Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Flotillin-1 (FLOT1)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_645

Synonyms

Historical Background

Flotillin-1 was originally identified in 1997, when Bickel et al. analyzed caveolin-rich membrane fractions from mouse lung and isolated a 45 kDa protein that was microsequenced. Degenerate primers were designed from the peptide sequences and used for PCR of mouse lung cDNA. The PCR-product was then used as a probe for screening of a 3T3-L1 adipocyte cDNA library. The characterized sequence showed similarity to the ESA protein. An antipeptide antibody identified a 47 kDa protein on Western blots. Because this protein was detected in floating, caveolin-rich, Triton X-100-insoluble membrane fractions, it was termed “flotillin-1.” The similar ESA protein (47% identity) was renamed as “flotillin-2.” Also in 1997, Schulte et al. identified and characterized two proteins that were induced in regenerating fish retinal ganglion cell axons after injury and thus were named “reggie-1” and “reggie-2.” In 1998, Lang et al. cloned the rat reggie proteins and found that rat reggie-1 is 99% identical to human flotillin-2, while rat reggie-2 is 98% identical to human flotillin-1. Reggie-2/flotillin-1 was identified at the plasma membrane of neurons and nonneuronal cells in noncaveolar micropatches coclustering with its homologue reggie-1/flotillin-2 and GPI-anchored cell adhesion molecules. These micropatches localize along the axon and in lamellipodia and filopodia of growth cones. In 1999, Volonté et al. presented co-immunoprecipitation data that identified flotillin-1, flotillin-2, caveolin-1, and caveolin-2 in one complex. This led to the hypothesis that flotillins are resident components of caveolae; however, this hypothesis could not be verified by several groups, as reviewed in Langhorst et al. (2005). In 2001, Stuermer et al. identified clusters of reggies/flotillins with GPI-anchored proteins and Fyn kinase in noncaveolar plasma membrane microdomains of neurons and astrocytes. These microdomains had a diameter of less than 100 nm. In Jurkat cells, cross-linking of Thy-1 induced colocalization of Thy-1, reggie/flotillin proteins, GM1, the T cell receptor, and Fyn kinase, suggesting the formation of a raft-associated signal transduction center. Reggies/flotillins were also found in lysosomes, suggesting that the signaling complex becomes subject to degradation, as reviewed in Langhorst et al. (2005). More details on the historical background can be found in Salzer and Prohaska (2011).

Protein Structure and Domain Organization

Mammalian flotillin-1 is a highly conserved, 48 kDa monotopic integral membrane protein of 427 amino acids that is divided into two large domains of similar size, an N-terminal “stomatin/prohibitin/flotillin/HflC/K” (SPFH) domain, also known as “prohibitin homology domain” (PHB) or “band 7 domain,” and a C-terminal “Flotillin domain” (Morrow and Parton 2005; Rivera-Milla et al. 2006; Babuke and Tikkanen 2007). The N-terminus of flotillin-1 contains two hydrophobic regions, residues 10–35 and 134–151, respectively, that are thought to be associated with the cytoplasmic face of the bilayer but not spanning it. Both flotillins are not cleaved when proteinases are added to intact cells (Morrow and Parton 2005). It is not known if these hydrophobic regions adopt “hairpin loops” similar to caveolin-1 or the related stomatin. Such structures appear less likely in flotillin-1 due to interspersed charged and hydrophilic residues. For additional hydrophobic interaction, the N-terminal region contains three cysteine residues, Cys-5, Cys-17, and Cys-34 that are likely to be palmitoylated (Rivera-Milla et al. 2006). In Vero cells, palmitoylation of Cys-34 was shown to be essential for plasma membrane targeting along a Golgi-independent pathway and for providing the necessary affinity for raft association (Morrow et al. 2002). In differentiated 3 T3-L1 adipocytes, mutation of Cys-34 to Ala did not affect plasma membrane targeting, whereas the second hydrophobic region was found to be essential for plasma membrane localization (Liu et al. 2005). Intact Cys-34 was, however, suggested to play a role in lipid raft association. It is possible that cell type-specific differences are the causes of these differing results. The structure of the SPFH domain of the mouse homologue flotillin-2 was determined by NMR analysis in solution (PDB ID: 1WIN; MMDB ID: 30602) and revealed a compact ellipsoid structure built from four β-strands on one side and four α-helices on the other. The crystal structure of the SPFH domain of an archaeal stomatin is surprisingly similar and both structures can be superposed very well (PDB ID: 3BK6; MMDB ID: 62565), showing the high conservation of this domain from archaea to mammals. This evolutionary conservation suggests an important function, but this is currently not clear. Because many SPFH-domain proteins are lipid raft associated, it is possible that this domain itself mediates raft association (Morrow and Parton 2005). The crystal structure shows that the membrane-facing surface of this domain contains hydrophobic patches that would favor interactions with membrane lipids. Moreover, the interaction with cortical actin of one SPFH-domain α-helix has been predicted and identified (Rivera-Milla et al. 2006). The C-terminal flotillin domain is characterized by three contiguous blocks of coiled-coil heptad motifs that contain multiple GluAla repeats as well as charged residues at the heptad positions 2, 3, and 5 (Rivera-Milla et al. 2006). This coiled-coil domain is predicted to form dimers, trimers, and oligomers. Using chemical cross-linking, it was determined that flotillins form homo- or heterotetramers as building blocks of larger oligomeric complexes (Solis et al. 2007). Moreover, it was shown that flotillin-1 is stabilized by flotillin-2 in hetero-oligomeric complexes. Near the C-terminal end, a conserved PDZ3-binding consensus was recognized that could be involved in regulating oligomerization of both flotillins (Rivera-Milla et al. 2006). A schematic model of the flotillin/reggie structural and functional domains (Rivera-Milla et al. 2006; Stuermer 2010) is shown in the flotillin-2 entry.

