There are two homologous members of the flotillin family which have been designated as flotillin-1/reggie-2 and flotillin-2/reggie-1, owing to their almost simultaneous discovery by two separate research groups [1, 2]. In 1997, these proteins were described under the name “reggies”, since they were found to be upregulated during the regeneration of retinal ganglion cells of the goldfish, following an optic nerve lesion . In the same year, Bickel
Biological membranes are complex structures composed of numerous proteins and lipids. As shown in Figure 1A, flotillin-1 and flotillin-2 are both constitutively associated with sphingolipid and cholesterol enriched membrane microdomains, known as lipid rafts [1, 3-5]. Especially flotillin-1 is often considered as a
The first description of flotillin-2 actually took place in 1994, when the group of Madeleine Duvic cloned an N-terminally truncated version of flotillin-2 from human epidermal keratinocytes and named it epidermal surface antigen (ESA) . However, in later studies by the same group, it turned out that the true ESA (ECS-1 antigen) is actually distinct from flotillin-2 . The flotillin-2 cDNA originally cloned by Schroeder
Both flotillin-1 and flotillin-2 show a constitutive expression in almost all mammalian cell types and are highly conserved from fly to man (reviewed in 16). Furthermore, flotillin-like proteins are also present in bacteria , plants , fungi and metazoans but are absent in yeast and
2. Modifications, oligomerization and trafficking of flotillins
Flotillin-1 and flotillin-2 are ubiquitously expressed proteins and exhibit a relatively complementary tissue distribution. Northern blot analysis showed a high tissue-specific expression of flotillin-1 in murine brain, fat and heart tissue . High protein levels of flotillin-1 and flotillin-2 were found, e.g. in murine lung, thymus, bladder, cerebrum and hypothalamus tissue lysates (24, our unpublished results). Additionally, it has been shown that the expression of flotillin-2 increases during differentiation and myogenesis of C2C12 cells, whereas flotillin-1 increases during 3T3-L1 cell adipogenesis  and osteoclastogenesis . In contrast, during differentiation of rat pheochromocytoma PC12 cells, no change in the expression of flotillin-1 or flotillin-2 was observed .
The subcellular localization of flotillin-1 and flotillin-2 is as diverse as their tissue expression pattern and seems to be cell type specific. In glandular cervical cancer HeLa cells, PC12 cells, human hepatocellular carcinoma HepG2 cells, Chinese hamster ovary (CHO) cells and human breast cancer MCF-7 cells, flotillins mainly reside at the inner leaflet of the plasma membrane and to a minor extent in vesicular structures. A major localization in vesicular structures is found in astrocytes, the human HaCaT keratinocyte cell line and mouse embryonic fibroblast (MEF) cells. Colabeling experiments and immunogold electron microscopy studies identified the vesicular structures as endosomes and lysosomes in astrocytes, Jurkat, PC12 and HeLa cells [13, 14, 27-30]. In line with the previous results, endogenous labeling of flotillin-2 in MCF10A cells shows a more prominent localization at the plasma membrane, whereas flotillin-1 is found in vesicular structures in non-confluent cells. However, with increasing confluency and advanced differentiation, flotillin-1 translocates to the plasma membrane (our unpublished results), a feature already observed in differentiating mouse 3T3-L1 cells . Rajendran
Total internal reflection fluorescence (TIRF) microscopy revealed that flotillin-1 and flotillin-2 exhibit a vesicular cycling at the plasma membrane , which seems to be independent of clathrin and caveolin [9, 13, 34, 35]. Microtubule disruption in HeLa cells results in the accumulation of flotillin-1/flotillin-2 positive vesicles in the cytosol , while the same treatment in murine neuroblastoma N2a cells did not affect flotillin-2 localization . Manipulation of the actin cytoskeleton might also have a cell type dependent influence on the mobility of flotillins. In FRAP (fluorescence recovery after photobleaching) experiments, an increased lateral mobility of flotillin-2 after disruption of the actin cytoskeleton with cytochalasin D in N2a cells was observed, while treatment with the actin stabilizing compound jasplakinolide reduced the lateral mobility. On the contrary, the use of cytochalasin D in HeLa cells had no significant effect on flotillin-2 mobility . Recently, Affentrager
Localization and trafficking of flotillins in the Golgi apparatus is still controversial. Nevertheless, evidence suggests that the involvement of Golgi in flotillin trafficking is also a cell type-specific process. In non-differentiated PC12, normal rat kidney (NRK), CHO and HeLa cells, flotillin-1 localizes to the Golgi complex [44-46]. Also in rat brain tissue sections, flotillin-1 was found in the Golgi . In contrast, Morrow
