FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1982142716> ?p ?o ?g. }

• W1982142716 endingPage "49944" @default.
• W1982142716 startingPage "49935" @default.
• W1982142716 abstract "The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C δ1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the β1-loop1-β2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 arebona fide PH domain proteins, with broad specificity and moderate affinity for PIPs. The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C δ1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the β1-loop1-β2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 arebona fide PH domain proteins, with broad specificity and moderate affinity for PIPs. The Ipl gene (also known as Tssc3) is highly expressed in the placenta, where its effective dosage is regulated by parental imprinting. Mouse concepti inheriting Ipldeletions from their mothers show placentomegaly, implicatingIpl in restraining placental growth (1Frank D. Fortino W. Clark L. Musalo R. Wang W. Saxena A. Li C.M. Reik W. Ludwig T. Tycko B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7490-7495Crossref PubMed Scopus (186) Google Scholar). However, the biochemical basis for this genetic effect is not known. The Ipl protein contains a single predicted pleckstrin homology (PH) 1The abbreviations used are: PH, pleckstrin homology; PHD, PH domain; BTK, Bruton's tyrosine kinase; CH1, cytohesin1; DAPP1, dual adaptor for phosphoinositides and phosphotyrosine; DYN1, Dynamin; GRP1, general receptor for phosphoinositides-1; IPL, gene imprinted in placenta and liver; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PLS-N, N-terminal PH domain from pleckstrin; SH2, Src homology domain2; Tih1, TDAG51/IPL homologue 1; PIP, phosphatidyl-inositol phosphate lipid, PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P2, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns, phosphatidylinositol; GST, glutathioneS-transferase; DTT, dithiothreitol; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP; EGF, epidermal growth factor; BSA, bovine serum albumin; TRITC, tetramethylrhodamine isothiocyanate; CMV, cytomegalovirus; PMSF, phenylmethylsulfonyl fluoride; ts, temperature-sensitive1The abbreviations used are: PH, pleckstrin homology; PHD, PH domain; BTK, Bruton's tyrosine kinase; CH1, cytohesin1; DAPP1, dual adaptor for phosphoinositides and phosphotyrosine; DYN1, Dynamin; GRP1, general receptor for phosphoinositides-1; IPL, gene imprinted in placenta and liver; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PLS-N, N-terminal PH domain from pleckstrin; SH2, Src homology domain2; Tih1, TDAG51/IPL homologue 1; PIP, phosphatidyl-inositol phosphate lipid, PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P2, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns, phosphatidylinositol; GST, glutathioneS-transferase; DTT, dithiothreitol; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP; EGF, epidermal growth factor; BSA, bovine serum albumin; TRITC, tetramethylrhodamine isothiocyanate; CMV, cytomegalovirus; PMSF, phenylmethylsulfonyl fluoride; ts, temperature-sensitive domain, with short N- and C-terminal extensions. It shares this structure with its closest relative, Tih1, an even smaller protein, which is broadly expressed and not regulated by imprinting (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar).The PH domain is a ∼100-amino acid module defined by sequence homology, which has a conserved tertiary structure of a β-sandwich of 3 + 4 stranded β sheets with variable intervening loops, followed by a single amphipathic α helix (3Lemmon M.A. Ferguson K.M. Curr. Top. Microbiol. Immunol. 1998; 228: 39-74PubMed Google Scholar). Most PH domain-containing proteins can bind to certain phosphatidylinositol phosphate lipids (PIPs), but their binding affinities for the specific products of phosphoinositide metabolism vary greatly (4Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar). For example, the PH domains from phospholipase C δ1 (PLCδ1) bind strongly to PtdIns(4,5)P2, but not to the downstream metabolites of this compound, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which are produced by the action of phosphatidylinositol 3-kinase (PI3K). Other PH domains, typified by that of the general receptor for phosphoinositides (GRP1), show the opposite pattern of binding affinity, with high affinity for PtdIns(3,4,5)P3 and only low affinity for PtdIns(4,5)P2. Based on in vitro and in vivo assays, Kavran et al. (5Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) classified PH domains into two groups: a small class of PH domains with high affinity and specificity for particular PIPs and a much larger class showing low affinity promiscuous binding to various PIPs. More recently, Maffucci and Falasca (6Maffucci T. Falasca M. FEBS Lett. 2001; 506: 173-179Crossref PubMed Scopus (107) Google Scholar) have proposed a further division of PH domains into three groups. In this scheme, Group 1 proteins are those with high affinities for specific PIPs; Group 2 proteins have lower but still measurable affinity for phosphoinositides, and show less ability to discriminate among the various PIPs; and Group 3 proteins have minimal affinity for phosphoinositides, with no detectable specificity.Consistent with their varying interactions with phosphoinositides, and with their occurrence alongside a variety of other modules, PH domains have diverse functions. Among these are roles in signal transduction downstream of membrane receptors, control of membrane vesicle trafficking in the cell, targeting of phospholipid-modifying enzymes to cellular membranes, and control of cytoskeleton-membrane interactions (3Lemmon M.A. Ferguson K.M. Curr. Top. Microbiol. Immunol. 