Flotillin-1Mediates PrP^C Endocytosis In The Cultured Cells During Cu²⁺ Stimulation Through Molecular Interaction

Prion diseases, also named as transmissible spongiform encephalopathies (TSEs). are a class of fatal neurodegenerative diseases which can infect central nervous system (CNS) of humans and animals, including Creutzfeld-Jakob disease (CJD), Kuru, fatal familial insomnia (FFI) and Gerstmann-Straussler-Scheinker syndrome (GSS) in human, bovine spongiform encephalopathy in cattle, scrapie in sheep and goat, chronic wasting disease (CWD) in elk and deer. The pathogenic agents of prion diseases are known as the conformations conversion of PrPC into an abnormal conformer PrPSc which is relatively insoluble, protease resistant and infectious, although the mechanism of conversion is still unclear.The normal cellular PrPC is a kind of ubiquitous glycoprotein which especially expressed in neurons of central nervous system (CNS) and predominantly anchored at the plasma membrane via a glycosylphosphatidylinositol (GPI) moiety. The founctions of PrPC are still not clearly documented. Many evidences show that PrPC may play roles in anti-oxidant systems, protection of nervous system and anti-cell apoptosis. The process of PrPC endocytosis is believed to be very important in achieving some physiological functions in cell, such as signal transduction and cellular copper metabolism, moreover, the endocytic process may relate to the conversion of PrPC into PrPSc. But the mechanism of PrPC endocytosis is still not very clear, which may involve in several different processes. Some membrane-associated proteins, such as clathrin caveolae and Arf6show abilities to bind with PrPC, which might participate in the endocytosis of PrPC.Flotillins are membrane-association proteins consisting of two homologous members, flotillin-1(Flot-1) and-2(Flot-2), which belong to flotillin proteins family of SPFH (stomatin, prohibitin, flotillin, HflK/C). They define a clathrin-independent endocytic pathway in mammal cells, which are also distinct from some other endocytosis mechanisms such as clathrin-, caveolin-and Arf6-dependent endocytosis. The implicated cargoes of the flotillin-dependent pathway are mainly some GPI-anchored proteins, such as CD59and Thy-1, which are positionally colocalize with flotillins at the plasma membrane microdomains.In this study, we use copper ions to stimulate PrPC endocytosis and explore whether Flot-1participating in the endocytosis of PrPC. The main results of our reaserch are as follows:1. In the stimulation of copper ions, the interaction between PrPCand Flot-1was detected by immunoprecipitation in SK-N-SH cells. Such Cuz+induced interaction between PrPc with Flot-1showed dose-and time-dependent manners.2. Screening of the influences of other divalent cations, including Fe+, Mn2+, Zn2+, Ni2+and Ca2+, on the interaction between PrPC and Flot-1by immunoprecipitation demonstrated that only Cu2+and Zn2+possessed the capacity of inducing the protein complexes between PrPC and Flot-1in the cultured cells.3. Double stained IFA and confocal microscopy assays identified that during the Cu2+stimulated endocytosis of PrPC, Flot-1colocalized well with PrPC in cytoplasm.4. Knockdown of cellular Flot-1with the Flot-1specific RNAi significantly reduced the Cu+induced endocytosis of PrPC in the assays of immunofluorescence and confocal microscopy.5. Challenge of Cu2+on HEK293cells that was previously verified not containing detectable endogenous PrPC did not induce Flot-1shift that was observed in SK-N-SH cells.6. Stimulation of Cu2+induced obvious PrPC-Flot-1interaction and PrPC endocyiosis in the HEK293cells expressing human wild-type PrP (PrP-PG5). but failed in the cells expressing human PrP deleted octarepeats (PrP-PGO), demonstrating that the octarepeats are critical for the Cu2+induced interaction between PrPC and Flot-1.In conclusion, we have demonstrated that in the stimulation of copper, Flot-1definitely participates in the endocytosis of PrPC in SK-N-SH cells, probably via forming PrP-Flot-1complex. Knockdown of the expression of cellular Flot-1largely abolishes Cu2+induced PrPC endocytosis. The octarepeats within PrP are critical for the Cu2+induced interaction between PrPC and Flot-1. Based on our data here and many other relevant evidences, we may conclude that the common feature of PrPC role in copper binding and trafficking appears to be the maintenance of cellular homeostasis, in which PrPC functions as a receptor for copper to maintain metal balances in cells under some special stress situations and Flot-1serves as a trafficking protein to help cells complete this activity.