Chemokines, growth factors and certain proteins can affect the localization and the trafficking of flotillin proteins. In Satb2-negative neuronal cells, flotillin-1 becomes more clustered in response to semaphorin 3a treatment, which acts as a potent guidance cue presented to cortical axons
Posttranslational modifications of eukaryotic proteins are often important cues for localization, trafficking and membrane association of proteins . Two important modifications are N-myristoylation and S-palmitoylation. N-myristoylation is an irreversible
acylation reaction at an N-terminal glycine residue, which is catalyzed by the enzyme N-myristoyl transferase . S-palmitoylation is a reversible reaction, in which the acyl group is transferred to cysteine residues by palmitoyl acyltransferases, referred to as the DHHC family [52, 53]. In some proteins, e.g. Src-related protein tyrosine kinases, which are both myristoylated and palmitoylated, the myristoylation reaction is a prerequisite for the palmitoylation to occur . Our experiments with overexpressed flotillin-2 constructs in HeLa cells showed that flotillin-2 is myristoylated at Gly2 and palmitoylated at Cys4, Cys19 and Cys20, and Cys4 seems to be the major palmitoylation site . The myristoylation at Gly2 is essential for membrane targeting of flotillin-2, as the mutant flotillin-2 Gly2Ala remains fully soluble upon overexpression. Furthermore, the mutation of the cysteine residues to alanine and the concomitant loss of palmitoylation sites resulted in more soluble flotillin-2 mutants . In the study by Li
Oligomeric proteins are prevalent in nature and comprise approximately one third of all known proteins. From the perspective of protein evolution, oligomerization may be of advantage for a functional control such as allosteric regulation . In the case of flotillin proteins, oligomerization is an essential feature for membrane raft association, endocytosis and EGF signaling [30, 56]. Flotillin-1 and flotillin-2 form both homo- and hetero-oligomers [4, 5, 23, 34], and Solis
Results of the Stuermer group have shown that ectopic expression of the presumed oligomerization domain of flotillin-2 comprising the amino acids 184 to 390 in Jurkat T cells interferes with function of flotillins in these cells and prevents the formation of macrodomains upon crosslinking of GM1 by cholera toxin . Hence, this fragment, termed R1EA, most likely prevents the proper oligomerisation of flotillins, thus functioning in a trans-negative manner. Upon overexpression of R1EA, insulin-like growth factor induced neurite outgrowth was abrogated in N2a neuroblastoma cells, and axon differentiation was impaired in hippocampal neurons . In addition, in R1EA expressing cells, recruitment of CAP/ponsin, which interacts with flotillin-1, to focal contacts was affected, resulting in imbalanced activation of Rho family GTPases Rac1 and Cdc42. Interestingly, focal adhesion kinase (FAK) activity was increased by R1EA expression. However, other signaling pathways such as ERK1/2, PKC, and PKB signals were unaltered . In line with this, previous studies have shown that upon growth factor stimulation of PC12 cells, protein kinase Pyk2, ubiquitin ligase Cbl and the CAP homolog ArgBP-2 are recruited to lipid rafts . Moreover, cholesterol depletion resulted in a diminished neurite outgrowth, suggesting involvement of membrane rafts . It has been proposed that ArgBP-2 associates with Cbl and Pyk2 via its SH3 domain and recruits them to lipid rafts by interacting with flotillin-1 through its SoHo domain . These findings point towards a vital role of flotillins in cytoskeletal remodeling and neurite growth at least in cultured cells. However, neither the flotillin-1 knockout mouse model  nor flotillin-2 deficient mice (our unpublished data) show a major neuronal phenotype. It thus remains to be clarified if flotillins are only required under conditions of neuronal regeneration or if they are generally important for the physiology of neurons in mammals.
3. In search of the molecular function of flotillins
As can be expected from such highly conserved and ubiquitous proteins, flotillins have been shown to play a role in a large number of vital cellular processes. Their original discovery as “reggies” suggested a function in neuronal regeneration in the goldfish [2, 45], which has been later verified by further studies from the same group [57, 60, 61]. However, the regenerative capacity of the optic nerve is poorly conserved in mammals, making a generalization of the regenerative function of flotillins in the nervous system of mammals difficult. Recent studies have confirmed the relevance of flotillins for axon regeneration in zebrafish as well as in mammalian cell cultures. Apparently, downregulation of flotillins by flotillin specific morpholinos in the zebrafish resulted in a massive reduction in the number of regenerating axons . Since the role of flotillins in neuronal regeneration has recently been directly reviewed, we here omit a long discussion of the topic. Instead, the reader is referred to the recent review of C. Stuermer .