1998; 228: 39-74PubMed Google Scholar, 7Rebecchi M.J. Scarlata S. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 503-528Crossref PubMed Scopus (248) Google Scholar). These functions are unified by the ability of PH domains to associate with the plasma membrane and/or intracellular membranes through their high or low affinity binding to phosphoinositide lipids. Certain PH domains can also mediate protein-protein interactions, for example that which occurs between the beta-adrenergic receptor protein kinase and the βγ subunit of heterotrimeric G-proteins (8Inglese J. Koch W.J. Touhara K. Lefkowitz R.J. Trends Biochem. Sci. 1995; 20: 151-156Abstract Full Text PDF PubMed Scopus (165) Google Scholar). But the protein-binding site is distant from the phosphoinositide binding region, and a PH domain-phosphoinositide interaction may be simultaneously required for physiological function (9Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar).Interactions of PH domains with phosphoinositides have been examinedin vitro and in vivo by several complementary approaches. These include direct binding of GST-PH domain fusion proteins to phosphoinositides in lipid vesicles or immobilized on nitrocellulose membranes (5Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 10Dowler S. Currie R.A. Downes C.P. Alessi D.R. Biochem. J. 1999; 342: 7-12Crossref PubMed Scopus (127) Google Scholar, 11Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Biochem. J. 2000; 351: 19-31Crossref PubMed Scopus (473) Google Scholar, 12Thomas C.C. Dowler S. Deak M. Alessi D.R. van Aalten D.M. Biochem. J. 2001; 358: 287-294Crossref PubMed Scopus (80) Google Scholar, 13Razzini G. Brancaccio A. Lemmon M.A. Guarnieri S. Falasca M. J. Biol. Chem. 2000; 275: 14873-14881Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Sankaran V.G. Klein D.E. Sachdeva M.M. Lemmon M.A. Biochemistry. 2001; 40: 8581-8587Crossref PubMed Scopus (62) Google Scholar, 15Fleming I.N. Gray A. Downes C.P. Biochem. J. 2000; 351: 173-182Crossref PubMed Scopus (107) Google Scholar), and analysis in vivoof the movement of GFP-tagged PH domains from the cytosol to the plasma membrane in response to growth factors that activate PI3K (5Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 13Razzini G. Brancaccio A. Lemmon M.A. Guarnieri S. Falasca M. J. Biol. Chem. 2000; 275: 14873-14881Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,15Fleming I.N. Gray A. Downes C.P. Biochem. J. 2000; 351: 173-182Crossref PubMed Scopus (107) Google Scholar, 16Currie R.A. Walker K.S. Gray A. Deak M. Casamayor A. Downes C.P. Cohen P. Alessi D.R. Lucocq J. Biochem. J. 1999; 337: 575-583Crossref PubMed Scopus (272) Google Scholar, 17Gray A. Van Der Kaay J. Downes C.P. Biochem. J. 1999; 344: 929-936Crossref PubMed Scopus (181) Google Scholar, 18Anderson K.E. Lipp P. Bootman M. Ridley S.H. Coadwell J. Ronnstrand L. Lennartsson J. Holmes A.B. Painter G.F. Thuring J. Lim Z. Erdjument-Bromage H. Grewal A. Tempst P. Stephens L.R. Hawkins P.T. Curr. Biol. 2000; 10: 1403-1412Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 19Bobe R. Wilde J.I. Maschberger P. Venkateswarlu K. Cullen P.J. Siess W. Watson S.P. Blood. 2001; 97: 678-684Crossref PubMed Scopus (40) Google Scholar, 20Chen R.H. Corbalan-Garcia S. Bar-Sagi D. EMBO J. 1997; 16: 1351-1359Crossref PubMed Scopus (115) Google Scholar, 21Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 22Watton S.J. Downward J. Curr. Biol. 1999; 9: 433-436Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 23Varnai P. Rother K.I. Balla T. J. Biol. Chem. 1999; 274: 10983-10989Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 24Venkateswarlu K. Gunn-Moore F. Tavare J.M. Cullen P.J. J. Cell Sci. 1999; 112: 1957-1965PubMed Google Scholar, 25Langille S.E. Patki V. Klarlund J.K. Buxton J.M. Holik J.J. Chawla A. Corvera S. Czech M.P. J. Biol. Chem. 1999; 274: 27099-27104Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). A third assay tests the ability of the PH domain of interest, fused to activated mutant Ras (Ras*), to rescue Saccharomyces cerevisiae that are defective in the Ras pathway due to a temperature-sensitive (ts) CDC25 mutation (26Isakoff S.J. Cardozo T. Andreev J. Li Z. Ferguson K.M. Abagyan R. Lemmon M.A. Aronheim A. Skolnik E.Y. EMBO J. 1998; 17: 5374-5387Crossref PubMed Scopus (281) Google Scholar). Because yeast do not make PtdIns(3,4,5)P3, PI3K-dependent and PI3K-independent rescue can be separately evaluated, by co-transformation