Interactions

Flotillin-1 interacts with a variety of proteins in different tissues. It appears that most interactions described for flotillin-1 actually involve the hetero-oligomers of flotillin-1 and flotillin-2; however, the relevance of this hetero-oligomerization has been recognized only recently and was ignored in the past. A list of described interaction partners is shown in Table 1.
Flotillin-1 (FLOT1), Table 1

Flotillin-1 interaction partners

Interacting protein

Location

Function

Remarks

Flotillin-2

Plasma membrane lipid rafts

Hetero-oligomerization

Flotillin-2 is essential for flotillin-1 stability

Fyn kinase

Lipid rafts

Tyr phosphorylation

Induces endocytosis

Lyn kinase

Basophilic leukemia cells

Enhances kinase activity

Involved in IgE-receptor signaling

NPC1L1

Plasma membrane lipid rafts

Cholesterol endocytosis and transport

Involves clathrin heavy chain and AP2

CAP/ponsin

Plasma membrane lipid rafts

Insulin signaling via c-Cbl, CrkII, and C3G

Binding of flotillin-1 to the SoHo domain of CAP

Focal adhesion sites

Regulation of actin cytoskeleton

Vinexin α

Cell adhesion sites

Integrin-mediated cell adhesion

Binding to the SoHo domain of vinexin α

ArgBP2

Lipid rafts

Regulation of the actin cytoskeleton

Binding to the SoHo domain of ArgBP2

PTOV1 (Prostate tumor overexpressed gene 1)

Plasma membrane rafts and nucleus

Mitogenic complex

Recruitment to the nucleus in S phase

Aurora B kinase

Nucleus

Mitotic regulator

Flotillin-1 regulates Aurora B abundance and activity

Neuroglobin

Lipid rafts

Protects neurons from hypoxia

Inhibits Gα proteins

NMDAR ( N-methyl- d-aspartate receptor)

Neuronal plasma membrane

Glutamate-binding calcium channel

NR2A/B subunits bind to SPFH domain

Gαq

Lipid rafts

Gαq-induced p38 MAPK activation

Depends on Src-family kinases

APP (Amyloid precursor protein)

Lipid rafts

APP recruitment and processing in lipid rafts

APP binds to flotillin-1 C-terminus

Myocilin

Eye

Mutated in primary open-angle glaucoma

Binding to myocilin C-terminal domain

TRIM5α

Cytoplasmic bodies

Antiviral tripartite motif family

Dependent on cholesterol

LGI3 (Leucine-rich glioma inactivated 3)

