title: FYVE and coiled-coil domains determine the specific localisation of Hrs to early endosomes.
abstract: Hrs, an essential tyrosine kinase substrate, has been implicated in intracellular trafficking and signal transduction pathways. The protein contains several distinctive domains, including an N-terminal VHS domain, a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding FYVE domain and two coiled-coil domains. Here we have investigated the roles of these domains in the subcellular localisation of Hrs. Hrs was found to colocalise extensively with EEA1, an established marker of early endosomes. While the membrane association of EEA1 was abolished in the presence of a dominant negative mutant of the endosomal GTPase Rab5, the localisation of Hrs to early endosomes was Rab5 independent. The VHS-domain was nonessential for the subcellular targeting of Hrs. In contrast, the FYVE domain as well as the second coiled-coil domain, 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 mutation (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 additional domain. We speculate that binding to PtdIns(3)P and a SNAP-25-related molecule may target Hrs specifically to early endosomes.
authors.affiliation: Department of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
authors.forename: C
authors.initials: C
authors.lastname: Raiborg
authors.forename: B
authors.initials: B
authors.lastname: Bremnes
authors.forename: A
authors.initials: A
authors.lastname: Mehlum
authors.forename: D J
authors.initials: DJ
authors.lastname: Gillooly
authors.forename: A
authors.initials: A
authors.lastname: D'Arrigo
authors.forename: E
authors.initials: E
authors.lastname: Stang
authors.forename: H
authors.initials: H
authors.lastname: Stenmark
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050825:Synaptosomal-Associated Protein 25; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000502:physiology
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C494208:SNAP25 protein, human; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate; D015544:Inositol 1,4,5-Trisphosphate; D020696:rab5 GTP-Binding Proteins