Flotillins also appear to be involved in membrane trafficking processes such as endocytosis and phagocytosis [13, 34, 42], and they have been suggested to be involved in the regulation the actin cytoskeleton [4, 11, 57]. One emerging important function of flotillins, especially of flotillin-1, is the regulation of membrane receptor signaling, e.g. through mitogen activated protein kinases (MAPK) [27, 30, 43, 56, 63-67]. Below, we have summarized the data on the function of flotillins in some of these processes.
Flotillins. in endocytosis
During their discovery, flotillins were described to be associated with membrane rafts [1, 24]. First findings suggested that flotillins would be integral membrane proteins of the cave-like invaginations called caveolae [1, 24], but these findings have later been disputed by several other studies [3, 9, 13, 34]. However, the raft association of flotillins appears to be important for their function, and flotillin-1 has even been suggested to define a novel non-clathrin, non-caveolin endocytosis pathway that would operate via membrane rafts [13, 34]. In eukaryotic cells, uptake of membrane bound molecules such as receptors can take place by means of several different pathways. The best characterized and the most common pathway for endocytosis is mediated by clathrin coated vesicles which are formed at the plasma membrane by the assembly of a coated pit which then forms a clathrin coated vesicle. However, recent research from several groups has revealed that endocytosis can be divided into numerous pathways, many of which do not require clathrin, and are thus designated as clathrin-independent. For a general introduction on endocytosis, the reader is referred to these excellent reviews [68-70]. Some factors that regulate the clathrin independent pathways have been identified in the recent years, and it is clear that small GTPases such as Cdc42, Rho A or Arf (ADP ribosylation factor) family are involved, each determining their own endocytic pathway. The role of flotillins in endocytosis has recently been reviewed in detail , and we thus here only present a brief overview and a summary of the very recent studies not included in the review of Otto and Nichols.
Early after their discovery, flotillins were shown to associate and colocalize with GPI-anchored proteins. A role in the internalization of GPI-anchored proteins such as CD59 and of the glycosphingolipid GM1 has been demonstrated [13, 34, 72]. Flotillins have also been suggested to be involved in the endocytosis of the GPI-linked Nodal coreceptor cripto . Although flotillins are affirmed regulators of cholera toxin subunit B endocytosis [13, 72], which is mediated by the binding of the toxin to GM1, no evidence for a direct role of flotillins in the endocytosis of the Shiga toxin or the plant toxin ricin was found . However, flotillins were shown to control other trafficking steps of these toxins, most likely a retrograde transport step towards Golgi/ER. Intriguingly, depletion of flotillins resulted in a higher degree of mannosylation of ricin, and the toxicity of both ricin and Shiga toxin was increased . Thus, flotillins are important factors in controlling not only the endocytosis but also other steps of the cellular trafficking of various molecules.
In addition to GPI-anchored proteins, flotillins have recently been shown to participate in the protein kinase C (PKC) induced endocytosis of neurotransmitter receptors such as the dopamine transporter DAT (also designed as solute carrier family 6, member 3 or SLC6A3) and glial glutamate transporter, GLT1 . PKC was shown to phosphorylate flotillin-1 at a Ser residue, and this phosphorylation was required for the endocytosis of DAT . Recent publications have revealed a role for flotillins in the endocytosis of the Alzheimer amyloid precursor protein (APP), the amyloidogenic processing of which requires its endocytosis . In addition, Niemann-Pick C1-like 1 (NPC1L1), an important mediator of the uptake of dietary cholesterol, relies on flotillins for its endocytosis, and depletion of flotillins drastically reduces cholesterol uptake in cultured cells . Curiously, APP, NPC1L1 and DAT are all endocytosed by means of clathrin mediated endocytosis, whereas flotillins as raft associated proteins have been rather suggested to define a non-clathrin endocytosis pathway, and there is very little colocalization between clathrin and flotillins . Thus, it is possible that rather than directly mediating raft dependent endocytosis through flotillin microdomains, flotillins might exert their function by first facilitating the uptake of some cargo molecules into rafts and their efficient clustering, but the act of endocytosis may only take place after transfer of the cargo into clathrin coated pits. Hence, it will be important in the future to study the exact relationship of flotillins and clathrin dependent endocytosis.