Endosomes

Vesicle trafficking and endosome formation

Regulates APP trafficking

PSGL-1 (P-selectin glycoprotein ligand 1)

Plasma membrane

Involved in the polarization of neutrophils

Both flotillins and PSGL-1 accumulate in uropods

Arginase

Erythrocyte membrane

Involved in NO signaling

Upregulates arginase activity

ABCA1 (ATP-binding cassette transporter A1)

Plasma membrane rafts, phagosomes

Cellular cholesterol efflux, phagocytosis

Complex with ABCA1 and syntaxin 13

Syntaxin 13

Plasma membrane rafts, endosomes, phagosomes

Phagocytosis

Complex with ABCA1 and syntaxin 13

PrP c (Prion protein)

Plasma membrane, lipid-rich vesicles

Clustering at cell-cell contacts, focal adhesions

Complex with both flotillins

DAT (Dopamine transporter)

Lipid rafts

PKC-triggered endocytosis of DAT-flotillin-1 complex

Flotillin-1 residues Cys-34 and Ser-315 are essential

EGF-receptor

HeLa cells

Activation of EGFR

Regulation of MAPK pathway activation

cRAF, MEK1, ERK, and KSR1

HeLa cells

MAP kinase signaling complex

Flotillin-1 knockdown leads to inactivation of ERK1/2

BACE1

Endosomes

Endosomal sorting

Binding of flotillins to the di-Leu sorting motif of BACE1

Gamma-catenin

Adherence junctions

Cell-cell adhesion

Binding of flotillins to the Armadillo domains 6-8

Cadherin complexes

Cell-cell junctions

Cell-cell adhesion

Binding to large cadherin complexes and associated F-actin

Rab11a and SNX4

Recycling endosomes

Regulation of the recycling compartment

Direct interaction with Rab11a and SNX4

FRS2 (fibroblast growth factor receptor substrate 2)

Plasma membrane

Regulation of receptor tyrosine kinase signaling

Flotillin-1 binds to the phosphotyrosine binding domain of FRS2

cSrc kinase

Mitochondria

Phosphorylation at Tyr56 and Tyr149

Phosphorylation required for interaction with CxII complex and reduction of ROS

Desmoglein-3

Desmosomes of human keratinocytes

Desmosomal adhesion complex

Binding to the cytoplasmic tails

IGF-1R

Plasma membrane

IGF-1R activation in response to IGF-1

Palmitoylated flotillin-1 is a major regulatory component needed for lipid raft targeting of IGF-1R

Kv2.1

Cerebellar granule neurons (CGN),

HEK-293 cells

Flot-1 decreases Kv2.1 current amplitude

Flot-1 may regulate neural excitability and development by modulation of ion channels

EphrinB2

Xenopus oocytes and embryos, plasma membrane

Regulation of ephrinB2 levels through ADAM10

Required for neural tube morphogenesis in Xenopus

Flotillin-1 preferentially forms hetero-oligomers with flotillin-2 by interacting with the C-terminal coiled-coil domains (Solis et al. 2007). Hetero-oligomerization is essential for flotillin-1 stability, because flotillin-2 depletion induces flotillin-1 degradation by proteasomes (Solis et al. 2007). Flotillin-1 defines a specific, clathrin-independent endocytic pathway in mammalian cells; however, hetero-oligomerization with flotillin-2 is required for this clathrin- and caveolin-independent endocytosis (Frick et al. 2007). This process is regulated by Fyn kinase, which phosphorylates Tyr-160 in flotillin-1 and Tyr-163 in flotillin-2 (Riento et al. 2009). The region between the two N-terminal hydrophobic domains of flotillin-1 appears to be necessary for the interaction with Fyn (Liu et al. 2005). An important interaction of flotillin-1 with the multifunctional adapter protein c-Cbl-associated protein (CAP) and c-Cbl in a ternary complex was found to induce a second signaling pathway required for insulin-stimulated glucose transport via GLUT4 (Baumann et al. 2000). An outline of these insulin signaling pathways is shown in Fig. 1. The flotillin-1 interaction site on CAP was identified as the sorbin homology (SoHo) domain. This domain is also present in vinexin α and ArgBP2, enabling these proteins to interact with flotillin-1 in the same way. All three members of this SoHo family of adaptor proteins contain three SH3 domains within their C-termini. The functional significance of these interactions can be seen in the recruitment of signaling proteins to flotillin-specific lipid rafts (Babuke and Tikkanen 2007; Stuermer 2010).
Flotillin-1 (FLOT1), Fig. 1

