Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rat gene encoding the class theta glutathione S-transferase (GST) subunit Yrs has been isolated from a rat lambda EMBL4 DNA library and completely sequenced. The gene spans approximately 4 kb comprising 5 exons separated by 4 introns, the smallest number of the gene components among known rat genes encoding GST subunits. Sequence analysis of the 5' flanking region of the GST Yrs gene indicated the absence of both TATA and CAAT boxes. However, potential binding sites for the transcription factors SP1, PU1, PEA3, and AP-2 were present in this region. Primer extension and 5'-rapid amplification of cDNA ends studies demonstrated the existence of multiple transcription initiation sites distributed over 58 bp in the upstream sequence from the translation initiation codon. The present study provides the first evidence for the primary structure of the gene encoding a class theta GST subunit.
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PMID:Isolation and characterization of the gene encoding rat class theta glutathione S-transferase subunit yrs. 780 57

Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.
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PMID:Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain. 916 77

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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PMID:Pan1p, yeast eps15, functions as a multivalent adaptor that coordinates protein-protein interactions essential for endocytosis. 953 49

The Ets gene family has a complex evolutionary history with many family members known to regulate genetic programs essential for differentiation and development, and some known for their involvement in human tumorigenesis. To understand the biological properties associated with a recently described epithelium-restricted Ets factor ESX, an 11 kb fragment from the 1q32.2 genomically localized human gene was cloned and analysed. Upstream of the ESX promoter region in this genomic fragment lies the terminal exon of a newly identified gene that encodes a ubiquitin-conjugating enzyme variant, UEV-1. Tissues expressing ESX produce a primary 2.2 kb transcript along with a 4.1 kb secondary transcript arising by alternate poly(A) site selection and uniquely recognized by a genomic probe from the 3' terminal region of the 11 kb clone. Endogenous expression of ESX results in a 42 kDa nuclear protein having fivefold greater affinity for the chromatin-nuclear matrix compartment as compared to other endogenous transcription factors like AP-2 and the homologous Ets factor, ELF-1. Exon mapping of the modular structure inferred from ESX cDNA and construction of GAL4(DBD)-ESX expression constructs were used to identify a transactivating domain encoded by exon 4 having comparable potency to the acidic transactivation domain of the viral transcription factor, VP16. This exon 4-encoded 31 amino acid domain in ESX was shown by mutation and deletion analysis to possess a 13 residue acidic transactivation core which, based on modeling and circular dichroism analysis, is predicted to form an amphipathic alpha-helical secondary structure. Using recombinant GST-ESX (exon 4) fusion proteins in an in vitro pull-down assay, this ESX transactivation domain was shown to bind specifically to one component of the general transcription machinery, TATA-binding protein (TBP). Transient transfection experiments confirmed the ability of this TBP-binding transactivation domain in ESX to squelch heterologous promoters independent of any promoter binding as efficiently as the transactivation domain from VP16.
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PMID:Exon 4-encoded acidic domain in the epithelium-restricted Ets factor, ESX, confers potent transactivating capacity and binds to TATA-binding protein (TBP). 1039 76

LTC4S conjugates reduce glutathione to LTA4 and is positioned as the pivotal and only committed enzyme involved in the formation of cysteinyl LTs. Despite its function as an enzyme that conjugates glutathione to LTA4, it is abundantly clear that LTC4S differs from the classic glutathione S-transferase (GST) families. This distinction is based on narrow substrate specificity, inability to conjugate GSH to xenobiotics, differential susceptibility to inhibitors, lack of homology, and failure to be immunorecognized by specific microsomal GST antibodies. The presence of LTC4S protein is restricted to a limited number of hematopoietic cells to include mast cells, eosinophils, basophils, monocytes/macrophages, and platelets, with the platelet being unique in its lack of the complete biosynthetic pathway for cysteinyl LTs. The purification of the protein and the cloning of the cDNA have demonstrated that the kinetic parameters of LTC4S are similar for the isolated natural or recombinant proteins. The protein is an 18-kDa integral perinuclear membrane enzyme, which is functional as a homodimer. The cDNA encodes a 150 amino-acid polypeptide monomer with three hydrophobic domains interspersed by two hydrophilic loops. Homology and secondary structural predictions have revealed that LTC4S is a member of a novel gene family that includes FLAP, mGST II, and mGST III. Each of these molecules is an integral membrane protein with the capacity to participate in LT biosynthesis: LTC4S as the terminal and only committed enzyme in cysteinyl LT formation, FLAP as an arachidonic acid presentation protein, and mGST II and mGST III as unique dual-function enzymes with primary detoxification functions. Site directed mutagenic studies of LTC4S have revealed that two residues, R51 and Y93, are involved in the acid and base catalysis, respectively, of LTA4 and GSH. Alignment of molecules with LTA4 conjugating ability demonstrates conservation of amino acid residues R51 and Y93, which appear necessary for this specific enzymatic function. The 2.5-Kb gene for human LTC4S contains five small exons and four introns, and the 5' UTR contains consensus sequences for AP-1 and AP-2 sites as well as an SP-1 site. The chromosomal localization of this gene is 5q35, distal to that of cytokine, growth factor, and receptor genes that have relevance to the development of allergic inflammation. Furthermore, there is genetic linkage of this region of human chromosome 5 to atopy and asthma, whereas no linkage exists for the chromosomal localization of the other family members, FLAP and mGST II, distinguishing LTC4S as a unique member of the novel gene family. LTC4S is profoundly overexpressed in the aspirin-induced asthmatic phenotype and correlates with overproduction of cysteinyl LTs and bronchial hyperreactivity to lysine aspirin. Ongoing studies are directed to the genomic regulation and additional polymorphisms within the gene of this pivotal enzyme, as well as to further identification of the amino acid residues central to its catalytic function.
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PMID:LTC4 synthase. Enzymology, biochemistry, and molecular characterization. 1043 63

Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.
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PMID:Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites. 1069 52

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, (1424)YDSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes have been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the mu subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of mu 2. Furthermore, isolated mu 2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that mu 2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative mu 2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared with wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the mu 2 subunit of AP-2 and mutations in mu 2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.
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PMID:Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway. 1156 Sep 23

The cystic fibrosis transmembrane conductance regulator (CFTR) functions at the apical membrane of epithelial cells to regulate chloride permeability. Recent studies have shown that CFTR is rapidly and efficiently internalized from the plasma membrane. We have shown that such internalization is mediated solely by clathrin-coated pathways, and that other pathways, such as caveolae, exclude CFTR. Moreover, CFTR co-precipitates with alpha-adaptin, a component of the endocytic adaptor complex (AP-2). The goal of our current studies was to elucidate further the molecular mechanisms that facilitate entry of CFTR into endocytic clathrin-coated vesicles. Protein-protein interactions generated by incubation of full-length in-vitro-translated CFTR with partially purified bovine brain adaptor complexes were evaluated following immunoprecipitation using an antibody against the alpha-adaptin subunit of the AP-2 complex. Such studies revealed co-immunoprecipitation of alpha-adaptin with full-length but not partially translated CFTR, suggesting that the C-terminus of CFTR may be responsible for this interaction. To test this hypothesis a C-terminal GST fusion protein (amino acids 1404-1480; CF-GST) was used in a "pull-down" assay with purified adaptor complexes. CF-GST sepharose was able to pull-down AP-2 endocytic adaptor complexes, as determined by immunoblot analyses of the precipitates using antibodies directed against alpha-adaptin. In contrast, CF-GST sepharose was unable to pull-down gamma-adaptin, a component of the Golgi-derived AP-1 clathrin adaptor complex. Thus, we demonstrate that CFTR is endocytosed via clathrin-coated vesicles, and that targeting of CFTR to these structures is mediated by binding of the AP-2 adaptor complex to the C-terminal domain of CFTR.
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PMID:Endocytic adaptor complexes bind the C-terminal domain of CFTR. 1184 7

The cytoplasmic tail of the invariant chain contains two leucine-based sorting signals, and each of those seems sufficient to route the invariant chain to its intracellular destination in either normal or polarized cells. It is believed that the intracellular routing of the invariant chain is mediated by its interactions with the clathrin-associated adaptor protein complexes AP1 and AP2. We () have previously demonstrated the in vitro interactions between the cytoplasmic tail of the invariant chain and AP1/AP2 complexes. These interactions were specific and depended on the critical leucine residues in the invariant chain's sorting signals. In the present study, we decided to investigate the molecular mechanism of these interactions. To this end, we constructed a set of glutathione S-transferase fusion proteins that contained the intact cytoplasmic tail of the invariant chain and its various mutants to define residues important for its interactions with AP1 and AP-2. Our results demonstrated the importance of several residues other than the critical leucine residues for such interactions. A strong correlation between in vitro binding of AP2 to the invariant chain and in vivo internalization of the invariant chain was observed, confirming the primary role of AP2 in recognition of endocytic signals. In addition, we demonstrated different requirements for AP1 and AP2 binding to cytoplasmic tail of the invariant chain, which may reflect that the different sorting pathways mediated by AP1 and AP2 involve their recognition of the primary structure of the sorting signal.
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PMID:Mechanism of interaction between leucine-based sorting signals from the invariant chain and clathrin-associated adaptor protein complexes AP1 and AP2. 1185 3

The subcellular localization of Ocrl, the inositol polyphosphate 5-phosphatase that is mutated in Lowe syndrome, was investigated by fluorescence microscopy. Ocrl was localized to endosomes and Golgi membranes along with clathrin, giantin, the mannose 6-phosphate receptor, transferrin, and the early endosomal antigen 1 endosomal marker in fixed cells. The endosomal localization of Ocrl was confirmed by live-cell time-lapse microscopy in which we monitored the dynamics of Ocrl on endosomes. GST binding assays show that Ocrl interacts with the clathrin terminal domain and the clathrin adaptor protein AP-2. Our findings suggest a role for Ocrl in endosomal receptor trafficking and sorting.
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PMID:The inositol polyphosphate 5-phosphatase Ocrl associates with endosomes that are partially coated with clathrin. 1535


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