title: Flotillin-1 mediates PrPc endocytosis in the cultured cells during Cu²⁺ stimulation through molecular interaction.
abstract: Flotillins are membrane association proteins consisting of two homologous members, flotillin-1 (Flot-1) and flotillin-2 (Flot-2). They define a clathrin-independent endocytic pathway in mammal cells, which are also distinct from some other endocytosis mechanisms. The implicated cargoes of the flotillin-dependent pathway are mainly some GPI-anchored proteins, such as CD59 and Thy-1, which positionally colocalize with flotillins at the plasma membrane microdomains. To see whether flotillins are involved in the endocytosis of PrP(C), the potential molecular interaction between PrP(C) and flotillins in a neuroblastoma cell line SK-N-SH was analyzed. Co-immunoprecipitation assays did not reveal a detectable complex in the cell lysates of a normal feeding situation. After stimulation of Cu(2+), PrP(C) formed a clear complex with Flot-1, but not with Flot-2. Immunofluorescent assays illustrated that PrP(C) colocalized well with Flot-1, and the complexes of PrP(C)-Flot-1 shifted from the cell membrane to the cytoplasm along with the treatment of Cu(2+). Down-regulating the expression of Flot-1 in SK-N-SH cells by Flot-1-specific RNAi obviously abolished the Cu(2+)-stimulated endocytosis process of PrP(C). Moreover, we also found that in the cell line human embryonic kidney 293 (HEK293) without detectable PrP(C) expression, the distribution of cellular Flot-1 maintained almost unchanged during Cu(2+) treatment. Cu(2+)-induced PrP(C)-Flot-1 molecular interaction and endocytosis in HEK293 cells were obtained when expressing wild-type human PrP (PrP(PG5)), but not in the preparation expressing octarepeat-deleted PrP (PrP(PG0)). Our data here provide direct evidences for the molecular interaction and endocytosis of PrP(C) with Flot-1 in the presence of copper ions, and the octarepeat region of PrP(C) is critical for this process, which strongly indicates that the Flot-1-dependent endocytic pathway seems to mediate the endocytosis process of PrP(C) in the special situation.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Chang-Bai Rd 155, Beijing, 102206, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Ren
authors.forename: Chen
authors.initials: C
authors.lastname: Gao
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.forename: Yin
authors.initials: Y
authors.lastname: Xu
authors.forename: Shao-Bin
authors.initials: SB
authors.lastname: Wang
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Chan
authors.initials: C
authors.lastname: Tian
authors.forename: Qi
authors.initials: Q
authors.lastname: Shi
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Dong
mesh_terms: D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003300:Copper / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D018621:PrPC Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D018621:PrPC Proteins; D034741:RNA, Small Interfering; C106354:flotillins; D003300:Copper; D015032:Zinc