Role of flotillin-1/reggie-2 in insulin signaling. In adipocytes and muscle cells, insulin receptor activation triggers the classic signaling cascade through phosphatidylinositol 3-kinase, PI3K. A second signaling route proceeds through membrane rafts and involves a ternary complex c-Cbl/CAP/reggie-2. Both pathways mediate the recruitment of the glucose transporter GLUT4 to the plasma membrane and uptake of glucose into the cell (Figure reprinted from Babuke and Tikkanen (2007) with permission from Elsevier)

Flotillin-1 and -2 were found to associate with Niemann-Pick C1-like 1 (NPC1L1) in a complex with clathrin heavy chain (CHC) and AP2 subunit μ2 (Ge et al. 2011). The localization of this complex is cholesterol-dependent. Cholesterol depletion causes transport of flotillins and NPC1L1 to the plasma membrane, whereas cholesterol replenishment leads to endocytosis of both. Thus, flotillins play a role in NPC1L1-mediated cellular cholesterol uptake and regulation of lipid levels. Knockdown of flotillin-1, flotillin-2, or both, dramatically decreased NPC1L1 endocytosis and cholesterol uptake and reduced the association between the CHC-AP2 complex and NPC1L1. Because the knockdown of CHC had no effect on the flotillin-NPC1L1 association, it appears that flotillins are required for recruitment of the CHC-AP2 complex to NPC1L1 and thus act upstream to mediate the internalization of NPC1L1 and cholesterol (Ge et al. 2011). The NPC1L1-binding drug Ezetimibe was found to disrupt the flotillin-NPC1L1 complex. This study reveals an important functional role of the flotillins as mediators of cholesterol absorption via flotillin-NPC1L1-generated, cholesterol-enriched membrane microdomains (Ge et al. 2011). Flotillins were also found to interact with the tetrameric N-methyl- d-aspartate receptor (NMDAR), an ionotropic glutamate receptor, with the subunits NR1, NR2A, and NR2B. The distal C-terminus of NR2B interacted with both flotillin-1 and flotillin-2, whereas the C-terminus of NR2A bound only flotillin-1. Both flotillins were found to colocalize with NMDARs in hippocampal neurons (Swanwick et al. 2009). Flotillins also interact with the guanine nucleotide-binding protein Gq subunit alpha (Gαq), independent of the Gαq nucleotide-binding state (Sugawara et al. 2007). The N-terminal regions of both flotillins are essential for Gαq binding. Knockdown of flotillins attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK). Because activation of MAPK depends on  Src-family kinases and intact lipid rafts, these results suggest that flotillins mediate Gαq-induced MAPK activation through  Src-kinases in lipid rafts (Sugawara et al. 2007). Flotillins form molecular complexes with EGFR in an EGF/EGFR kinase-independent manner, while flotillin-1 forms a complex with cRAF, MEK1, ERK, and KSR1 (kinase suppressor of RAS) suggesting that it may constitute a novel scaffolding protein in MAP kinase signaling (Amaddii et al. 2012). Conversely, flotillin-1 knockdown results in impaired activation of EGFR and MAP kinases. In leukocytes, flotillins interact with P-selectin glycoprotein ligand-1 (PSGL-1), also known as SELPLG or CD162 (Rossy et al. 2009). When neutrophils are activated, flotillins rapidly form caps and later accumulate in the uropods of the polarized cells. While PSGL-1 accumulates in the uropods concomitantly, other uropod components such as  CD43 and ERM-proteins accumulate at a slower pace. Flotillins accumulate in uropods even in the absence of PSGL-1, indicating that PSGL-1 accumulation depends on flotillins (Rossy et al. 2009). This study was extended by using neutrophils from the flotillin-1 knockout mouse (Ludwig et al. 2010). Flotillin-2 was affected by the lack of flotillin-1, showing only weak, uniform staining of the plasma membrane and absence from detergent-resistant membranes. Importantly, the recruitment of