We have recently shown that flotillins are involved in EGFR/MAP kinase signaling, and siRNA mediated downregulation of flotillin-1 results in severe impairment of both EGFR phosphorylation and MAPK signaling . Since flotillins also become phosphorylated upon EGFR signaling , it would not be surprising if they were involved in EGFR endocytosis, which has been shown to take place by means of both clathrin dependent and raft mediated endocytosis [77, 78]. However, according to our recent findings, flotillin-1 depletion does not significantly affect the EGF-induced endocytosis of EGFR . Furthermore, EGFR is efficiently endocytosed even in cells depleted of both clathrin and flotillin-1 (our unpublished data). Interestingly, recent findings from the Stuermer group have implicated that depletion of flotillin-2/reggie-1 may affect the endocytosis of EGFR . This finding is somewhat counter intuitive as flotillin-2 depletion induces a significant depletion of flotillin-1 as well, and flotillin-2 knockdown cells express only minor amounts of both flotillins. Flotillin-1 knockdown cells in turn still exhibit substantial flotillin-2 expression. Thus, these results imply that the flotillin-2 homo-oligomers that are still present in flotillin-1 knockdown cells may be capable of supporting normal EGFR endocytosis, whereas in the absence of flotillins, EGFR uptake from the plasma membrane is impaired. However, one also has to keep in mind some important methodological differences between our study  and that of Solis
One important open question on the role of flotillins in endocytosis is if the flotillin-mediated endocytosis of various cargo molecules is dependent on the GTPase dynamin or not, since the studies have resulted in contradictory results, depending on the cell system used and cargo molecules studied. Since flotillins are endocytosed from the plasma membrane as a result of EGF stimulation  but appear not to directly affect EGFR endocytosis , they may in some cases also themselves represent cargo molecules whose cellular localization needs to be regulated during signaling. It is possible that depending on the endocytic process, cargo and stimulus, mechanistically different pathways are used. For a further discussion of this idea, please refer to the recent review of Otto and Nichols . Another important question is how the modifications (such as phosphorylation) and oligomerization of flotillins affect the trafficking of their putative cargo molecules. This topic is discussed more in detail below in the chapter “
Functions. of flotillins in cell migration and adhesion
Already the discovery of flotillins by using antibodies directed against cell surface proteins led to the assumption of a functional role of flotillin proteins in cell adhesion [2, 82]. Up to date, much has been speculated about this attribute, but only a few studies actually showed an involvement of flotillins in the establishment and maintenance of cell-cell and cell-matrix adhesion structures. One of the first functional hints was obtained in the study by Hoehne
One major type of cell-cell contact in multicellular organisms is the adherens junction. The core of adherens junctions is composed of proteins of the cadherin and catenin family . Insights into a functional association of flotillins with this cell-cell contact type were obtained in the study of Bodrikov
The complexing of the junctional proteins p120-catenin (p120ctn) and N-Cadherin at cell-cell contact sites was shown to occur in cholesterol rich membrane domains in mouse C2C12 cells . A similar observation was made in differentiated human colon adenocarcinoma HT-29 cells, in which the interaction of E-Cadherin with p120ctn preferentially takes place in lipid rafts. A stable knockdown of flotillin-1 resulted in a diffuse localization of p120ctn and an impaired recruitment of p120ctn and E-Cadherin in lipid rafts. In addition, flotillin-1 depletion decreased the enzymatic activity of ALP and DPPIV during the enterocytic differentiation process . This study revealed a novel function of flotillin-1 in a lipid raft mediated maturation of adherens junctions and in intestinal cell differentiation.
Other studies implicated that flotillins seem to be of functional relevance for cell-matrix adhesion processes. We showed that the transient knockdown of flotillin-2 in HeLa cells impaired cell spreading on fibronectin, as compared to cells transfected with control siRNA . Overexpression of flotillin-2-EGFP enhanced cell spreading and induced filopodia-like protrusions in an expression-level dependent manner in several epithelial cells lines, e.g. HaCaT cells [4, 30]. The observed enrichment of flotillin-2 fusion proteins at cell-cell contact sites and the induction of filopodia suggest an active link to the cytoskeleton. Indeed, use of
Role. of flotillins in EGF receptor and mitogen activated protein kinase signaling
In the last 12 years, various studies have shown that flotillin-1 and flotillin-2 are physically and functionally linked to signal transduction pathways of several membrane receptors, especially receptor tyrosine kinases such as the insulin receptor (IR), the nerve growth factor (NGF) receptor, tropomyosin-related kinase A (TrkA), the polymeric Immunoglobulin E (IgE) receptor, fibroblast growth factor receptor (FGFR) and the epidermal growth factor receptor (EGFR) [27, 30, 63, 65, 89, 90]. However, the mechanisms how flotillins may regulate the signaling process have not been dissected in detail. Only recently, we have shed light on the molecular mechanisms of flotillin-1 function in EGFR/MAPK signaling [27, 89]. We showed that flotillin-1 actually plays a dual role by first regulating the clustering of EGFR in the plasma membrane after EGF stimulation and, later on during signaling, by functioning as a MAPK scaffolder protein . We also demonstrated that flotillin-1, but not flotillin-2, interacts with Fibroblast Growth Factor Receptor Substrates 2 and 3 (FRS2 and FRS3)  which have previously been shown to be involved in MAPK signaling [91-93]. (See Figure 3).