with active versus inactive constructs of the catalytic subunit of PI3K.As a first step in assessing the biochemical functions of Ipl and Tih1, we have used each of these approaches to compare the behavior of the PH domains from these proteins with several other PH domains. We have also carried out sequence comparisons with other PH domains. The data indicate that Ipl and Tih1 define an outlying phylogenetic branch of the PH domain superfamily, but that they are bona fide PIP binders, which fit best into the group with moderate affinity and poor selectivity for the known phosphoinositide lipids.DISCUSSIONThe Ipl gene was originally found in a chromosomal walk, which was undertaken to identify and characterize novel imprinted genes in a megabase-scale imprinted domain on human Chr11p15.5 and distal mouse Chr7 (35Qian N. Frank D. O'Keefe D. Dao D. Zhao L. Yuan L. Wang Q. Keating M. Walsh C. Tycko B. Hum. Mol. Genet. 1997; 6: 2021-2029Crossref PubMed Scopus (144) Google Scholar). Since then, substantial information has accrued concerning the tissue-specific expression and functional imprinting of this gene, and the creation of Ipl-knockout mice has established that at least one of its functions is to regulate the growth of the placenta (1Frank D. Fortino W. Clark L. Musalo R. Wang W. Saxena A. Li C.M. Reik W. Ludwig T. Tycko B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7490-7495Crossref PubMed Scopus (186) Google Scholar, 2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). Biochemical information is needed to explain this genetic finding, and a starting point is provided by the domain structure of the Ipl protein. This structure, which is shared by the closest Ipl homologue, Tih1, is remarkably simple, consisting of a single predicted PH domain with short N- and C-terminal extensions. The results shown here verify that this central region acts as a bona fide PH domain, which is capable of interacting with phosphoinositide lipids, both in direct binding assays and in membrane translocation assays, as well as functionally in a yeast growth rescue assay. Overall, the findings suggest that the Ipl and Tih1 PH domains bind to known PIPs with moderate affinity and low specificity. Whether this promiscuous binding reflects the true physiological targets of proteins in this group is not known, and they may have a high affinity for a yet unidentified lipid, or even protein, ligand. For example, recent work has shown that the tagged PH domain of human oxysterol binding protein accumulates at the yeast Golgi apparatus partly in response to the presence of PtdIns(4)P and partly in response to an unidentified factor that is dependent on the Golgi ATPase Arf1p (36Levine T.P. Munro S. Curr. Biol. 2002; 12: 695-704Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar).As summarized in Table I, our data also show clear differences in the behavior of the Ipl versus Tih1 PH domains. Consistent with these differences, Ipl and Tih1 have different patterns of tissue-specific expression (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar), and no detectable growth abnormality was found in Tih1-null concepti (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). In addition, the Ipl protein is highly conserved in vertebrate evolution, presumably reflecting a conserved biological function, but we have not been able to identify Tih1 orthologues in non-mammalian vertebrates.Further progress in understanding the biochemical functions of Ipl and Tih1 will depend on localizing the native proteins within the cell at high resolution and on identifying any proteins or other lipids with which they interact. For most PH domain-containing proteins, such protein-protein interactions do not occur via the PH domain, but instead involve different modules elsewhere in the protein. For example, Lnk associates with the ABP-280 actin binding protein via an interdomain stretch of 56 amino acids between its PH and SH2 domains (37He X. Li Y. Schembri-King J. Jakes S. Hayashi J. Mol. Immunol. 2000; 37: 603-612Crossref PubMed Scopus (21) Google Scholar), several GRB family proteins bind to cytoplasmic domains of growth factor receptors via their SH2 domains (38Han D.C. Shen T.L. Guan J.L. Oncogene. 2001; 20: 6315-6321Crossref PubMed Scopus (141) Google Scholar), and Gab1 binds to PI3K and intracellular domains of some growth factor receptors via phosphorylated tyrosine residues outside of its PH domain (39Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell. Biol. 2000; 20: 1448-1459Crossref PubMed Scopus (282) Google Scholar). Neither Ipl nor Tih1 show clear evidence for protein-protein binding motifs, except possibly for proline-containing sequences near their C termini. Additional work will be required to determine whether their short C- or N-terminal extensions, or their PH domains, are able to mediate protein-protein interactions. An alternative possibility, which is suggested by the lack of additional functional modules in the small Ipl and Tih1 proteins, is that these proteins may act as natural “dominant-negatives” to dampen some biological processes by