The B cell-specific major raft protein, Raftlin, is necessary for the integrity of lipid raft and BCR signal transduction

Abstract

Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identi®cation of proteins that co-puri®ed with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/ or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.

Toll-like receptor 3 recognizes incomplete stem structures in single-stranded viral RNA

“…TLR3 activation by poly(I:C) requires clathrin-mediated internalization of extracellular poly(I:C) 33 . In human myeloid DCs and epithelial cells such as HeLa cells, the cytoplasmic raft protein, raftlin, induces poly(I:C) uptake through interaction with clathrin-AP-2 complex 34,35 . To understand how extracellular PV-RNAs activate endosomal TLR3, we examined the requirement of raftlin in PV-RNA-induced TLR3 activation by gene silencing of raftlin with small interfering RNA (siRNA).…”

“…The anti-human raftlin polyclonal Ab (pAb) and anti-mouse TLR3 mAb were kindly provided by Dr K. Saeki (Kyushu University) and Dr D. M. Segal (National Institutes of Health, MD), respectively. 34,11 Anti-EEA1 pAb (PA1-063) was purchased from Affinity Bioreagents; anti-b-actin mAb (A2228) was purchased from Sigma; the anti-LAMP1 mAb (328601) was purchased from Biolegend; the anti-TICAM-1 pAb (4596) was purchased from Cell Signaling; and Alexa Fluor-488 and -633-conjugated secondary antibodies were purchased from Invitrogen. Poly(I:C) was purchased from Amersham Biosciences.…”

Expansion of Functionally Anergic CD21−/low Marginal Zone-like B Cell Clones in Hepatitis C Virus Infection-Related Autoimmunity

“…Finally, upregulation of RFNT1/Raftlin, the B cell-specific major raft protein that is necessary for the integrity of lipid rafts and BCR signal transduction, further highlights the related functional involvement of the genes of this transcriptional signature (33).…”

Myosin 1g regulates cytoskeleton plasticity, cell migration, exocytosis, and endocytosis in B lymphocytes

“…This region has been reported to be necessary for binding phosphoinositides such as those enriched in lipid rafts [20]. With this precedent and previous data reporting the inclusion of Myo1g in lipid rafts of neutrophils [21] and human B lymphocytes [22], we looked for the presence of this protein in these membrane regions of B cells. As defined by the colocalization analysis with GM1 ( Fig.…”