Anti-CD20 Antibody [2H7] (PerCP) (A85693)

CD20 is a B cell integral membrane protein capable of initiating growth-modulating signals in human B lymphocytes upon its engagement with monoclonal anti-CD20 antibodies. In this report, we demonstrate that treatment of B cells with CD20 antibodies induces rapid redistribution of CD20 into a detergent-insoluble membrane compartment. Redistribution is detected as early as 15 s, following antibody addition, and involves up to 95% of CD20 molecules, depending on the antibody used. All of the detergent-insoluble CD20 was found in the low density fractions of sucrose density gradients, indicating that CD20 redistributes to glycolipid-rich membrane domains, analogous to caveolae in some cell types. As CD20 has previously been shown to associate with Src family tyrosine kinases, their co-existence in these compartments suggests a link to the role of CD20 in signal transduction. This study provides insight into the mechanism by which CD20 communicates signals to the cell interior and indicates that the search for membrane-proximal intracellular signaling partners should be directed to the Triton-insoluble fraction.

CD20, a non-glycosylated cell-surface protein expressed exclusively on B lymphocytes, is one of a family of 4-pass transmembrane molecules that also includes the beta chain of the high affinity receptor for IgE. The precise function of CD20 is unknown, although in vitro effects of CD20-specific antibodies on resting B cells indicate that it is able to transduce an extracellular signal affecting the G0/G1 cell cycle transition. Previous studies have demonstrated that CD20-initiated intracellular signals involve tyrosine kinase activation and that CD20 is tightly associated with both serine and tyrosine kinases. Here, analysis of CD20-associated molecules has revealed that CD20 is associated with the Src family tyrosine kinases p56/53lyn, p56lck, and p59fyn and with 75/80-kDa proteins phosphorylated in vivo on tyrosine residues. Mutagenesis of CD20 was performed to define regions of CD20 involved in intermolecular interactions. Mutants were analyzed in the human T lymphoblastoid cell line Molt-4, in which ectopically expressed wild-type CD20 associated with p59fyn, p56lck, and 75/80-kDa phosphoproteins. Deletion of major portions of the cytoplasmic regions of CD20 did not abolish its association with either p75/80 or tyrosine kinases. The interaction between CD20 and the Src-related kinases is therefore likely to be independent of CD20 cytoplasmic domains and may occur indirectly. The interaction may be mediated by the p75/80 phosphoproteins, which were found to be tightly associated with the Src family kinases isolated from the CD20 complex.