binding to PIPs and thereby excluding PIP binding “effector” proteins. The Ipl gene (also known as Tssc3) is highly expressed in the placenta, where its effective dosage is regulated by parental imprinting. Mouse concepti inheriting Ipldeletions from their mothers show placentomegaly, implicatingIpl in restraining placental growth (1Frank D. Fortino W. Clark L. Musalo R. Wang W. Saxena A. Li C.M. Reik W. Ludwig T. Tycko B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7490-7495Crossref PubMed Scopus (186) Google Scholar). However, the biochemical basis for this genetic effect is not known. The Ipl protein contains a single predicted pleckstrin homology (PH) 1The abbreviations used are: PH, pleckstrin homology; PHD, PH domain; BTK, Bruton's tyrosine kinase; CH1, cytohesin1; DAPP1, dual adaptor for phosphoinositides and phosphotyrosine; DYN1, Dynamin; GRP1, general receptor for phosphoinositides-1; IPL, gene imprinted in placenta and liver; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PLS-N, N-terminal PH domain from pleckstrin; SH2, Src homology domain2; Tih1, TDAG51/IPL homologue 1; PIP, phosphatidyl-inositol phosphate lipid, PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P2, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns, phosphatidylinositol; GST, glutathioneS-transferase; DTT, dithiothreitol; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP; EGF, epidermal growth factor; BSA, bovine serum albumin; TRITC, tetramethylrhodamine isothiocyanate; CMV, cytomegalovirus; PMSF, phenylmethylsulfonyl fluoride; ts, temperature-sensitive1The abbreviations used are: PH, pleckstrin homology; PHD, PH domain; BTK, Bruton's tyrosine kinase; CH1, cytohesin1; DAPP1, dual adaptor for phosphoinositides and phosphotyrosine; DYN1, Dynamin; GRP1, general receptor for phosphoinositides-1; IPL, gene imprinted in placenta and liver; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; PLS-N, N-terminal PH domain from pleckstrin; SH2, Src homology domain2; Tih1, TDAG51/IPL homologue 1; PIP, phosphatidyl-inositol phosphate lipid, PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(4)P, phosphatidylinositol 4-phosphate; PtdIns(5)P, phosphatidylinositol 5-phosphate; PtdIns(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3, 4,5)P2, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate; PtdIns(3, 5)P2, phosphatidylinositol 3,5-bisphosphate; PtdIns, phosphatidylinositol; GST, glutathioneS-transferase; DTT, dithiothreitol; PBS, phosphate-buffered saline; GFP, green fluorescent protein; EGFP, enhanced GFP; EGF, epidermal growth factor; BSA, bovine serum albumin; TRITC, tetramethylrhodamine isothiocyanate; CMV, cytomegalovirus; PMSF, phenylmethylsulfonyl fluoride; ts, temperature-sensitive domain, with short N- and C-terminal extensions. It shares this structure with its closest relative, Tih1, an even smaller protein, which is broadly expressed and not regulated by imprinting (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). The PH domain is a ∼100-amino acid module defined by sequence homology, which has a conserved tertiary structure of a β-sandwich of 3 + 4 stranded β sheets with variable intervening loops, followed by a single amphipathic α helix (3Lemmon M.A. Ferguson K.M. Curr. Top. Microbiol. Immunol. 1998; 228: 39-74PubMed Google Scholar). Most PH domain-containing proteins can bind to certain phosphatidylinositol phosphate lipids (PIPs), but their binding affinities for the specific products of phosphoinositide metabolism vary greatly (4Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar). For example, the PH domains from phospholipase C δ1 (PLCδ1) bind strongly to PtdIns(4,5)P2, but not to the downstream metabolites of this compound, PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which are produced by the action of phosphatidylinositol 3-kinase (PI3K). Other PH domains, typified by that of the general receptor for phosphoinositides (GRP1), show the opposite pattern of binding affinity, with high affinity for PtdIns(3,4,5)P3 and only low affinity for PtdIns(4,5)P2. Based on in vitro and in vivo assays, Kavran et al. (5Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) classified PH domains into two groups: a small class of PH domains with high affinity and specificity for particular PIPs and a much larger class showing low affinity promiscuous binding to various PIPs. More recently, Maffucci and Falasca (6Maffucci T. Falasca M. FEBS Lett. 2001; 506: 173-179Crossref PubMed Scopus (107) Google Scholar) have proposed a further division of PH domains into three groups. In this scheme, Group 1 proteins are those with high affinities for specific PIPs; Group 2 proteins have lower but still measurable affinity for phosphoinositides, and show less ability to discriminate among the various PIPs; and Group 3 proteins have minimal affinity for phosphoinositides, with no detectable specificity. Consistent with their varying interactions with phosphoinositides, and with their occurrence alongside a variety of other modules, PH domains have diverse functions. Among these are roles in signal transduction downstream of membrane receptors, control of membrane vesicle trafficking in the cell, targeting of phospholipid-modifying enzymes to cellular membranes, and control of cytoskeleton-membrane interactions (3Lemmon M.A. Ferguson K.M. Curr. Top. Microbiol. Immunol. 