The lipid raft microdomain-associated protein reggie-1/flotillin-2 is expressed in human B cells and localized at the plasma membrane and centrosome in PBMCs.
Reggie-1/flotillin-2 is a plasma membrane-associated cytoplasmic protein, which defines non-caveolar raft microdomains. Reggie-1/flotillin-2 is enriched in detergent insoluble (TX100) membrane fractions (DIG), co-localizes with activated GPI-linked proteins and the fyn-kinase in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. In T cells activated by crosslinking the GPI-linked protein Thy-1 or by crosslinking the ganglioside GM1, reggie-1/flotillin-2 co-localizes with the T cell receptor. To determine whether reggie-1/flotillin-2 is also expressed in B cells, primary B cells from human blood and cell lines representing the developmental stages of pro, pre, mature and plasma B cells were analyzed by Western blotting, RT-PCR and immunofluorescence. Here, we show that reggie-1/flotillin-2 is expressed throughout B cell development, as well as in primary B cells, purified by cell sorting. On non-activated mature B cell Raji cell line we found reggie-1/flotillin-2 are exclusively in the detergent (TX100) insoluble membrane fractions that are staining positive for the raft marker GM1. Immunofluorescence microscopy showed that reggie-1/flotillin-2 is localized at the plasma membrane and marks intracellular spots in PBMCs. Confocal co-localization studies showed that reggie-1/flotillin-2 is associated with the plasma membrane, and the centrosomes (microtubule organizing centers) in these PBMCs. Comparison of reggie-1/flotillin-2 cDNA sequences with the genomic sequence database allowed us to determine the exon/intron structures in mouse and human. The gene organizations are highly conserved suggesting an important function of reggie-1/flotillin-2. Since reggie/flotillin proteins co-cluster with the T cell receptor and fyn kinases upon T cell stimulation, our findings of reggie-1/flotillin-2 in B cells suggest a similar role in B cell function.
Solomon
Samuel
S
Faculty of Sciences, Department of Biology, University of Konstanz, Germany.
Masilamani
Madhan
M
Rajendran
Lawrence
L
Bastmeyer
Martin
M
Stuermer
Claudia A O
CA
Illges
Harald
H
D000818:Animals; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D015153:Blotting, Western; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D018385:Centrosome / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D029941:Fish Proteins / Q000096:biosynthesis* / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000276:immunology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D029941:Fish Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C106354:flotillins