The two members of the flotillin/reggie protein family, flotillin- 1/reggie-2 (flot-1) and flotillin-2/reggie-1 (flot-2) are associated with specific membrane microdomains enriched in cholesterol and sphingolipids, also called rafts (For a review, see [21,22]). Their membrane association is mediated by palmitoylation (both flot-1 and flot-2) and myristoylation (flot-2 only), but neither protein contains a transmembrane domain [23,24]. Flotillins are widely expressed and well conserved between species, but their molecular function has remained somewhat enigmatic. Flotillins have been shown to participate in various signaling processes, including insulin and EGF receptor signaling [25,26,27,28], in endocytosis [29], phagocytosis [30] and cell adhesion [27]. In addition, a role in neuronal regeneration has been suggested in goldfish and zebrafish [31,32], but the evidence for such a role in mammalian animal models is lacking. Our earlier results show that flot-2 becomes Tyr phosphorylated by Src kinases and is endocytosed together with flot-1 in EGF stimulated cells [25,27]. Although functional implications for flotillins in diverse cellular processes have been accumulating, in many cases the exact molecular mechanisms of flotillin action and the interaction partners remain to be identified. One of the known interaction partners of flotillins is the Cbl-associated protein (CAP), also known as Ponsin, which is an adaptor protein containing multiple SH3 (Src homology 3) domains and one or two sorbin homology (SoHo) domains [33]. During insulin receptor signaling, CAP is responsible for recruiting the ubiquitin ligase Cbl into membrane rafts by means of interacting with flotillin-1 [26]. Furthermore, CAP has been shown to be localized in focal adhesions and to mediate processes requiring actin remodeling [34]. Our recent findings also suggest that CAP is involved in signaling and becomes phosphorylated by the Abl kinase [35]. Here, we have identified the PTB-domain containing adaptor proteins FRS2 and FRS3 as novel interaction partners of flot-1. Membrane association and cellular localization of FRS2 was shown to be partly dependent on flot-1. We observed an interaction between flot-1 and FRS2 in mouse tissues. Intriguingly, FRS2 appears to be more Tyr phosphorylated in flot-1 knockdown cells, indicating that the interaction with flot-1 regulates the downstream signaling of FRS2. Moreover, since flot-1 binds to the PTB domain of FRS2, it competes for the binding with the FGF receptor. Furthermore, flot-1 is necessary for the signaling induced recruitment of FRS2 into lipid rafts. In addition to flot-1, FRS2 was also found to interact with CAP, which has been linked to flot- 1 during insulin signaling [26]. The binding domains of these three proteins overlap, and thus flot-1 and CAP compete for the binding to FRS2. Here, we have dissected the putative role of these proteins during receptor tyrosine kinase signaling. Results In search of novel interaction partners of flotillins, we performed a yeast two-hybrid (Y2H) screen of a human brain library using rat flot-1 as a bait, in which several clones containing a partial sequence of FRS3 were obtained (data not shown). Since FRS3 shows a high homology to the more ubiquitously expressed FRS2, we tested the possibility of a direct interaction of flot-1 with FRS2 and characterized the interacting domains in a Y2H assay (Fig. 1A). The constructs used for this are shown in Suppl. Fig. S1A. Interaction was detected as growth of the transformed yeast colonies on nutrient deficient plates and blue colour (Fig. 1A). Full- length (FL) flot-1 was found to interact with the FL FRS2 and the PTB domain of FRS2, whereas no interaction was detected with the C-terminal part of FRS2. Interestingly, flot-2 did not interact with any of the FRS2 fragments. We also attempted to produce FRS2 Interaction with Flotillin-1 and CAP deletion fragments of flot-1 (see Suppl. Fig. S1A), but the C- terminal parts flot-1-CT (amino acids 226–428) and flot-1-CC (327–428) displayed autoactivity, resulting in blue colonies without any interaction partner. Furthermore, flot-1-NT (amino acids 1– 253) was poorly expressed, preventing any conclusions about its interaction with FRS2. However, a deletion mutant of flot-1 missing 100 amino acids from the C-terminal end (Flot-1- STOP328) was unable to interact with any of the FRS2 fragments, indicating that the interaction domain may reside in the far C- terminus of flot-1. Suppl. Fig. S1B shows the expression in the yeast of the constructs used in Fig. 1A and a test for autoactivation in the yeast. We next used GST-pulldown analysis to verify the interaction between FRS2 and flot-1 (Fig. 1B–C). FL FRS2, the PTB and CT domains were expressed as GST fusions and immobilized on glutathione beads (Fig. 1B). All three variants of FRS2 were capable of binding substantial amounts of flot-1 from HeLa cell lysates. To demonstrate a direct interaction, we used bacterially expressed, purified proteins. FL Flot-1-GST and Flot-1-CT-GST were immobilized on sepharose beads and tested for their binding to His-tagged FRS2 (Fig. 1C). FRS2 was clearly bound by FL and even more by the CT of flot-1. Thus, our yeast two-hybrid and pulldown data suggest that FRS2 interacts with flot-1 but not with flot-2, and the interaction involves both the PTB domain and C- terminus of FRS2 and the C-terminal part of flot-1. To verify the interaction between flot-1 and FRS2 in vivo, we performed coimmunoprecipitation from mouse tissues (Fig. 1D). FRS2 was immunoprecipitated using same amounts of homoge- nized mouse tissues which express the highest amounts of FRS2 (brain, liver and kidney; see right part of Fig. 1D for relative expression levels). Coimmunoprecipitation of flot-1 with FRS2 was seen from all three tissues, whereas no precipitation of flot-1 was detected when an isotype-matched control antibody was used. To study the possible colocalization of flot-1 with FRS2 in animal cells, we chose Hep3B cells which express high amounts of endogenous FRS2 and flotillins. Immunofluorescence staining of FRS2 and flotillins revealed that in serum-grown Hep3B cells, FRS2 was mainly localized at the plasma membrane with some diffuse and vesicle-like staining in the cytoplasm (Fig. 2). Flot-1 (Fig. 2, upper row) and flot-2 (Fig. 2, lower row) were both detected at the plasma membrane, where they colocalized with FRS2. However, little if any colocalization was detected in the endosomes, which contained high amounts of flotillins. Our previous findings have shown that upon knockdown of flot- 1 in HeLa cells, EGF-stimulated activation of MAP kinase signaling is inhibited (Amaddii et al., under revision). Furthermore, it has been shown that overexpression of FRS2 results in a dose- dependent enhancement of ERK activation upon EGF signaling [15]. Thus, we tested if overexpression of FRS2 would be able to compensate for the MAP kinase activation defects upon receptor tyrosine kinase stimulation in flot-1 knockdown cells. However, since the best characterized function of FRS2 lies within FGF receptor (FGFR) signaling, where it also mediates an increase of MAP kinase activation, we here used FGF stimulation instead of EGF. Phosphorylation of Akt and ERK2 after 5 min of FGF stimulation were found to be reduced in flot-1 knockdown cells as compared to the control cells (Fig. 3A), consistent with our previous findings (Amaddii et al., under revision). Overexpression of FRS2-CFP did not result in correction of the diminished phosphorylation of these proteins after FGF stimulation in flot-1 knockdown cells. Expression of FRS2-CFP and the knockdown efficiency of flot-1 were verified by means of Western Blot (Fig. 3A). We observed a considerable shift in the gel motility of FRS2 after FGF treatment, most likely due to phosphorylation, in both PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29739