1998; 228: 39-74PubMed Google Scholar, 7Rebecchi M.J. Scarlata S. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 503-528Crossref PubMed Scopus (248) Google Scholar). These functions are unified by the ability of PH domains to associate with the plasma membrane and/or intracellular membranes through their high or low affinity binding to phosphoinositide lipids. Certain PH domains can also mediate protein-protein interactions, for example that which occurs between the beta-adrenergic receptor protein kinase and the βγ subunit of heterotrimeric G-proteins (8Inglese J. Koch W.J. Touhara K. Lefkowitz R.J. Trends Biochem. Sci. 1995; 20: 151-156Abstract Full Text PDF PubMed Scopus (165) Google Scholar). But the protein-binding site is distant from the phosphoinositide binding region, and a PH domain-phosphoinositide interaction may be simultaneously required for physiological function (9Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Interactions of PH domains with phosphoinositides have been examinedin vitro and in vivo by several complementary approaches. These include direct binding of GST-PH domain fusion proteins to phosphoinositides in lipid vesicles or immobilized on nitrocellulose membranes (5Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 10Dowler S. Currie R.A. Downes C.P. Alessi D.R. Biochem. J. 1999; 342: 7-12Crossref PubMed Scopus (127) Google Scholar, 11Dowler S. Currie R.A. Campbell D.G. Deak M. Kular G. Downes C.P. Alessi D.R. Biochem. J. 2000; 351: 19-31Crossref PubMed Scopus (473) Google Scholar, 12Thomas C.C. Dowler S. Deak M. Alessi D.R. van Aalten D.M. Biochem. J. 2001; 358: 287-294Crossref PubMed Scopus (80) Google Scholar, 13Razzini G. Brancaccio A. Lemmon M.A. Guarnieri S. Falasca M. J. Biol. Chem. 2000; 275: 14873-14881Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 14Sankaran V.G. Klein D.E. Sachdeva M.M. Lemmon M.A. Biochemistry. 2001; 40: 8581-8587Crossref PubMed Scopus (62) Google Scholar, 15Fleming I.N. Gray A. Downes C.P. Biochem. J. 2000; 351: 173-182Crossref PubMed Scopus (107) Google Scholar), and analysis in vivoof the movement of GFP-tagged PH domains from the cytosol to the plasma membrane in response to growth factors that activate PI3K (5Kavran J.M. Klein D.E. Lee A. Falasca M. Isakoff S.J. Skolnik E.Y. Lemmon M.A. J. Biol. Chem. 1998; 273: 30497-30508Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 13Razzini G. Brancaccio A. Lemmon M.A. Guarnieri S. Falasca M. J. Biol. Chem. 2000; 275: 14873-14881Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,15Fleming I.N. Gray A. Downes C.P. Biochem. J. 2000; 351: 173-182Crossref PubMed Scopus (107) Google Scholar, 16Currie R.A. Walker K.S. Gray A. Deak M. Casamayor A. Downes C.P. Cohen P. Alessi D.R. Lucocq J. Biochem. J. 1999; 337: 575-583Crossref PubMed Scopus (272) Google Scholar, 17Gray A. Van Der Kaay J. Downes C.P. Biochem. J. 1999; 344: 929-936Crossref PubMed Scopus (181) Google Scholar, 18Anderson K.E. Lipp P. Bootman M. Ridley S.H. Coadwell J. Ronnstrand L. Lennartsson J. Holmes A.B. Painter G.F. Thuring J. Lim Z. Erdjument-Bromage H. Grewal A. Tempst P. Stephens L.R. Hawkins P.T. Curr. Biol. 2000; 10: 1403-1412Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 19Bobe R. Wilde J.I. Maschberger P. Venkateswarlu K. Cullen P.J. Siess W. Watson S.P. Blood. 2001; 97: 678-684Crossref PubMed Scopus (40) Google Scholar, 20Chen R.H. Corbalan-Garcia S. Bar-Sagi D. EMBO J. 1997; 16: 1351-1359Crossref PubMed Scopus (115) Google Scholar, 21Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (895) Google Scholar, 22Watton S.J. Downward J. Curr. Biol. 1999; 9: 433-436Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 23Varnai P. Rother K.I. Balla T. J. Biol. Chem. 1999; 274: 10983-10989Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 24Venkateswarlu K. Gunn-Moore F. Tavare J.M. Cullen P.J. J. Cell Sci. 1999; 112: 1957-1965PubMed Google Scholar, 25Langille S.E. Patki V. Klarlund J.K. Buxton J.M. Holik J.J. Chawla A. Corvera S. Czech M.P. J. Biol. Chem. 1999; 274: 27099-27104Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). A third assay tests the ability of the PH domain of interest, fused to activated mutant Ras (Ras*), to rescue Saccharomyces cerevisiae that are defective in the Ras pathway due to a temperature-sensitive (ts) CDC25 mutation (26Isakoff S.J. Cardozo T. Andreev J. Li Z. Ferguson K.M. Abagyan R. Lemmon M.A. Aronheim A. Skolnik E.Y. EMBO J. 1998; 17: 5374-5387Crossref PubMed Scopus (281) Google Scholar). Because yeast do not make PtdIns(3,4,5)P3, PI3K-dependent and PI3K-independent rescue can be separately evaluated, by co-transformation with active versus inactive constructs of the catalytic subunit of PI3K. As a first step in assessing the biochemical functions of Ipl and Tih1, we have used each of these approaches to compare the behavior of the PH domains from these proteins with several other PH domains. We have also carried out sequence comparisons with other PH domains. The data indicate that Ipl and Tih1 define an outlying phylogenetic branch of the PH domain superfamily, but that they are bona fide PIP binders, which fit best into the group with moderate affinity and poor selectivity for the known phosphoinositide lipids. DISCUSSIONThe Ipl gene was originally found in a chromosomal walk, which was undertaken to identify and characterize novel imprinted genes in a megabase-scale imprinted domain on human Chr11p15.5 and distal mouse Chr7 (35Qian N. Frank D. O'Keefe D. Dao D. Zhao L. Yuan L. Wang Q. Keating M. Walsh C. Tycko B. Hum. Mol. Genet. 