The gap junction protein Cx43 regulates B-lymphocyte spreading and adhesion.

The gap junction protein connexin43 (Cx43) is widely expressed in mammalian cells and forms intercellular channels for the transfer of small molecules between adjacent cells, as well as hemichannels that mediate bidirectional transport of molecules between the cell and the surrounding environment. Cx43 regulates cell adhesion and migration in neurons and glioma cells, and we now show that Cx43 influences BCR-, LFA-1- and CXCL12-mediated activation of the Rap1 GTPase. Using shRNA knockdown of Cx43 in WEHI 231 cells, we show that Cx43 is required for sustained Rap1 activation and BCR-mediated spreading. To determine the domains of Cx43 that are important for this effect, Cx43-null J558 µm3 B cells (which express a wild-type IgM BCR) were transfected with wild-type Cx43-GFP or a C-terminal-truncated Cx43 (Cx43ΔT-GFP). Expression of wild-type Cx43-GFP, but not Cx43ΔT-GFP, was sufficient to restore sustained, BCR-mediated Rap1 activation and cell spreading. Cx43, and specifically the C-terminal domain, was also important for LFA-1- and CXCL12-mediated Rap1 activation, spreading and adhesion to an endothelial cell monolayer. These data show that Cx43 has an important and previously unreported role in B-cell processes that are essential to normal B-cell development and immune responses.

Mutations of Cx43 that affect B cell spreading in response to BCR signaling.

The 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.

The role of Ig-α/ß in B cell antigen receptor internalization.

The B cell antigen receptor (BCR) is expressed on the surface of B lymphocytes where it can bind antigen then transmit signals which regulate activation, growth, and differentiation. These signals can induce a number of cytoskeletal rearrangements leading to dynamic cellular processes including internalization of the bound antigen which is then processed and presented to T cells on MHC II. The relative importance of regions within the Igα and Igß cytoplasmic domains has been well studied in terms of signaling but their roles in BCR internalization and trafficking are less clear. We hypothesize that the Igα and Igß cytoplasmic domains is important for normal internalization and trafficking of the 4 chain BCR. An Igα and Igß deficient murine lymphoid cell line was used to express mIgM along with a panel of Igα and Igß mutants in order to compare their internalization and subcellular localization. Here we show that the Igα and Igß cytoplasmic domains are each sufficient for internalization, though Igα is dominant in this process. We also show that the internalization signal is contained in a region past the first cytoplasmic tyrosine residue of Igα and Igß, Y176 and Y195 respectively. We also show that a 4 amino acid motif normally contained within the Igα ITAM is sufficient to rescue aberrant internalization. In terms of receptor trafficking, each cytoplasmic domain is sufficient for trafficking to lysosomal compartments but that a normal rate of trafficking likely requires the tandem effects of both Igα and Igß.