FYVE and coiled-coil domains determine the specific localisation of Hrs toearly endosomes

Hrs, an essential tyrosine kinase substrate, has been implicated in intrace llular trafficking and signal transduction pathways, The protein contains s everal distinctive domains, including an N-terminal VHS domain, a phosphati dylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coi l domains. Here we have investigated the roles of these domains in the subc ellular localisation of Hrs. Hrs was found to colocalise extensively with E EA1, an established marker of early endosomes, While the membrane associati on of EEA1 was abolished in the presence of a dominant negative mutant of t he endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Ra b5 independent. The VHS-domain was nonessential for the subcellular targeti ng of Hrs. In contrast, the FYVE domain as well as the second coiled-coil d omain, which has been shown to bind to SNAP-25, were required for targeting of Hrs to early endosomes, A small construct consisting of only these two domains was correctly localised to early endosomes, whereas a point mutatio n (R183A) in the PtdIns(3)P-binding pocket of the FYVE domain inhibited the membrane targeting of Hrs, Thus, like EEA1, the endosomal targeting of Hrs is mediated by a PtdIns(3)P-binding FYVE domain in cooperation with an add itional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-relat ed molecule may target Hrs specifically to early endosomes.