flotillin-1-deficient neutrophils toward the chemoattractant fMLP was strongly reduced. Immunoisolation of tagged flotillin-2 followed by mass spectrometry of co-isolated proteins revealed the association of flotillins with cytoskeletal proteins, such as α- and β-spectrin and  myosin IIa (Ludwig et al. 2010). The authors conclude that this association is important for neutrophil migration, uropod formation, and regulation of  myosin Iia. Recently, flotillin-1 was found to be required for protein kinase C (PKC)-regulated internalization of the dopamine transporter (DAT) and the glial glutamate transporter EAAT2 (Cremona et al. 2011). Flotillin-1 associates with DAT in plasma membrane lipid rafts and is required to maintain DAT in rafts. For internalization of the DAT-flotillin-1 complex, palmitoylation of flotillin-1 at Cys-34 and phosphorylation at Ser-315 is essential (Cremona et al. 2011); however, flotillins may rather modulate the functional association of DAT with plasma membrane rafts than mediate DAT endocytosis (Sorkina et al. 2013). Palmitoylation of flotillin-1 at Cys-34 is essential for plasma membrane localization of flotillin-1 and IGF-1R. The IGF-1-dependent depalmitoylation and repalmitoylation of flotillin-1 sustains tyrosine kinase activation of the plasma-membrane-targeted IGF-1R. Thus, flotillin-1 palmitoylation is a new mechanism by which the intracellular localization and activation of IGF-1R are controlled (Jang et al. 2015). Flotillin-1 changes the endosomal sorting of BACE1 by binding to the dileucine motif on the cytoplasmic tail of BACE1 (John et al. 2014). Both flotillins directly interact with gamma-catenin, specifically with the Armadillo domains 6-8, and thereby regulate epithelial cell-cell adhesion (Kurrle et al. 2013). Flotillin-1 and -2 are also partners of large cadherin complexes; they are localized to cell-cell junctions in a cadherin-dependent manner and associate with F-actin bundles (Guillaume et al. 2013). The role of flotillins in intercellular adhesion was recently reviewed (Bodin et al. 2014). The interaction of flotillins with Rab11a and SNX4 corroborates cell biological evidence that flotillins are regulating the tubulovesicular recycling compartment (Solis et al. 2013). Flotillin-1 and the cbl-associated protein/ponsin (CAP) are interacting partners of the fibroblast growth factor receptor substrate 2 (FRS2), which is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. Due to overlapping binding domains, CAP and flotillin-1 appear to compete for binding to FRS2, thus regulating receptor tyrosine kinase signaling (Tomasovic et al. 2012). Flotillin-1 is phosphorylated by mitochondrial c-Src at residues Tyr56 and Tyr149, and these phosphorylations are required for interaction with the respiratory complex CxII and prevention of ROS production (Ogura et al. 2014). In human keratinocyte desmosomes, flotillins interact with the cytoplasmic tails of desmogleins, particularly desmoglein-3, while loss of flotillin expression results in weakened desmosomal adhesion (Vollner et al. 2016). When flotillin-1 was coexpressed with the Voltage-gated potassium channel subunit Kv2.1 of the delayed rectifier outward potassium channel (IK), the amplitude of K+ current was downregulated. Co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) proved the direct interaction of flotillin-1 and Kv2.1 suggesting a novel role for flotillin-1 as a scaffolding protein affecting the regulation of membrane ion channel function (Liu et al. 2016). Flotillin-1 interacts with ephrinB2, which plays a key role in Xenopus development and neural tube morphogenesis. Absence of flotillin-1 leads to loss of ephrinB2 due to cleavage by the metalloprotease ADAM10. Thus, flotillin-1 regulates ephrinB2 levels through ADAM10 (Ji et al. 2014). More details on flotillin-1 interactions are described in Salzer and Prohaska (2011).

