as a transient species which rapidly forms (10 ms) and dissipates. Recently, however, it was shown that the I′-intermediate also forms under equilibrium conditions. To elucidate the factors which control the formation of the I′-intermediate, a detailed series of equilibrium and slow kinetic (>2-s) experiments were performed. Equilibrium pH titrations reveal that the I′-intermediate forms at successively higher pH as the ionic strength increases. pH-jump experiments (pH 6.9 to 3.2 and pH 4.4 to 3.2) indicate that the rate of formation of the intermediate is dramatically affected by the ionic strength conditions. If the ionic strength is held constant during the pH-jump, the I′-intermediate forms slowly (∼ 35 s) and the formation rate is independent of ionic strength. If the ionic strength is jumped from low to high values during the pH-jump, the formation rate of the I′-intermediate monotonically increases. Conversely, if the ionic strength is jumped from high to low values during the pH-jump, the rate monotonically decreases. The former result explains the finding of early pH-jump experiments wherein the I′-intermediate was found to form very rapidly. In these experiments, the ionic strength was also jumped from low to very high values during the pH-jump. In both types experiments where the pH and ionic strength are simultaneously jumped, the rate of formation of the I′-intermediate is independent of the initial and final ionic strength and depends only on the difference. The kinetic and equilibrium data are well accounted for with a simple three-state model in which the N-form is transformed into the I′-form via a single transition (T) state, and the free energy of the various forms depends linearly on the ionic strength. The model predicts that both the N-form and the T-state are stabilized with increasing ionic strength and that the extent of stabilization is approximately the same for both (−4.84 cal/mol per mM). The I′-form is also stabilized with increasing ionic strength; however, the extent of stabilization is greater than for the N-form. This picture is qualitatively consistent with a simple Born model which predicts that a medium with higher dielectric constant should impart greater stabilization to a species with higher overall charge. The I′-form is stabilized relative to the N-form at higher ionic strength (higher dielectric constant) because it is formed in a pH region where several of the histidine residues in the protein titrate, thus increasing the net positive charge on the protein relative to the N-form at neutral pH. Collectively, the studies provide a self-consistent picture of the factors which control the acid-induced transformation of deoxyMb from the N- to I′-forms. © 1997 John Wiley & Sons, Inc. Biospect 3: 17–29, 1997
Article
The importance of lateral inhibition mediated by NOTCH signaling is well demonstrated to control neurogenesis both in invertebrates and vertebrates. We have identified the chicken homolog of Drosophila numb, which suppresses NOTCH signaling. We show that chicken NUMB (c-NUMB) protein is localized to the basal cortex of mitotic neuroepithelial cells, suggesting that c-NUMB regulates neurogenesis by the modification of NOTCH signaling through asymmetrical cell division. Consistent with this suggestion, we show (1) that c-NUMB interferes with the nuclear translocation of activated c-NOTCH-1 through direct binding to the PEST sequence in the cytoplasmic domain of c-NOTCH-1 and (2) that c-NUMB interferes with c-NOTCH-1-mediated inhibition of neuronal differentiation.
Article
The sorting of specific proteins into clathrin-coated pits and the mechanics of membrane invagination are determined by assembly of the clathrin lattice. Recent structures of a six-fold barrel clathrin coat at 21 Å resolution by electron cryomicroscopy and of the clathrin terminal domain and linker at 2.6 Å by X-ray crystallography together show how domains of clathrin interact and orient within the coat and reveal the strongly puckered shape and conformational variability of individual triskelions. The β propeller of the terminal domain faces the membrane so that recognition segments from adaptor proteins can extend along its lateral grooves. Clathrin legs adapt to different coat environments in the barrel by flexing along a segment at the knee that is free of contacts with other molecules.
Article
We have identified a ∼140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N-terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N-x11–13-V-x2-A-T-x34–36-R-x7–8-W-R-x3-K-x12-G-x-E-x15-L-x11–12-D-x-G-R-x11-D-x7-R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N-terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.
Article
The endocytic protein Eps15 contains three copies of the EH domain, a protein module thought to function in protein-protein interactions. Using overlay assays with an Eps15 EH domain fusion protein, we have now identified a protein of 95 kDa (p95) as a major EH domain-binding partner in a wide variety of tissues. The amino acids asparagine-proline-phenylalanine (NPF) form the core of an EH domain-binding motif and three NPF repeats are found in the endocytic protein synaptojanin-170. We have confirmed previous studies indicating that synaptojanin-170 is an EH domain-binding protein, and have used peptide blocking experiments to demonstrate that the interaction is mediated through the NPF repeats. Interestingly, the same peptide also blocks EH domain-binding to p95. Finally, we have shown that p95 is enriched on clathrin-coated vesicles, suggesting an endocytic role for the protein. These data support an important role for EH domain-NPF motif interactions in endocytosis.
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Some acute lymphocytic leukaemias, particularly those in young children, are associated with a t(4;11)(q21;q23) reciprocal translocation. We have cloned the translocation breakpoint on chromosome 11q23 and isolated corresponding RNA transcripts from this region. The translocation occurs within a cluster of Alu repetitive elements located within an intron of a gene that gives rise to 11.5 (kb) transcript spanning the translocation breakpoint. The 11.5 kb transcript encodes a protein that is highly homologous to the Drosophila trithorax gene, a developmental regulator. An analysis of a series of leukaemic patients carrying t(4;11) and t(9;11) translocations indicate that the majority of breakpoints in infant leukaemias lie within a 5 kb region.
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The neuronal protein NP185 is a neural tissue-specific protein isolated from clathrin-coated vesicles in brain. Using 8G8, a monoclonal antibody (MAb) characterized in our laboratory, we studied the expression and distribution of neuronal protein NP185 in developing avian cerebellum and in mature murine cerebellum. Furthermore, we compared these parameters to that of synapse-specific neuronal protein, synaptophysin, and an axon-specific (i.e., non-synaptic) neuronal protein, neurofilament NF68. We found that NP185 expression temporally and spatially corresponds to avian cerebellar synaptogenesis. In addition, NP185 distribution parallels synaptophysin distribution throughout development, while differing from that of either unassembled or filamentous forms of NF68. The evidence also suggests that embryonic NP185 expression coincides with synaptogenesis, and that NP185 remains concentrated in the terminal boutons of mature neurons. The synapse specificity of NP185 and the recent biochemical properties reported for this protein support the postulate that this molecule may trigger synaptic events and distinguish structurally and functionally active synapses.