Assembly of hepatic gap junctions. Topography and distribution of connexin 32 in intracellular and plasma membranes determined using sequence-specific antibodies

The subcellular distribution in rat liver and the topography in intracellular and plasma membranes of connexin 32, a major protein component of gap junctions, was studied using sequence-specific anti-peptide antibodies generated to extracellular and intracellular domains of the protein. The distribution of connexin 32 in liver analyzed using SDS-polyacrylamide gel electrophoresis and Western blotting showed the relative protein levels in the subcellular fractions to be: lateral plasma membranes > Golgi membranes > sinusoidal plasma membranes > lysosomes. Low amounts of connexin 32 were detected in microsomes, endosomes, and bile canalicular plasma membranes. Six highly conserved cysteine residues are located in the amino acid sequences comprising the two extracellular loops of all connexins thus far isolated, and these loops are positioned to extend the channel in the lipid bilayers across the intercellular region of the gap junction. In the present work, the intramolecular disulfide bonds linking the extracellular loops in gap junctions were shown to be present in connexins located in plasma membranes, Golgi, and a microsomal fraction, and it was concluded that the disulfide linkages were formed in the endoplasmic reticulum. In addition, immature configurations of connexin 32, probably occurring during membrane insertion, were detected in liver microsomal fractions. The results contribute to charting of the biogenetic routes followed by connexins in hepatocytes and the general mechanisms of gap junction assembly.     

Contributions of cytoplasmic domains of desmosomal cadherins to desmosome assembly and intermediate filament anchorage

To examine the potential of cytoplasmic portions ("tails") of desmosomal cadherins for assembly of desmosome plaque structures and anchorage of intermediate filaments (IFs), we transfected cultured human A-431 carcinoma cells, abundant in desmosomes and cytokeratin IFs, with constructs encoding chimeric proteins in which the transmembranous region of connexin 32 had been fused with tails of desmocollin (Dsc) or desmoglein (Dsg). The results show that the tail of the long splice form a of Dsc, but not its shorter splice form b, contains sufficient information to recruit desmoplakin and plakoglobin to connexon membrane paracrystals (gap junctions) and to form a novel kind of plaque at which cytokeratin IFs attach. By contrast, chimeras containing a Dsg tail, which accumulated in the plasma membrane, showed a dominant-negative effect: they not only were unable to form gap junction structures and plaques but also led to the disappearance of all endogenous desmosomes and the detachment of IFs from the plasma membrane.     

Degradation of connexin43 gap junctions involves both the proteasome and the lysosome

Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1-3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin-proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of approximately 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions.     

Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase

Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.     

In vitro studies of cochlear excitation

Cells in tissues coordinate their activity by sharing ions, second messengers, and small metabolites through clusters of intercellular channels called gap junctions. The thyroid hormones 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) are capable of modulating gap junctional communication (GJC) as are 1,25-dihydroxyvitamin D3, retinoic acid, and other nuclear receptor ligands. T3 and T4 were found to stimulate GJC in WB-F344 rat liver epithelial cells dose-dependently at concentrations between 1 nM and 0.1 microM, assayed by the dye transfer method using Lucifer Yellow CH. The stimulation of cell-cell communication was preceded by an increase in connexin43 mRNA levels and was accompanied by an accumulation of connexin43 protein measurable 2 days after incubation with these compounds. These observations establish a novel role of thyroid hormones in the regulation of gap junctional intercellular communication via connexin43 gene expression.     