1997; 6: 2021-2029Crossref PubMed Scopus (144) Google Scholar). Since then, substantial information has accrued concerning the tissue-specific expression and functional imprinting of this gene, and the creation of Ipl-knockout mice has established that at least one of its functions is to regulate the growth of the placenta (1Frank D. Fortino W. Clark L. Musalo R. Wang W. Saxena A. Li C.M. Reik W. Ludwig T. Tycko B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7490-7495Crossref PubMed Scopus (186) Google Scholar, 2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). Biochemical information is needed to explain this genetic finding, and a starting point is provided by the domain structure of the Ipl protein. This structure, which is shared by the closest Ipl homologue, Tih1, is remarkably simple, consisting of a single predicted PH domain with short N- and C-terminal extensions. The results shown here verify that this central region acts as a bona fide PH domain, which is capable of interacting with phosphoinositide lipids, both in direct binding assays and in membrane translocation assays, as well as functionally in a yeast growth rescue assay. Overall, the findings suggest that the Ipl and Tih1 PH domains bind to known PIPs with moderate affinity and low specificity. Whether this promiscuous binding reflects the true physiological targets of proteins in this group is not known, and they may have a high affinity for a yet unidentified lipid, or even protein, ligand. For example, recent work has shown that the tagged PH domain of human oxysterol binding protein accumulates at the yeast Golgi apparatus partly in response to the presence of PtdIns(4)P and partly in response to an unidentified factor that is dependent on the Golgi ATPase Arf1p (36Levine T.P. Munro S. Curr. Biol. 2002; 12: 695-704Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar).As summarized in Table I, our data also show clear differences in the behavior of the Ipl versus Tih1 PH domains. Consistent with these differences, Ipl and Tih1 have different patterns of tissue-specific expression (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar), and no detectable growth abnormality was found in Tih1-null concepti (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). In addition, the Ipl protein is highly conserved in vertebrate evolution, presumably reflecting a conserved biological function, but we have not been able to identify Tih1 orthologues in non-mammalian vertebrates.Further progress in understanding the biochemical functions of Ipl and Tih1 will depend on localizing the native proteins within the cell at high resolution and on identifying any proteins or other lipids with which they interact. For most PH domain-containing proteins, such protein-protein interactions do not occur via the PH domain, but instead involve different modules elsewhere in the protein. For example, Lnk associates with the ABP-280 actin binding protein via an interdomain stretch of 56 amino acids between its PH and SH2 domains (37He X. Li Y. Schembri-King J. Jakes S. Hayashi J. Mol. Immunol. 2000; 37: 603-612Crossref PubMed Scopus (21) Google Scholar), several GRB family proteins bind to cytoplasmic domains of growth factor receptors via their SH2 domains (38Han D.C. Shen T.L. Guan J.L. Oncogene. 2001; 20: 6315-6321Crossref PubMed Scopus (141) Google Scholar), and Gab1 binds to PI3K and intracellular domains of some growth factor receptors via phosphorylated tyrosine residues outside of its PH domain (39Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell. Biol. 2000; 20: 1448-1459Crossref PubMed Scopus (282) Google Scholar). Neither Ipl nor Tih1 show clear evidence for protein-protein binding motifs, except possibly for proline-containing sequences near their C termini. Additional work will be required to determine whether their short C- or N-terminal extensions, or their PH domains, are able to mediate protein-protein interactions. An alternative possibility, which is suggested by the lack of additional functional modules in the small Ipl and Tih1 proteins, is that these proteins may act as natural “dominant-negatives” to dampen some biological processes by binding to PIPs and thereby excluding PIP binding “effector” proteins. The Ipl gene was originally found in a chromosomal walk, which was undertaken to identify and characterize novel imprinted genes in a megabase-scale imprinted domain on human Chr11p15.5 and distal mouse Chr7 (35Qian N. Frank D. O'Keefe D. Dao D. Zhao L. Yuan L. Wang Q. Keating M. Walsh C. Tycko B. Hum. Mol. Genet. 