The Stomatin/Prohibitin/Flotillin/HflK/C Domain of Flotillin-1 Contains Distinct Sequences That Direct Plasma Membrane Localization and Protein Interactions in 3T3-L1 Adipocytes

Abstract

Flotillin-1 is a lipid raft-associated protein that has been implicated in various cellular processes. We examined the subcellular distribution of flotillin-1 in different cell types and found that localization is cell type-specific. Flotillin-1 relocates from a cytoplasmic compartment to the plasma membrane upon the differentiation of 3T3-L1 adipocytes. To delineate the structural determinants necessary for its localization, we generated a series of truncation mutants of flotillin-1. Wild type flotillin-1 has two putative hydrophobic domains and is localized to lipid raft microdomains at the plasma membrane. Flotillin-1 fragments lacking the Nterminal hydrophobic stretch are excluded from the lipid raft compartments but remain at the plasma membrane. On the other hand, mutants with the second hydrophobic region deleted fail to traffic to the plasma membrane but are instead found in intracellular granule-like structures. Flotillin-1 specifically interacts with the adaptor protein CAP, the Src family kinase Fyn, and cortical F-actin in lipid raft microdomains in adipocytes. Furthermore, CAP and Fyn associate with different regions in the N-terminal sequences of flotillin-1. These results furthered our understanding for how flotillin-1 can function as a molecular link between lipid rafts of the plasma membrane and a multimeric signaling complex at the actin cytoskeleton.

Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain

“…1), suggesting the maintenance of an important function in both types of structural element. Recent reports suggest that reggies/flotillins interact with F-actin [9], and that small and hydrophobic residues within a helices from actin-binding proteins are likely to interact with actin [34]. We analyzed the a helices within .…”

“…Although they were initially thought to be components of caveolae [4], subsequent biochemical and microscopic characterization revealed that these 48-kDa proteins are distinctive non-caveolar markers of detergent-resistant microdomains (DRMs) [3,6]. In humans, reggie-1 and reggie-2 polypeptides show similar length, primary sequence composition and biochemical profiles, with a conserved protein architecture at the N terminus containing one myristoylation site (Gly-2, only in reggie-1) and four palmitoylation sites (Cys-5, Cys-19, Cys-20, Cys-38), which are thought to assist in membrane association and lipid raft targeting [7][8][9]. Reggies/flotillins do not have typical transmembrane domains, but harbor two conserved hydrophobic stretches that allow potential interactions with the inner leaflet of the plasma membrane, and possibly protein-protein interactions [7,8].…”

“…At LM and EM levels, reggies/flotillins co-cluster with various specific surface proteins and intracellular signal transduction components like the cellular prion protein (PrP C ), Thy-1, activated cell adhesion molecules (CAMs), Src family kinases (e.g. lck and fyn) and interact with actin cytoskeleton-associated protein (vinexins, CAP/ponsin and ArgBP2) [6,9,10,[13][14][15][16]. The induction of signal cascades is demonstrated by the elevation of intracellular Ca 2+ and MAP kinase phosphorylation during PrP C capping in T cells [10], by actin cytoskeletal changes [9,13,15,16] and by relocation of glucose transporter 4 to the plasma membrane in adipocytes [9].…”

“…lck and fyn) and interact with actin cytoskeleton-associated protein (vinexins, CAP/ponsin and ArgBP2) [6,9,10,[13][14][15][16]. The induction of signal cascades is demonstrated by the elevation of intracellular Ca 2+ and MAP kinase phosphorylation during PrP C capping in T cells [10], by actin cytoskeletal changes [9,13,15,16] and by relocation of glucose transporter 4 to the plasma membrane in adipocytes [9]. Additional lines of evidence such as co-immunoprecipitation, cross-linking and microscopy confirm the idea that reggie/ flotillin microdomains play an important role in cell-cell recognition, adhesion and signaling events [reviewed in refs.…”

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

“…In addition, ␤1-integrin recruitment can lead to an activation of Rac1 in HeLa cells (O'Toole et al, 2011). Since reggies interact with Rab11a and Src (Liu et al, 2005;Solis et al, 2012;Stuermer et al, 2001Stuermer et al, , 2004 it is possible that reggies at vesicles and at the plasma membrane coordinate the interactions, underlying FA formation and turnover. Or FAK activation is caused by defective integrin recycling in consequence of loss of reggie-1 or both.…”