Expression of gap junctions in cultured rat L6 cells during myogenesis

Although gap junctions are absent from adult skeletal muscle, they have been described in embryonic and neonatal rat skeletal muscle and in cultured rat myoblasts. In order to determine the precise developmental expression and molecular composition of gap junctions during myogenesis, RNA was isolated from cultures of rat L6 myoblasts and examined using Northern blot analysis with cDNA probes specific for connexin32 and connexin43. Connexin32 mRNA could not be detected in rat myoblast and myotube samples. However, connexin43 mRNA was expressed at high levels in cycling L6 myoblasts and this expression decreased by approximately 60% in L6 myotubes following fusion. Immunofluorescent localization with an antibody specific for connexin43 confirmed the accumulation of connexin43 protein in membranes shared between adjacent myoblasts at 12 hr of culture. By 24 hr of culture, connexin43 disappeared from most cells, only to reappear at 36 hr at a low level that was maintained through 72 hr in culture. Although most myoblasts in these cultures expressed connexin43, myotubes expressed little or no membrane-associated connexin43. Dye transfer experiments established that, at 12 hr of culture, the majority of myoblasts were dye coupled suggesting that connexin43 protein is assembled into functional gap junctions. At 24 hr, the number of coupled cells decreased slightly, while at 48 hr, most of the myoblasts were not dye coupled. These results demonstrate that the expression of connexin43 is temporally correlated with myoblast fusion and may play a role in this process.     

Direct high affinity modulation of connexin channel activity by cyclic nucleotides

Protonated aminosulfonate compounds directly inhibit connexin channel activity. This was demonstrated by pH-dependent connexin channel activity in Good's pH buffers (MES (4-morpholineethanesulfonic acid)), HEPES, and TAPS (3-({[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]amino]-1-propanesulfonic acid)) that have an aminosulfonate moiety in common and by the absence of pH-dependent channel activity in pH buffers without an aminosulfonate moiety (maleate, Tris, and bicarbonate). The pH-activity relation was shifted according to the pKa of each aminosulfonate pH buffer. At constant pH, increased aminosulfonate concentration inhibited channel activity. Taurine, a ubiquitous cytoplasmic aminosulfonic acid, had the same effect at physiological concentrations. These data raise the possibility that effects on connexin channel activity previously attributed to protonation of connexin may be mediated instead by protonation of cytoplasmic regulators, such as taurine. Modulation by aminosulfonates is specific for heteromeric connexin channels containing connexin-26; it does not occur significantly for homomeric connexin-32 channels. The identification of taurine as a cytoplasmic compound that directly interacts with and modulates connexin channel activity is likely to facilitate understanding of cellular modulation of connexin channels and lead to the development of reagents for use in structure-function studies of connexin protein.     

Insulin-sensitive targeting of the GLUT4 glucose transporter in L6 myoblasts is conferred by its COOH-terminal cytoplasmic tail

The GLUT4 glucose transporter appears to be targeted to a unique insulin-sensitive intracellular membrane compartment in fat and muscle cells. Insulin stimulates glucose transport in these cell types by mediating the partial redistribution of GLUT4 from this intracellular compartment to the plasma membrane. The structural basis for the unique targeting behavior of GLUT4 was investigated in the insulin-sensitive L6 myoblast cell line. Analysis of immunogold-labeled cells of independent clonal lines by electron microscopy indicated that 51-53% of GLUT1 was present in the plasma membrane in the basal state. Insulin did not significantly affect this distribution. In contrast, only 4.2-6.1% of GLUT4 was present in the plasma membrane of basal L6 cells and insulin increased this percentage by 3.7-6.1-fold. Under basal conditions and after insulin treatment, GLUT4 was detected in tubulovesicular structures, often clustered near Golgi stacks, and in endosome-like vesicles. Analysis of 25 chimeric transporters consisting of reciprocal domains of GLUT1 and GLUT4 by confocal immunofluorescence microscopy indicated that only the final 25 amino acids of the COOH-terminal cytoplasmic tail of GLUT4 were both necessary and sufficient for the targeting pattern observed for GLUT4. A dileucine motif present in the COOH-terminal tail of GLUT4 was found to be necessary, but not sufficient, for intracellular targeting. Contrary to previous studies, the NH2 terminus of GLUT4 did not affect the subcellular distribution of chimeras. Analysis of a chimera containing the COOH-terminal tail of GLUT4 by immunogold electron microscopy indicated that its subcellular distribution in basal cells was very similar to that of wild-type GLUT4 and that its content in the plasma membrane increased 6.8-10.5-fold in the presence of insulin. Furthermore, only the chimera containing the COOH terminus of GLUT4 enhanced insulin responsive 2-deoxyglucose uptake. GLUT1 and two other chimeras lacking the COOH terminus of GLUT4 were studied by immunogold electron microscopy and did not demonstrate insulin-mediated changes in subcellular distribution. The NH2-terminal cytoplasmic tail of GLUT4 did not confer intracellular sequestration and did not cause altered subcellular distribution in the presence of insulin. Intracellular targeting of one chimera to non-insulin-sensitive compartments was also observed. We conclude that the COOH terminus of GLUT4 is both necessary and sufficient to confer insulin-sensitive subcellular targeting of chimeric glucose transporters in L6 myoblasts.     

