The association of Lyn (or GM1) with rafts in the presence of 1% MCD was only slightly affected, indicating that Lyn associates with a higher affinity to lipid rafts than CD20. of Rituxan, CD20 exhibits a low affinity to Chlormadinone acetate lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca2+ levels and downstream apoptotic signalling. Keywords: B-cell specific antigen, CD20, Rituxan, store-operated calcium channel, lipid rafts Introduction Rituxan, a chimeric anti-CD20 antibody (Rituximab, Mabthera, C2B8), is in clinical use for non-Hodgkin’s B-cell lymphoma and has also shown excellent efficacy in inducing clinical improvement and remission in rheumatoid arthritis patients [1C4]. The effectiveness of Rituxan-based therapy is achieved by B-cell depletion. Several mechanisms have been proposed to be responsible for the therapeutic activity of Rituxan, including antibody-dependent cell cytotoxicity (ADCC), activation of the complement system, and CD20-mediated regulation of the cell cycle and apoptosis [5C7]. Crosslinking CD20 with anti-CD20 monoclonal antibodies like Rituxan, 2H7 and 1F5 triggers cell-cycle block at the G1 phase and inhibits B-cell differentiation and EBV or pokeweed mitogen-induced Ig secretion [8]. CD20 belongs to the MS4A gene family, which consists of at least 25 members clustered on human chromosome 11q12C131 [9,10]. The MS4A family has a predicted tetraspanning membrane topology with an N- and C-terminal cytoplasmic domain. CD20 is the best studied member of this family and is specifically expressed on the surface of B-cells and cells from most B-cell lymphoproliferative disorders [11]. Different isoforms of CD20 (33, 35, 37 kD) result from multiple phosphorylation of serine and threonine residues in the cytoplasmic domains, implying that CD20 is highly regulated by phosphorylation. Stimulation of Chlormadinone acetate the B-cell receptor induces depletion of intracellular calcium stores which in turn results in the activation of store-operated calcium channels at the plasma membrane. A sustained influx of Chlormadinone acetate extracellular calcium ensures the progression of calcium-dependent signalling processes such as transcriptional control, cell cycle progression or apoptosis. The induction of apoptosis is blocked by chelating intracellular or extracellular calcium [12,13]. Studies using cell lines transfected with CD20 show an increased calcium conductance across the plasma Chlormadinone acetate membrane, strongly suggesting that CD20 functions as a calcium channel important for regulating cell cycle progression and calcium homeostasis [14,15]. Furthermore, it was reported that reduced expression levels of CD20 in B-cell lines, achieved by antisense CD20 sequence, result in a significantly decreased calcium entry across the plasma membrane [15,16]. These results provide the first evidence that CD20 functions as a store-operated calcium channel [17]. However, the mechanism of how the decrease in luminal calcium concentration causes an activation of store-operated calcium entry at the plasma membrane is still not understood. Hypercrosslinking of CD20 antibodies bound to Chlormadinone acetate the cell surface results in an increase in calcium conductivity without preceding depletion of intracellular calcium stores, uncoupling the store-operated channel activity from regulation via intracellular calcium levels [14]. Binding of antibodies to CD20 is also reported to cause a rapid redistribution of CD20 molecules to lipid rafts, which represent specialized microdomains of the plasma membrane, highly enriched in sphingolipids and cholesterol [18]. Lipid rafts are implicated in the organization of numerous membrane-associated signalling pathways providing a platform for the scaffolding of messenger molecules [19,20]. Truncation of the CD20 cytoplasmic domain (219-225) abolished CD20 lipid raft association and significantly decreased the calcium influx downstream of B-cell receptor-stimulated calcium mobilization from intracellular stores [15]. The current study was initiated to investigate the role of CD20 lipid raft localization for CD20 calcium channel activity by directly crosslinking CD20 by Rituxan. The data Rabbit polyclonal to Lamin A-C.The nuclear lamina consists of a two-dimensional matrix of proteins located next to the inner nuclear membrane.The lamin family of proteins make up the matrix and are highly conserved in evolution. described here provides evidence that agents disturbing the raft integrity inhibit Rituxan-induced translocation of CD20 into lipid rafts as well as Rituxan-induced calcium influx and subsequent caspase-mediated apoptosis. Materials and methods Cells, antibodies and reagents Ramos B cells were maintained in culture in RPMI 1640 supplemented with foetal calf serum (10%), HEPES (100 m m), sodium pyruvate (1 m.
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