Depletion of flotillin-1 in HeLa cells results in a decreased tyrosine phosphorylation of the EGFR especially at Tyr1173 already after a short EGF stimulation, and the reduced phosphorylation persists up to a time point where no phosphorylation of EGFR can be detected . By means of TIRF microscopy, we showed that the formation of EGFR clusters at the cell surface upon EGF stimulation is impaired after flotillin-1 knockdown. This fits well with the findings of Schneider
contact . This model is well in line with our earlier findings showing that upon EGF stimulation, the molecular mass of flotillin oligomers increases, most likely due to their coalescence into larger complexes .
We also observed a reduced downstream signaling towards the canonical MAPK pathway and Protein Kinase B/Akt after stimulation with EGF or basic fibroblast growth factor (bFGF) upon flotillin-1 knockdown , implicating that flotillin-1 is not only involved in EGFR but also in FGFR signaling. Similar results with EGF have later on been obtained by Solis
Flotillin-1 depletion also resulted in reduced activation of the MAPK pathway, especially of ERK kinases, which are required for transmitting the MAPK signal into the nucleus . Although flotillin-1 depletion clearly impairs an early step, EGFR clustering, during the signaling, which alone would be sufficient to inhibit MAPK signaling, this appears not to be the only reason for the reduced ERK activity. Incubation of the cells with phorbol myristate acetate (PMA) results in the activation of PKC, which in turn can active the cRAF kinase in the absence of an upstream EGFR signal [95, 96]. Our attempt to overcome the downstream ERK inhibition by PKC-mediated activation of cRAF resulted in normal activation of cRAF and MEK kinases, whereas the ERK phosphorylation could not be rescued in flotillin-1 knockdown cells . This demonstrates that flotillin-1 must play a role not only at the plasma membrane but also at the level of ERK activation. Accordingly, we showed that flotillin-1 directly interacts with several MAPK pathway proteins, namely cRAF, MEK1 and ERK2. This interaction was not dependent on the MAPK scaffolder Kinase Suppressor of Ras (KSR), since flotillin-1 was found to coprecipitate with the MAPK component also in KSR1 depleted cells . Thus, these results suggest that flotillin-1 is indeed a
The role of flotillins in EGFR signaling is supported by our recent findings in stable flotillin-1 and flotillin-2 knockdown human breast cancer MCF-7 cells (Figure 4). Comparable to HeLa cells, in MCF7 cells the stability of flotillin-1 is strongly dependent on flotillin-2, since the knockdown of flotillin-2 resulted also in a loss of flotillin-1. However, knockdown of flotillin-1 had no effect on flotillin-2 expression. Immunoblot analysis showed that the amount of EGFR is increased in flotillin-1 and flotillin-2 knockdown cells (Figure 4A). This effect is only visible in stable knockdown cells and not in transient knockdown cells, most likely due to an adaption of the EGFR amount to overcome the decreased EGFR-MAPK signaling in flotillin knockdown cells. We have previously observed this effect also in HeLa cells and other cell lines (our unpublished results). The EGFR, also known as ErbB1/Her1, is one of the four known members of the ErbB/Her protein-tyrosine kinase family (reviewed in ). Analysis of the protein amount of ErbB2/Her2 and ErbB3/Her3 showed no detectable
change in the protein level (data not shown), which indicates that flotillin depletion specifically affects the protein level of the EGFR. In general, the endogenous EGFR is not detectable by immunofluorescence in MCF-7 cells, whereas in flotillin-1 depleted MCF-7 cells, a prominent staining at the plasma membrane is visible (Figure 4B). Stimulation with EGF for 120 minutes results in a translocation of the EGFR in vesicular structures in these cells. This is in line with our previous results in HeLa cells, indicating that the endocytosis of the EGFR is not affected by flotillin-1 depletion .
