Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP.
The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.
Reczek
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Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Bretscher
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D000595:Amino Acid Sequence; D000818:Animals; D064194:Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D018076:DNA, Complementary; D010450:Endopeptidases; D005260:Female; D005656:Fungal Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011257:Pregnancy Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D017923:Sodium-Hydrogen Exchangers; D014018:Tissue Distribution; D043222:Ubiquitin Thiolesterase; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D000099149:Ezrin
D064194:Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D010450:Endopeptidases; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion; D010455:Peptides; D010750:Phosphoproteins; D011257:Pregnancy Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers; D043222:Ubiquitin Thiolesterase; D020691:rab GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D000099149:Ezrin; C504466:ARHGAP17 protein, human; C504467:Arhgap17 protein, mouse; C070351:BUB2 protein, S cerevisiae; C577395:CDC16 protein, human; C577396:Cdc16 protein, mouse; C425805:TBC1D10A protein, human; C093376:sodium-hydrogen exchanger regulatory factor; C473330:USP6 protein, human