Cellular and Subcellular Localization

Both flotillins are expressed ubiquitously in virtually all tissues and cell types but have been studied particularly in neurons, hematopoietic cells, and adipocytes (Babuke and Tikkanen 2007; Stuermer 2010). Gene expression analyses by quantitative PCR of human and mouse (Mouse Genome Informatics MGI:1100500) tissues have shown flotillin-1 expression in all tissues analyzed. In cells, flotillin-1 is localized to the plasma membrane and internal vesicles such as Golgi vesicles, lipid droplets, recycling endosomes, multivesicular bodies, exosomes, late endosomes, lysosomes, phagosomes, and centrosomes, respectively (Langhorst et al. 2005). Moreover, it can translocate to the nucleus when interacting with the mitogenic protein PTOV1 (Prostate tumor overexpressed gene 1) or with the mitotic regulator Aurora B kinase. At the plasma membrane of neuronal cells, flotillin-1 is found in micropatches along the axons, in filopodia and lamellipodia of growth cones, and at synapses (Stuermer 2010). Flotillin hetero-oligomers accumulate at cell-cell contacts of many cell types (Langhorst et al. 2005; Stuermer 2010). These hetero-oligomers in plasma membrane microdomains are eventually endocytosed (Frick et al. 2007; Babuke et al. 2009; Riento et al. 2009) and apparently recycle back to the plasma membrane. Endocytosis takes place either in a clathrin- and caveolin-independent way (Riento et al. 2009) or in association with clathrin and NPC1L1 (Ge et al. 2011). In hematopoietic cells such as the B cell lines Raji and Ramos, T leukemic Jurkat cells, and the promonocytic cell line U937, both flotillins are asymmetrically localized in stable, preassembled platforms of the plasma membrane, to which signaling components are recruited on activation (Rajendran et al. 2003). An example for this extraordinary phenomenon is shown in Fig. 2.
Flotillin-1 (FLOT1), Fig. 2

Flotillin preassembled platforms. Lipid raft clustering recruits raft-associated signaling molecules to the preassembled platforms and activates T cells. Non-cross-linked T cells (left) show uniform distribution of GM1 (Cholera toxin-B, red), CD3, Thy-1, and CD55 (green) but very polarized expressions of flotillins-1 and -2. GM1 cross-linking (right) by Cholera toxin-B (red) induces patching and recruits signaling molecules CD3 and Thy-1, but not CD55, (green) to preassembled flotillin platforms (Figure reprinted from Rajendran et al. (2003) with permission from PNAS)

In T lymphocytes, one stimulus for T cell receptor (TCR) complex association with the preassembled platform is the clustering of GPI-anchored proteins by antibody cross-linking. Thy-1 or cellular prion protein (PrP) cross-linking leads to their selective association with the flotillin platform. This induces the MAPK pathway and calcium signaling, resulting in recruitment of the TCR-CD3 complex. Thus, the flotillin membrane domains serve as platforms for cluster formation, signaling, cytoskeletal rearrangement, and recruitment of  CD3 (Stuermer 2010). In chemoattracted neutrophils, flotillin microdomains accumulate rapidly in the uropod (Rossy et al. 2009) due to specific interaction with the cytoskeletal proteins α- and β-spectrin and  myosin II (Ludwig et al. 2010). Uropod formation in chemokine-stimulated T-cells requires coexpression of flotillin-1 and -2. These hetero-oligomers are present in both resting and stimulated human T-cells and cluster upon cell activation, thus contributing to the structuring of the T-cell uropod (Baumann et al. 2012). More details on flotillin-1 localizations are described in Salzer and Prohaska (2011). The role of flotillin membrane microdomains in signaling, cytoskeletal regulation, and endocytosis was reviewed and discussed recently (Otto and Nichols 2011).