Flotillins. and other receptor tyrosine kinases
The first experimental evidence for a functional role of flotillins in insulin signaling was presented by the Saltiel group . They demonstrated that flotillin-1 forms a ternary complex with c-Cbl-associated protein (CAP) and the E3 ubiquitin protein ligase Cbl upon insulin stimulation (Figure 5). This was shown to be important for lipid raft association, downstream signaling of the CAP-Cbl complex and finally for glucose uptake by the glucose transporter GLUT4 in 3T3-L1 adipocytes . Previously, the interaction of CAP with Cbl was shown to be independent of insulin stimulation
Receptor tyrosine kinases of the Trk receptor family regulate various cellular processes, such as proliferation and differentiation, through several cellular signal cascades. Three types of Trk receptors exist in humans, including TrkA, TrkB and TrkC, which have varying affinities to neutrotrophins such as NGF. TrkA is specifically activated by NGF (reviewed in [104, 105]. Downstream signaling by TrkA seems to be dependent on the lipid raft association of TrkA. Upon NGF stimulation, TrkA is recruited to lipid rafts in PC12 cells, a process dependent on its association with CAP, which in turn binds to flotillin-1 . The deletion of the CAP SoHo domain abolishes the lipid raft association of TrkA and further downstream signaling events such as ERK1/2 phosphorylation . (See Figure 5)
Recently, we have demonstrated that flotillin-1 directly interacts
Animal. models for the function of flotillins
Flotillins are highly conserved proteins which are assumed, mainly on the basis of cell culture studies, to play multifaceted roles in developmental regulation. Hoehne
Flotillins are also implicated to regulate axon regeneration in a zebrafish model. As a consequence of gene duplication in fish, there are two copies of each flotillin gene. To understand the essential role of flotillins in axon regeneration, specific morpholino (Mo) antisense nucleotides were used to transiently downregulate flotillin-1a, flotillin-1b and flotillin-2a in zebrafish . Interestingly, the number of retinal ganglion cells (RGC) undergoing axon regeneration was reduced by 69% in flotillin-Mo treated eyes after 7 days . Recently, a zebrafish model was employed to study the role of flotillins in cholera intoxication. Consequently, flotillin-1 and flotillin-2 knockdown prevented cholera intoxication in zebrafish embryos. Moreover, cholera toxin subunit B (CTxB) colocalized with flotillins at the plasma membrane and in endosomes in COS1 cells . Thus, these results suggest flotillins as essential elements for axon regeneration and indicate a novel cholera intoxication
Cell migration is an essential mechanism during the development and maintenance of multicellular organisms. It requires the constant turnover of cell-matrix adhesion structures, e.g., integrin-based focal contacts. Results presented by Ludwig
4. Functional significance of phosphorylation and oligomerization of flotillins
Recent data from several groups have revealed flotillins as important regulators of vital cellular processes such as signaling and endocytosis. One major challenge in the future will be to dissect how the oligomerization and the covalent modifications of flotillins affect their function during these processes. Endocytosis of flotillins appears to be influenced by upstream signaling [30, 49, 56] and requires clustering of flotillin-1 and flotillin-2 molecules [34, 56]. We have shown that EGF stimulation results in uptake of flotillins hetero-oligomers from the plasma membrane, and that hetero-oligomerization appears to be required for the endocytosis [30, 56]. Mutation of a single Tyr residue, Tyr163, in flotillin-2 is sufficient to prevent the EGF-induced endocytosis of flotillins , and this mutation impairs the hetero-oligomerization of flotillin-2 with flotillin-1 . Flotillins are phosphorylated by the Src family kinases cSrc and Fyn [4, 49], but the direct role of Tyr phosphorylation in the endocytosis is still under debate. Our data show that inhibition of cSrc kinase, the activity of which is necessary for flotillin-2 phosphorylation , does not impair the endocytosis of flotillin-2 , whereas the mutation of the single Tyr residue Y163 does . Thus, it is more likely that this mutation affects the oligomerization of flotillins and thereby also endocytosis. However, phosphorylation of flotillins by the Fyn kinase appears to be sufficient to induce endocytosis of flotillins , suggesting that the Src family kinases might play different roles in terms of flotillin function. It is also important to keep in mind that PKC has recently been shown to phosphorylate flotillin-1 in Ser315, and that this phosphorylation is required for the endocytosis of the dopamin transporter DAT . Thus, phosphorylation seems to be an important direct or indirect regulator of flotillin trafficking.
Not only phosphorylation but also covalent modifications with fatty acids myristate and palmitate take place in the case of flotillins [3, 4, 53]. Mutation of Gly2 prevents the myristoylation and the subsequent palmitoylation of flotillin-2, thus also inhibiting its membrane association and rendering the protein soluble . Recently, the palmitoyl transferase DHHC5 was shown to palmitoylate flotillin-2 . Interestingly, growth factor withdrawal, which induces neuronal differentiation, resulted in rapid degradation of DHHC5 , implicating that palmitoylation might be important for the well established function of flotillins in neurite outgrowth in cultured neuronal cells. Since the soluble, non-modified flotillin-2 with Gly2Ala mutation seems to function as a dominant negative protein during cell-matrix adhesion , and successive removal of the three palmitoylation sites affect the membrane association of flotillin-2 , the fatty acid modifications, especially the reversible palmitoylation, are most likely important for the regulation of flotillin function. Accordingly, we have observed that flotillin-2 expression is necessary for the membrane association of flotillin-1, which is not myristoylated and only palmitoylated in a single residue. Thus, ectopic expression of flotillin-1 in flotillin-2 depleted cells results in a soluble protein that is not capable of exerting its function (our unpublished data).