1997; 6: 2021-2029Crossref PubMed Scopus (144) Google Scholar). Since then, substantial information has accrued concerning the tissue-specific expression and functional imprinting of this gene, and the creation of Ipl-knockout mice has established that at least one of its functions is to regulate the growth of the placenta (1Frank D. Fortino W. Clark L. Musalo R. Wang W. Saxena A. Li C.M. Reik W. Ludwig T. Tycko B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7490-7495Crossref PubMed Scopus (186) Google Scholar, 2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). Biochemical information is needed to explain this genetic finding, and a starting point is provided by the domain structure of the Ipl protein. This structure, which is shared by the closest Ipl homologue, Tih1, is remarkably simple, consisting of a single predicted PH domain with short N- and C-terminal extensions. The results shown here verify that this central region acts as a bona fide PH domain, which is capable of interacting with phosphoinositide lipids, both in direct binding assays and in membrane translocation assays, as well as functionally in a yeast growth rescue assay. Overall, the findings suggest that the Ipl and Tih1 PH domains bind to known PIPs with moderate affinity and low specificity. Whether this promiscuous binding reflects the true physiological targets of proteins in this group is not known, and they may have a high affinity for a yet unidentified lipid, or even protein, ligand. For example, recent work has shown that the tagged PH domain of human oxysterol binding protein accumulates at the yeast Golgi apparatus partly in response to the presence of PtdIns(4)P and partly in response to an unidentified factor that is dependent on the Golgi ATPase Arf1p (36Levine T.P. Munro S. Curr. Biol. 2002; 12: 695-704Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). As summarized in Table I, our data also show clear differences in the behavior of the Ipl versus Tih1 PH domains. Consistent with these differences, Ipl and Tih1 have different patterns of tissue-specific expression (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar), and no detectable growth abnormality was found in Tih1-null concepti (2Frank D. Mendelsohn C.L. Ciccone E. Svensson K. Ohlsson R. Tycko B. Mamm. Genome. 1999; 10: 1150-1159Crossref PubMed Scopus (85) Google Scholar). In addition, the Ipl protein is highly conserved in vertebrate evolution, presumably reflecting a conserved biological function, but we have not been able to identify Tih1 orthologues in non-mammalian vertebrates. Further progress in understanding the biochemical functions of Ipl and Tih1 will depend on localizing the native proteins within the cell at high resolution and on identifying any proteins or other lipids with which they interact. For most PH domain-containing proteins, such protein-protein interactions do not occur via the PH domain, but instead involve different modules elsewhere in the protein. For example, Lnk associates with the ABP-280 actin binding protein via an interdomain stretch of 56 amino acids between its PH and SH2 domains (37He X. Li Y. Schembri-King J. Jakes S. Hayashi J. Mol. Immunol. 2000; 37: 603-612Crossref PubMed Scopus (21) Google Scholar), several GRB family proteins bind to cytoplasmic domains of growth factor receptors via their SH2 domains (38Han D.C. Shen T.L. Guan J.L. Oncogene. 2001; 20: 6315-6321Crossref PubMed Scopus (141) Google Scholar), and Gab1 binds to PI3K and intracellular domains of some growth factor receptors via phosphorylated tyrosine residues outside of its PH domain (39Rodrigues G.A. Falasca M. Zhang Z. Ong S.H. Schlessinger J. Mol. Cell. Biol. 2000; 20: 1448-1459Crossref PubMed Scopus (282) Google Scholar). Neither Ipl nor Tih1 show clear evidence for protein-protein binding motifs, except possibly for proline-containing sequences near their C termini. Additional work will be required to determine whether their short C- or N-terminal extensions, or their PH domains, are able to mediate protein-protein interactions. An alternative possibility, which is suggested by the lack of additional functional modules in the small Ipl and Tih1 proteins, is that these proteins may act as natural “dominant-negatives” to dampen some biological processes by binding to PIPs and thereby excluding PIP binding “effector” proteins. Anti-peptide antibody against β1-integrin was generously provided by E. Marcantonio. We thank the Confocal Microscopy facility at the Columbia University Comprehensive Cancer Center for expert advice and assistance." @default.
• W1982142716 created "2016-06-24" @default.
• W1982142716 creator A5030528219 @default.
• W1982142716 creator A5032589906 @default.
• W1982142716 creator A5051475582 @default.
• W1982142716 creator A5069882244 @default.
• W1982142716 creator A5070082045 @default.
• W1982142716 creator A5073959537 @default.
• W1982142716 creator A5075565429 @default.
• W1982142716 date "2002-12-01" @default.
