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/lgA/ZO-1 -like) domains followed by a COOH-terminal sequence that binds to active ER M 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 pr eferentially to the first PDZ domain of EBP50. as well as to E3KARP, and th at 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 th at contains a Tre/Bub2/ Cdc16 (TBC)/rab GTPase-activating protein (GAP) dom ain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ do mains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colo calizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell micro villi, 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 isofor m of nadrin, a rhoGAP domain-containing protein implicated in regulating ve sicular transport. Nadrin binds EBP50 directly probably through its COOH-te rminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.