Our recent findings revealed flotillin-1 as a MAPK regulator . However, no data are so far available on the role of flotillin modifications such as phosphorylation in the regulation of MAPK signaling. Intriguingly, another SPFH/PHB family protein, prohibitin-1, has been shown to regulate an earlier step during MAPK signaling, namely signaling by the small GTPase Ras and RAF kinase [114, 115]. Although the most evident cellular localization of prohibitin is the inner mitochondrial membrane, various studies have now shown that prohibitin is a substrate of several cytoplasmic kinases such as Akt, and it becomes Tyr phosphorylated upon insulin stimulation of cells [116, 117]. Thus, at least a fraction of prohibitin must be found in another cellular localization such as the plasma membrane. Importantly, prohibitin has been shown to be essential for cell migration that is induced by the Ras-Raf-MAPK pathway , and very recent findings show that phosphorylation of prohibitin, which takes place in a raft domain, is necessary for the Ras-dependent enhancement of cellular metastasis , verifying the results of Rajalingam
5. Flotillins in human diseases
Role. of flotillins in cancers
Early findings already indicated that flotillins may be involved in the regulation of the actin cytoskeleton. Overexpression of flotillin-2 was found to result in induction of filopodia-like protrusions and changes in the cytoskeleton [4, 11]. Furthermore, flotillins have been shown to be important for the polarization of various immune cells such as T cells and neutrophils [29, 32, 33, 37, 110, 111], and to be involved in the regulation of the activity of various small GTPases that control e.g. the remodeling of the actin cytoskeleton [36, 57]. In fact, flotillin-2 has even been suggested to directly interact with actin , although this has not been verified in further studies. Thus, there are very strong implications that flotillins are required for a proper regulation of the actin cytoskeleton, which is necessary for e.g. the control of cell migration. Consequently, taking also into account the numerous reports on the involvement of flotillins in signaling, it is evident that flotillins are critical factors that regulate the cell physiology and cell growth. Thus, the findings showing that flotillins are involved in some types of cancer are well in accordance with this function.
The strongest evidence for the role of flotillins in cancer to date comes from studies on human malignant melanoma. The direct association of flotillins with human cancers was shown in 2004 when the group of M. Duvic demonstrated that flotillin-2 is highly expressed in various melanoma cell lines, and that flotillin-2 expression correlated with the progression of human melanoma . Furthermore, the thickness of primary melanoma lesions, so-called Breslow depth, which is a prognostic marker for the malignancy of a melanoma lesion, showed a correlation with the increased flotillin-2 expression . Importantly, benign, non-malignant cells could be converted into tumorigenic, metastatic ones upon overexpression of flotillin-2 . Furthermore, flotillin-2 was shown to be associated with the thrombin receptor PAR1, the expression of which was dependent on the level of flotillin-2 protein .
Although flotillins evidently play a role in cancer and their overexpression is associated with malignancy, very little is known about their transcriptional regulation. Flotillin-2 was shown to be a transcriptional target of the p53 family transcription factors p63 and p73 but not of p53 . We have recently addressed this question more in detail and shown that flotillins are transcriptionally regulated by the Extracellularly Regulated Kinase signaling and by the Retinoid X Receptor (Banning
Implications. of flotillins in Alzheimer’s Disease and other neurodegenerative disorders
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder whose most prominent pathological hallmarks include neuronal loss, neurofibrillary tangle formation and senile plaques throughout the brain cortex. The major component of the senile plaques is the amyloid β peptide (Aβ) comprising of 40-42 residues, which is generated by a proteolytic cleavage of a large transmembrane precursor protein named amyloid β precursor protein (APP). APP is subjected to proteolytic processing at several sites by a group of proteases called secretases. Sequential cleavage of APP by β-secretase (β–site APP cleaving enzyme or BACE) and γ-secretases generates Aβ and is hence termed the amyloidogenic pathway (reviewed in [123-126]). APP can also be processed through a non-amyloidogenic pathway, in which the α-secretase cleaves within the Aβ sequence, thereby precluding the formation of Aβ. It has been strongly implicated that the amyloidogenic processing of APP takes place in lipid rafts, which is supported by the findings showing that APP can be detected in rafts isolated from cortical neurons . Besides APP, other AD associated raft resident proteins include BACE-1 , endoproteolytic fragments of presenilin-1 (PS-1) and the presenilins [128, 129]. Compelling evidence from various research groups shows that the flotillin family of proteins is partially associated with AD pathology and trafficking [47, 130, 131]. Flotillins are considered as classic raft markers in neuronal tissue since they show an abundant expression in the brain, typically in pyramidal neurons and astrocytes. Kokubo
Parallel studies in brain sections from AD, Down syndrome and non-demented subjects with plaques showed enhanced flotillin-1 expression with the progression of AD . Further insight was provided by Rajendran
Flotillins and other lipid raft-associated proteins might also be involved in other human neuronal diseases. The study by Shin
Parkinson’s disease (PD) is one of the most common, progressive neurological disorders. The pathological hallmark of PD is the extensive loss of dopamine secreting cells within the substantia nigra, particularly affecting the ventral component of the pars compacta (reviewed in ), which can lead to uncontrolled muscle contraction and movement, dementia, depression and anxiety. Flotillin-1 staining in rat brain sections showed a prominent labeling in the cytoplasm of catecholamine releasing cells (dopamine, norepinephrine, and epinephrine) and an upregulated gene expression of flotillin-1 in the substantia nigra of Parkinson’s brain by RT-PCR . Although the molecular function of flotillin-1 in Parkinson’s disease is still not known, this study implies that flotillin-1 may be involved in the neuronal changes occurring during Parkinson’s disease.
