Gap junction assembly: PTX-sensitive G proteins regulate the distribution of connexin43 within cells.

Cells expressing connexin43 are able to upregulate gap junction (GJ) communication by enhancing the assembly of new GJs, apparently through increased connexin trafficking. Because G proteins are known to regulate different aspects of protein trafficking, we examined the effects of pertussis toxin (PTX; a specific inhibitor of certain G proteins) on GJ assembly. Dissociated Novikoff hepatoma cells were reaggregated for 60 min to form nascent junctions. PTX inhibited GJ assembly, as indicated by a reduction in dye transfer. Electron microscopy also revealed a 60% decrease in the number of GJ channels per cell interface. Importantly, PTX blocked the twofold enhancement in GJ assembly found in the presence of low-density lipoprotein. Two G(i alpha) proteins (G(i alpha 2) and G(i alpha 3)), which have been implicated in the control of membrane trafficking, reacted with PTX in ADP-ribosylation studies. PTX and/or the trafficking inhibitors, brefeldin A and monensin, inhibited GJ assembly to comparable degrees. In addition, assays for GJ hemichannels demonstrated reduced plasma membrane levels of connexin43 following PTX treatment. These results suggest that PTX-sensitive G proteins regulate connexin43 trafficking, and, as a result of inhibition with PTX, the number of plasma membrane hemichannels available for GJ assembly is reduced.

Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.

The differential effects of 12-O-tetradecanoylphorbol-13-acetate on the gap junctions and connexins of the developing mammalian lens.

Epithelial cells in primary ovine lens cultures express the gap junction proteins connexin43 (Cx43) and connexin49 (Cx49; a.k.a. MP70), a homologue of mouse connexin50. In contrast, lens cultures of differentiated, fiber-like cells (termed lentoid cells) express Cx49 and connexin46 (Cx46), but not Cx43. To investigate the regulation of lens cell gap junctions by protein kinase C (PKC), differentiating lens cultures were treated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate (beta-TPA). Within 10 min, beta-TPA significantly inhibited the transfer of Lucifer Yellow dye between epithelial, but not lentoid, cells. This inhibition was correlated with the phosphorylation of Cx43 and was followed by the gradual disappearance of Cx43 from cell interfaces. The protein kinase inhibitor staurosporine prevented Cx43 phosphorylation and the loss of Cx43 from intercellular junctions. Following treatment of cultures with beta-TPA for 2-6 hr, Cx49 disappeared from epithelial cell interfaces, and by 24 hr of beta-TPA treatment, levels of Cx49 detected on immunoblots of purified epithelial membrane fractions had also diminished significantly. The beta-TPA-induced loss of Cx49 both from regions of epithelial cell contact and from isolated membranes was correlated with the disappearance of Cx49 mRNA. In contrast to the epithelial connexins, the lentoid connexins Cx49 and Cx46 were unaffected by even extended beta-TPA treatment. In spite of lentoid dye transfer being refractory to beta-TPA, significant levels of PKC-alpha (a beta-TPA-sensitive isoform) were detected in the lentoid cell. The response of lens gap junctions to beta-TPA depends upon the stage of differentiation and the complement of connexins expressed. The contrasting effects of beta-TPA on Cx43 and Cx49 in lens epithelial cells indicate a fundamental difference in the regulation of these connexin proteins in the developing mammalian lens.

Cell-to-cell communication in a differentiating ovine lens culture system.

PURPOSE: This study was performed to determine whether the junctions between both the epithelial and the differentiating fiber-like cells of ovine lens cultures, like gap junctions in other tissues, exhibit cell-to-cell communication that is inhibited by n-octanol, and to determine whether lens connexins and the fiber cell membrane proteins MP20 and MP26 are expressed by these ovine lens cell cultures. METHODS: Cells were injected with Lucifer yellow CH to measure cell-to-cell communication. Antibodies to connexin-related lens membrane protein MP70, connexin 43 (Cx43), and connexin 46 (Cx46) and to membrane proteins MP20 and MP26 were used to immunofluorescently label lens cultures and probe Western blots of membranes isolated from lens cultures. RESULTS: Both epithelial cells and differentiating clear cells exhibited cell-to-cell transfer of Lucifer yellow that was inhibited by n-octanol. Although a Cx43 antibody immunofluorescently labeled small plaques between the epithelial cells, an MP70 antibody labeled large plaques as well as small punctate areas of the differentiating fiber-like cells. It is interesting that Cx43 and MP70 were frequently present in the same plaques at cell interfaces between epithelial cells as well as some of the larger plaques on the differentiating fiber-like cells. Cx46 and MP70 antibodies labeled the same plaques in membranes of differentiating fiber-like cells and late-stage epithelial cells. The electrophoretic mobility of all three connexin proteins was modified after treatment with alkaline phosphatase. Immunohistochemical staining of these differentiating regions and Western immunoblotting of purified membranes derived from differentiated cultures also showed the presence of MP20 and MP26. CONCLUSIONS: The different cell types in the ovine lens culture exhibit gap junction-mediated cell-to-cell communication that is likely effected by one or more of the connexin proteins.

Identification of an 18,000-dalton protein in mammalian lens fiber cell membranes.

Monoclonal antibodies have been produced against electrophoretically purified MP18, a major calf lens membrane Mr = 18,000 substrate for cAMP-dependent protein kinase. One of these antibodies (monoclonal antibody 2D10) cross-reacted with both native MP18 in lens membranes, and sodium dodecyl sulfate-denatured, electrophoretically purified MP18. In immunoblots, this antibody recognized MP18 in pig, sheep, rat, human, but not chicken lens membranes, indicating the similarity of this protein in mammalian lenses. Amino acid sequencing revealed that the N-terminal sequence of MP18 is identical in these five different mammalian species and is unrelated to any previously sequenced lens or junctional proteins. Electron microscopic examination of monoclonal antibody 2D10-labeled bovine, pig and rat lens membranes indicated that MP18 is localized exclusively to the thicker 16-17 nm junctions in isolated preparations of lens fiber cell membranes. These results provide evidence of a role for MP18 in mammalian lens fiber cell junctional organization.