• W1982142716 modified "2023-10-16" @default.
• W1982142716 title "Phosphoinositide Binding by the Pleckstrin Homology Domains of Ipl and Tih1" @default.
• W1982142716 cites W1951003906 @default.
• W1982142716 cites W1963825704 @default.
• W1982142716 cites W1963941913 @default.
• W1982142716 cites W1967352670 @default.
• W1982142716 cites W1972974751 @default.
• W1982142716 cites W1974530349 @default.
• W1982142716 cites W1978410319 @default.
• W1982142716 cites W1982996449 @default.
• W1982142716 cites W1983127375 @default.
• W1982142716 cites W1993995347 @default.
• W1982142716 cites W2005511164 @default.
• W1982142716 cites W2016441216 @default.
• W1982142716 cites W2029282790 @default.
• W1982142716 cites W2031474998 @default.
• W1982142716 cites W2044005459 @default.
• W1982142716 cites W2046826693 @default.
• W1982142716 cites W2047439787 @default.
• W1982142716 cites W2060378031 @default.
• W1982142716 cites W2064242629 @default.
• W1982142716 cites W2067051871 @default.
• W1982142716 cites W2068488447 @default.
• W1982142716 cites W2076211600 @default.
• W1982142716 cites W2083592023 @default.
• W1982142716 cites W2102436071 @default.
• W1982142716 cites W2106882534 @default.
• W1982142716 cites W2138195153 @default.
• W1982142716 cites W2141206157 @default.
• W1982142716 cites W2148612336 @default.
• W1982142716 cites W2148915142 @default.
• W1982142716 cites W2150597600 @default.
• W1982142716 cites W2152515453 @default.
• W1982142716 cites W2154284350 @default.
• W1982142716 cites W2154817329 @default.
• W1982142716 cites W2168721537 @default.
• W1982142716 cites W4239979702 @default.
• W1982142716 cites W4247210984 @default.
• W1982142716 cites W4249232512 @default.
• W1982142716 cites W4376453135 @default.
• W1982142716 doi "https://doi.org/10.1074/jbc.m206497200" @default.
• W1982142716 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12374806" @default.
• W1982142716 hasPublicationYear "2002" @default.
• W1982142716 type Work @default.
• W1982142716 sameAs 1982142716 @default.
• W1982142716 citedByCount "47" @default.
• W1982142716 countsByYear W19821427162012 @default.
• W1982142716 countsByYear W19821427162014 @default.
• W1982142716 countsByYear W19821427162015 @default.
• W1982142716 countsByYear W19821427162017 @default.
• W1982142716 countsByYear W19821427162018 @default.
• W1982142716 countsByYear W19821427162019 @default.
• W1982142716 countsByYear W19821427162020 @default.
• W1982142716 countsByYear W19821427162021 @default.
• W1982142716 countsByYear W19821427162023 @default.
• W1982142716 crossrefType "journal-article" @default.
• W1982142716 hasAuthorship W1982142716A5030528219 @default.
• W1982142716 hasAuthorship W1982142716A5032589906 @default.
• W1982142716 hasAuthorship W1982142716A5051475582 @default.
• W1982142716 hasAuthorship W1982142716A5069882244 @default.
• W1982142716 hasAuthorship W1982142716A5070082045 @default.
• W1982142716 hasAuthorship W1982142716A5073959537 @default.
• W1982142716 hasAuthorship W1982142716A5075565429 @default.
• W1982142716 hasBestOaLocation W19821427161 @default.
• W1982142716 hasConcept C104317684 @default.
• W1982142716 hasConcept C107824862 @default.
• W1982142716 hasConcept C12554922 @default.
• W1982142716 hasConcept C165525559 @default.
• W1982142716 hasConcept C167625842 @default.
• W1982142716 hasConcept C185592680 @default.
• W1982142716 hasConcept C2779315201 @default.
• W1982142716 hasConcept C49658373 @default.
• W1982142716 hasConcept C515207424 @default.
• W1982142716 hasConcept C55493867 @default.
• W1982142716 hasConcept C62478195 @default.
• W1982142716 hasConcept C70721500 @default.
• W1982142716 hasConcept C71240020 @default.
• W1982142716 hasConcept C86803240 @default.
• W1982142716 hasConcept C95444343 @default.
• W1982142716 hasConceptScore W1982142716C104317684 @default.
• W1982142716 hasConceptScore W1982142716C107824862 @default.
• W1982142716 hasConceptScore W1982142716C12554922 @default.
• W1982142716 hasConceptScore W1982142716C165525559 @default.
• W1982142716 hasConceptScore W1982142716C167625842 @default.
• W1982142716 hasConceptScore W1982142716C185592680 @default.
• W1982142716 hasConceptScore W1982142716C2779315201 @default.
• W1982142716 hasConceptScore W1982142716C49658373 @default.
• W1982142716 hasConceptScore W1982142716C515207424 @default.

• next