Flotillins. and pathogens
During evolution, many pathogens have developed mechanisms to use host-cell lipid rafts as signaling and entry platforms to escape the host immune system (reviewed in [140, 141]]. Several studies demonstrated that lipid rafts are often used as entry sites for bacteria, e.g.,
Not only bacteria use lipid raft domains as preferred entry sites in host-cells, but also various enveloped and non-enveloped viruses are dependent on an intact lipid raft structure and the presence of cholesterol for successful virus entry (reviewed in ]. In the case of the retrovirus human immunodeficiency virus-1 (HIV-1), flotillin-1 was suggested to play a role of in the cellular response to the viral infection since the protein level of flotillin-1 was increased in peripheral blood mononuclear cells after treatment with the HIV-1 component gp120 . The primary HIV-1 receptor CD4 and the co-receptor CCR5, both of which are important for HIV-1 host-cell entry, were also shown to associate with flotillin-1 containing lipid rafts in monocytes . Recently, flotillin-1 was also identified as an interaction partner of the overexpressed rhesus monkey TRIM5α . TRIM5α is a member of tripartite motif (TRIM) protein family, and plays an important role during host cell defense against retroviruses such as HIV-1 especially in old world monkeys, but not in humans (reviewed in ). The function of flotillin-1/TRIM5α interaction is still unknown, as flotillin-1 knockdown or overexpression did not affect HIV-1 transduction efficiency, leading to the suggestion that flotillin-1 at least plays no role in the post-entry restriction of HIV-1 in fetal rhesus monkey kidney (FRhK4) cells.
Flotillins. and diabetes mellitus
Flotillins seem to be of functional relevance in insulin signaling (see above) and the metabolic disease diabetes mellitus, as first demonstrated by Baumann
Lipid rafts also play a role in diabetic xerostomia, or dry mouth. This disease, when associated with diabetes mellitus, is caused by degenerative changes in the salivary glands leading to increased infectious conditions in the oral cavity and in the long-range aggravates the risk of atherosclerosis and cardiovascular diseases (reviewed in ). Wang
It has become evident that flotillins play a key role in the regulation of cellular signaling and membrane trafficking. Although flotillins are not essential for life in metazoans, including mammals, as shown by the recent studies in flotillin knockout mice, they appear to be extremely important for many processes in cultured cells. This would suggest that especially mammals are capable compensating for the loss of flotillins by upregulating the expression of other proteins and thus overcoming the defects in e.g. signaling and cell adhesion. This feature is observed in both our flotillin-2 knockout mice and cultured cells with a constitutive flotillin knockdown (our unpublished data). In the future, it will be important to study what the molecular mechanisms of the compensation of loss of flotillin function might be. It will also be of major interest to study if the original function of flotillins in neuronal regeneration can also be observed in the mammalian knockout model. Furthermore, the functional significance of flotillin modifications can now be studied in the context of mammalian organisms when knockout/knock-in mice with specific mutations become available. Thus, the recent development of flotillin-1 and flotillin-2 knockout mice has brought the studies of flotillin functions to a new exciting era, and important data on the physiological function of flotillins can be expected in the near future.
The research in the Tikkanen lab is supported by the German Research council DFG (Grants Ti291/6-1 and Ti291/6-2), the Von Behring-Röntgen Foundation and the state of Hessen (LOEWE program “Non-neuronal Cholinergic Systems“), whose support is gratefully acknowledged.