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J. Biol. Chem., Vol. 283, Issue 25, 16971-16984, June 20, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/25/16971    most recent M708459200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Walshe, C. A. Articles by Cragg, M. S. PubMed PubMed Citation Articles by Walshe, C. A. Articles by Cragg, M. S. Social Bookmarking                      What's this?

Induction of Cytosolic Calcium Flux by CD20 Is Dependent upon B Cell Antigen Receptor Signaling^*

From the Cancer Sciences Division, General Hospital, Southampton University School of Medicine, Southampton SO16 6YD, United Kingdom

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.

Received for publication, October 11, 2007 , and in revised form, April 3, 2008.

^* This work was supported in part by the Association of International Cancer Research, Leukemia Research Fund, and Tenovus, Cardiff. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ These authors contributed equally to the project and should both be considered first authors.

² To whom correspondence should be addressed: Tenovus Laboratory, Cancer Sciences Division, School of Medicine, Southampton General Hospital, Tremona Rd., Southampton, SO16 6YD, UK. Fax: 44-0-23-80704-061; E-mail: msc{at}soton.ac.uk.

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