Abstract
ABSTRACTThe gap junction (GJ) protein connexin 43 (Cx43) is both necessary and sufficient for B cell receptor (BCR)-mediated cell spreading. To address how Cx43 mediates this effect, we blocked its function genetically, by expressing mutants of Cx43, and pharmacologically, by using chemical inhibitors. While various point mutations of Cx43 inhibited B cell spreading, treatment with channel blocking drugs did not, suggesting that this response was independent of channel function. The critical region of Cx43 appears to be the cytoplasmic carboxyl-terminal (CT) domain, which has previously been shown to be important for B cell spreading. Consistent with this, mutations of either tyrosine 247 or 265 found in the CT were sufficient to inhibit spreading. Thus Cx43 may influence B cell spreading by mechanisms requiring protein binding to, or modification of, these sites in the CT tail.
Highlights
Recognition of antigen (Ag) by the B cell receptor (BCR) leads to B cell proliferation and differentiation
The importance of associated membrane proteins for BCR signaling led us to consider other candidates that might influence BCR signaling and changes in the cytoskeleton. Using both loss-of-function and gain-of-function strategies, we previously showed the importance of connexin 43 (Cx43) for cell spreading in response to BCR signaling (Machtaler et al, 2011)
Overexpression of Cx43 in the B cell line J558mm3 results in hemichannel activity The J558mm3 plasmacytoma B cell line expresses a full 4 chain (IgM, Igl, Iga, and Igb) BCR at the plasma membrane and is ideal for the study of Cx43 mutants since it does not express endogenous Cx43, which could otherwise mask the effects of the expression of mutated Cx43 by forming heteromeric hexamers with WT Cx43
Summary
Recognition of antigen (Ag) by the BCR leads to B cell proliferation and differentiation. We have previously shown that the CT tail (amino acids 246– 382) was necessary for J558mm3 cell spreading in response to BCR-stimulation (Machtaler et al, 2011).
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