Crosslinking membrane immunoglobulin (mIg), the B-cell antigen receptor, stimulates tyrosine phosphorylation of a number of proteins. Since many receptors are phosphorylated after ligand binding, we asked ifcomponents of the mIg receptor complexes were tyrosine-phosphorylated after mIg crosslinking. Both mIgM and mIgD are noncovalently associated with at least two other proteins. mIgM Is associated with the MB-1 protein, which is disulfide-linked to a protein designated Ig-f3. mIgD is not associated with MB-1 but is with IgD-a, which is also disulfide-linked to Ig-fi. Using immunoprecipitation with a specific anti-MB-i antiserum followed by anti-phosphotyrosine immunoblotting, we found that crosslinking mIgM stimulated tyrosine phosphorylation of (5), were resuspended at 16.7 x 106 cells per ml in a modified Hepes-buffered saline solution (9). Cells were stimulated at 37°C, washed, and resuspended at 108 cells per ml in lysis buffer [20 mM Tris'HCI, pH 8/137 mM NaCl/10% (wt/vol) glycerol/1% Triton X-100/1 mM Na3VO4/2 mM EDTA/1 mM phenylmethylsulfonyl fluoride/20 ,uM leupeptin/0.15 unit of aprotinin per ml] as described (5). Detergent-insoluble material was removed by centrifugation, and protein concentrations were determined by the BCA (bicinchoninic acid) protein assay (Pierce).Anti-MB-i Antiserum. A 34-amino acid peptide from the putative cytoplasmic domain of the murine MB-1 protein [amino acids 187-220 (21) were collected on protein A-Sepharose for 1 hr. The beads were suspended in 0.5 ml of lysis buffer containing 50 ,uM Na3VO4, 0.4% deoxycholate, and 0.3% SDS (wash buffer), layered over 0.4 ml of 30%o sucrose in 0.5 x wash buffer, and centrifuged 5 min at 10,000 x g. The beads were then washed once each with wash buffer and 50 ,uM Na3VO4. Proteins were eluted by boiling in nonreducing SDS/PAGE sample Abbreviations: mlg, membrane immunoglobulin; anti-Tyr(P), antiphosphotyrosine.tTo whom reprint requests should be addressed.tThe first two authors contributed equally to the experiments in this paper. 3436The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Connexin43 (Cx43) has roles in cell-cell communication as well as channel independent roles in regulating motility and migration. Loss of function approaches to decrease Cx43 protein levels in neural cells result in reduced migration of neurons during cortical development in mice and impaired glioma tumor cell migration. In other cell types, correlations between Cx43 expression and cell morphology, adhesion, motility and migration have been noted. In this review we will discuss the common themes that have been revealed by a detailed comparison of the published results of neuronal cells with that of other cell types. In brief, these comparisons clearly show differences in the stability and directionality of protrusions, polarity of movement, and migration, depending on whether a) residual Cx43 levels remain after siRNA or shRNA knockdown, b) Cx43 protein levels are not detectable as in cells from Cx43(-/-) knockout mice or in cells that normally have no endogenous Cx43 expression, c) gain-of-function approaches are used to express Cx43 in cells that have no endogenous Cx43 and, d) Cx43 is over-expressed in cells that already have low endogenous Cx43 protein levels. What is clear from our comparisons is that Cx43 expression influences the adhesiveness of cells and the directionality of cellular processes. These observations are discussed in light of the ability of cells to rearrange their cytoskeleton and move in an organized manner. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.

SummaryThe 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 m3 B cells (which express a wild-type IgM BCR) were transfected with wildtype Cx43-GFP or a C-terminal-truncated Cx43 (Cx43T-GFP). Expression of wild-type Cx43-GFP, but not Cx43T-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.

Abstract. A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.

The B-cell antigen receptors consist of membrane immunoglobulins (mlgs) noncovalently associated with two accessory proteins, MB-i and Ig-P. We used transfection into a nonlymphoid cell line to test whether MB-1 and Ig-j3 were sufficient to promote cell surface expression of mlgM capable of signal transduction. Expression of MB-1 and Ig-P, but not MB-1 alone, allowed high-level surface expression of mIgM in the AtT20 endocrine cell line, which presumably lacks other B-cell-specific components. The reconstituted antigen receptor was capable of mediating some of the sgnalin reactions characteristic of mlgM in B lymphocytes. Crosslinking mIgM on transfected AtT20 cells stimulated tyrosine phosphorylation of MB-1 and Ig-. and also increased the amount of phosphatidylinositol 3-kinase activity that could be precipitated with anti-phosphotyrosine antibodies. When total cell lysates were analyzed by anti-phosphotyrosine immunoblotting, however, no induced phosphorylation of more abundant proteins was detected. Moreover, crosslinking of the receptor in AtT20 cells did not stimulate inositol phospholipid breakdown. Thus, the transfected B-cell antigen receptor could initiate some signal transduction events but AtT20 cells may lack components required for other sigalig events associated with mIgM.The antigen receptors on B lymphocytes initiate intracellular signals that regulate B-cell growth and differentiation. Crosslinking of the B-cell antigen receptors stimulates inositol phospholipid hydrolysis (1-3) and protein tyrosine phosphorylation (4-7). The B-cell antigen receptors consist of membrane immunoglobulin (mIg) that is noncovalently associated with at least two accessory proteins. Murine mIgM is associated with the 34-to 36-kDa MB-1 protein [the product ofthe mb-i gene (8-10)], which is disulfide linked to the 37-to 40-kDa Ig-,B protein [the product of the B29 gene (11,12)]. It is unclear whether there are additional mIg-associated proteins. The mIg-associated proteins could have two roles, facilitating cell surface expression of mIg and coupling mIg to its signal transduction pathways.When mIgM is expressed in the J558 plasmacytoma cell line, it is retained in the endoplasmic reticulum (ER) unless MB-1 is also expressed (13). J558 cells, however, express Ig-,B and may also express other lymphoid-specific proteins required for surface expression of mIgM. To investigate the functions of MB-1 and Ig-P, we expressed mIgM, MB-1, and Ig-,f in a nonlymphoid cell line and asked whether these components were sufficient for cell surface expression in the absence of other B-cell-specific components and sufficient to mediate signal transduction events characteristic of mIgM in B cells. pCMV was made by ligating the 0.68-kb HindIII-Sau3A cytomegalovirus enhancer/promoter element (17) from pRR23 (gift of W. Schaffner) to the 2.5-kb Sal I-Xho I fragment from pu (18). pCMVMB-1 was made by ligating an mb-i cDNA into the Xho I site of pCMV. To generate a construct containing the intact mb-i gene, mb-i cDNA sequences of pCMV...