Regulation of Expression and Activity

In fish and mammals, both flotillins are upregulated in retinal ganglion cells during axon regeneration after optic nerve section, as reviewed in Langhorst et al. (2005) and Stuermer (2010). Flotillin-1 stability depends on flotillin-2 expression (Solis et al. 2007). Conversely, flotillin-2 expression correlates with flotillin-1 expression (Ludwig et al. 2010). Both flotillins coassemble at the plasma membrane in roughly equal amounts and act together to generate microdomains (Frick et al. 2007), which are endocytosed after Fyn-dependent tyrosine phosphorylation together with GPI-anchored proteins (Riento et al. 2009). Mutation of flotillin-1 residue Tyr-160 and flotillin-2 residue Tyr-163 prevents Fyn-induced internalization and leads to reduced uptake of GPI-anchored proteins. Thus, the uptake of flotillin microdomains is established as a tyrosine-kinase-regulated, endocytic process (Riento et al. 2009). Deletion of flotillin-1 apparently results in complete destruction of flotillin microdomains (Ludwig et al. 2010). The localization of the flotillin-1 and -2 complex with NPC1L1 and clathrin heavy chain and AP2 μ2 is regulated by the cholesterol level in the plasma membrane. Cholesterol depletion causes transport to the plasma membrane, whereas cholesterol replenishment leads to endocytosis. Thus, flotillins play a role in NPC1L1-mediated cellular cholesterol uptake and regulation of lipid levels (Ge et al. 2011). The role of flotillins in endocytosis and endocytic trafficking of membrane-bound cargo was extensively reviewed (Meister and Tikkanen 2014).

For the PKC-triggered endocytosis of the lipid raft-associated dopamine transporter-flotillin-1 complex, palmitoylation of flotillin-1 at Cys-34 and phosphorylation at Ser-315 is essential (Cremona et al. 2011). In many biological processes, flotillins act as platforms associated with membrane receptors or transporters and the cytoskeleton to mediate signaling and endocytosis, as described above and reviewed in Morrow and Parton (2005), Langhorst et al. (2005), Babuke and Tikkanen (2007), Stuermer (2010), and Salzer and Prohaska (2011).

Flotillin-1 plays a direct role during the early and late phase of EGF signaling by activating EGFR and MAP kinases (Amaddii et al. 2012). This activating effect is also found in association with other receptor tyrosine kinases and may lead to formation of various forms of cancer, as reviewed by Banning et al. (2014). A large number of clinical studies have shown that tumors that are highly expressing flotillins have a tendency to generate metastases resulting in poor prognosis for patient survival. Flotillin-1 also plays a regulatory role in Xenopus embryo neural tube morphogenesis. Knockdown of flotillin-1 leads to reduced expression of ephrinB2, which is essential for neural tube closure. Reduction of ephrinB2 is caused by cleavage of the ectodomain by the metalloprotease ADAM10 (Ji et al. 2014).

In a learning-related paradigm study, when the mollusk Aplysia californica was treated with serotonin, flotillin-1 was found upregulated in the abdominal ganglia. Mice trained in the Morris water maze showed flotillin-1 upregulation in hippocampal tissue. Thus, a translational approach, from invertebrates to rodents, led to the identification of flotillin-1 as an evolutionary-conserved memory-related protein (Monje et al. 2013).

Summary

Flotillin-1, also known as reggie-2, is a highly conserved, oligomeric, lipid-raft-associated, integral membrane protein of 48 kDa that is widely expressed in different tissues and cell types. It is associated with the cytosolic side of the plasma membrane and endosomes due to hydrophobic interaction and palmitoylation. Flotillin-1 is mainly found in a hetero-oligomeric complex with its homologue flotillin-2, also known as reggie-1. Both flotillins/reggies are members of the SPFH (stomatin/prohibitin/flotillin/HflC/K) superfamily, more recently termed PHB (prohibitin homology domain) superfamily. Hetero-oligomerization is required for endocytosis of flotillin microdomains either in a clathrin- and caveolin-independent way, such as Fyn kinase-regulated endocytosis of GPI-anchored proteins, or in a clathrin-dependent way, such as cholesterol uptake mediated by Niemann-Pick C1-like 1 (NPC1L1). In neurons, flotillins/reggies control axon growth and growth cone elongation by coordinating the assembly of signaling complexes that regulate cortical cytoskeleton remodeling. Expression levels of flotillin-1 in mollusk and mouse ganglions after learning-related tasks show that flotillin-1 is an evolutionary-conserved memory-related protein upregulated in implicit and explicit learning paradigms. In neutrophils, flotillin interaction with cytoskeletal components is required for normal migration and uropod formation. Flotillin microdomains thus constitute scaffolding platforms interacting with signaling components, cytoskeleton, and/or membrane proteins in various cellular processes.

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Max F. Perutz Laboratories (MFPL)Medical University of ViennaViennaAustria