Reggie-1 and reggie-2 are highly conserved and widely expressed proteins associated with membrane rafts. The molecular function of reggies remains to be clarified, but recent data indicate that they are involved in various cellular processes such as insulin signaling, phagocytosis and actin remodeling. However, there is discrepancy in the literature if reggies are associated with caveolae or non-caveolar rafts. Reggies are expressed and raft associated also in many cells which do not contain caveolae, such as neurons and lymphocytes. However, it is not clear if the function or localization of reggies are dependent on the presence of caveolae and expression of caveolin-1 protein. In this study, we directly addressed this question in epithelial cells. We could show that ectopic expression of caveolin-1 does not result in any change in the cellular localization of reggie-1, which is present at the plasma membrane also in the absence of caveolin-1. On the other hand, caveolin-2, which localizes in caveolae, is dependent on caveolin-1 expression in order to be localized at the plasma membrane. Although reggie-1 and reggie-2 strongly interact with each other, we did not detect a direct interaction between caveolin-1 and reggies by means of a yeast two-hybrid assay, nor could reggies be co-immunoprecipitated with caveolin-1. Furthermore, endogenous reggie-1 and -2 were found not to colocalize with caveolin-1 in epithelial cells. Thus, our data indicate that reggies are localized in microdomains different from caveolae, and the function of reggies is different from and independent of caveolin-1.
"Flotillin-1 (also known as reggie-2) and flotillin-2 (also known as reggie-1) are membrane-associated oligomeric proteins that are ubiquitously expressed (Lang et al., 1998; Neumann-Giesen et al., 2004; Solis et al., 2007; Stuermer et al., 2001). The flotillins form homo-and hetero-oligomers (Neumann-Giesen et al., 2004; Solis et al., 2007), which assemble into clusters defining specialized flat membrane microdomains at the plasma membrane independently of caveolins and caveolae (Fernow et al., 2007; Frick et al., 2007; Lang et al., 1998; Stuermer et al., 2001). Flotillins have been shown to be involved in various cellular processes including cell proliferation, T-cell activation and phagocytosis (Dermine et al., 2001; Gómez et al., 2010; Santamaría et al., 2005; Stuermer et al., 2004), and have been implicated in signaling activation of IgE and TrkA, and epidermal growth factor, insulin and G-protein-coupled receptors (Amaddii et al., 2012; Baumann et al., 2000; Kato et al., 2006; Limpert et al., 2007; Wehmeyer et al., 2014). "
"Although Flot-2 is thought to locate mainly in planar lipid rafts while cav-1 locates in caveolae, no definite techniques can differentiate these two types of lipid rafts directly , . In fact, whether flotillins locate only in planar lipid rafts or locate in both types of rafts remain unclear , . At least one report has shown that Flot-1 locates also in caveolae , despite that no reports focus on the Flot-2 yet. "
[Show abstract][Hide abstract] ABSTRACT: Lipid rafts are subdomains of the cell membrane with distinct protein composition and high concentrations of cholesterol and glycosphingolipids. Raft proteins are thought to mediate diverse cellular processes including signal transduction. However, its cellular mechanisms remain unclear. Caveolin-1 (cav-1, marker protein of caveolae) has been thought as a switchboard between extracellular matrix (ECM) stimuli and intracellular signals. Flotillin-2/reggie-1(Flot-2) is another ubiquitously expressed raft protein which defines non-caveolar raft microdomains (planar raft). Its cellular function is largely uncharacterized. Our novel studies demonstrated that Flot-2, in conjunction with cav-1, played important functions on controlling cell death via regulating Fas pathways. Using Beas2B epithelial cells, we found that in contrast to cav-1, Flot-2 conferred cytoprotection via preventing Fas mediated death-inducing signaling complex (DISC) formation, subsequently suppressed caspase-8 mediated extrinsic apoptosis. Moreover, Flot-2 reduced the mitochondria mediated intrinsic apoptosis by regulating the Bcl-2 family and suppressing cytochrome C release from mitochondria to cytosol. Flot-2 further modulated the common apoptosis pathway and inhibited caspase-3 activation via up-regulating the members in the inhibitor of apoptosis (IAP) family. Last, Flot-2 interacted with cav-1 and limited its expression. Taken together, we found that Flot-2 protected cells from Fas induced apoptosis and counterbalanced the pro-apoptotic effects of cav-1. Thus, Flot-2 played crucial functions in cellular homeostasis and cell survival, suggesting a differential role of individual raft proteins.
PLoS ONE 10/2013; 8(10):e77519. DOI:10.1371/journal.pone.0077519 · 3.23 Impact Factor
"GST-fusion constructs of FRS2 were constructed by subcloning into vector pGEX-4T-1 (GE Healthcare), and FRS2-His was obtained by subcloning into pSCherry2 (Eurogentec, Seraing, Belgium). The flot-1 (NM_022701.2) constructs in pGBKT7 vector have been described previously , . Rat flotillin-1 in pET41a was generated by PCR cloning. "
[Show abstract][Hide abstract] ABSTRACT: Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway.
PLoS ONE 01/2012; 7(1):e29739. DOI:10.1371/journal.pone.0029739 · 3.23 Impact Factor
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