Gap junction change in supporting cells of the organ of Corti with ryanodine and caffeine

It has been demonstrated that the gap junctions of the supporting cells of the organ of Corti are controlled by H+ and Ca2+. Inside these cells there is a tubular structure. It is supposed that this network is endoplasmic reticulum. Calcium release from inside the cells, and the effect of calcium on the gap junctions of these cells, were investigated under whole cell clamping application of ryanodine and caffeine. Membrane capacitance and membrane resistance were calculated, with corrections for changes in whole cell parameters. Ryanodine-treated cells (1 microM-10 mM), caffeine-treated cells (5 mM 500 nM) and A23187-treated cells were uncoupled at their gap junctions. Therefore, Ca2+ plays a role in the uncoupling of the gap junctions in supporting cells of the organ of Corti from inside the cells.     

Regulation of connexin43 function by activated tyrosine protein kinases

Gap junctions are specialized membrane structures that are involved in the normal functioning of numerous mammalian tissues and implicated in several human disease processes. This mini-review focuses on the regulation of gap junctions through phosphorylation of connexin43 induced by the v-Src or epidermal growth factor receptor tyrosine kinases. These tyrosine kinases markedly disrupt gap junctional communication in mammalian cells. here, we describe work correlating the alteration of connexin43 function with the ability of the v-Src tyrosine kinase to phosphorylate connexin43 directly on two distinct tyrosine sites in mammalian cells (Y247 and Y265). We also present evidence that proline-rich regions and phosphotyrosine sites of connexin43 may mediate interactions with the SH3 and SH2 domains of v-Src. In contrast to v-Src, the activated epidermal growth factor receptor acts indirectly through activated MAP kinase which may stimulate phosphorylation of connexin43 exclusively on serine. This phosphorylation event is complex because MAP kinase phosphorylates three serine sites in connexin43 (S255, S279, and S282). These findings suggest novel interactions between connexin43, the v-Src tyrosine kinase, and activated MAP kinase that set the stage for future investigations into the regulation of gap junctions by protein phosphorylation.     

Reticulospinal actions on primary afferent depolarization of cutaneous and muscle afferents in the isolated frog neuraxis

The disposition and orientation of mouse ductin (the subunit c of the vacuolar H(+)-ATPase) in gap junctions has been examined. Like the Nephrops norvegicus (arthropod) form, mouse ductin in the intact junctional structure is resistant to high levels of nonspecific proteinase, suggesting that it is for the most part buried in the bilayer. Antisera to an octapeptide near the N-terminus cross-react with ductins in gap junction preparations from four different mouse tissues, from chicken and Xenopus laevis liver, and from N. norvegicus hepatopancreas. The antisera and antibodies, affinity purified against the octapeptide, agglutinate isolated gap junctions, suggesting that the N-terminus is located on the exposed surface, equivalent to the cytoplasmic face of an intercellular gap junction. The antibodies also block dye coupling when injected into cells in culture, confirming the cytoplasmic location of the epitope. The lipophylic reagent dicylohexyl carbodiimide (DCCD), which targets carboxyl groups within the membrane and selectively reacts with ductin in N. norvegicus gap junction preparations, rapidly inhibits junctional communication. Bafilomycin A1, which inhibits V-ATPase and stops vacuolar acidification, does not affect dye coupling, showing that the inhibition seen with antibodies and DCCD is not an indirect consequence of their action on the ductin of V-ATPase. Consistent with this interpretation the anti-peptide antibodies do not bind to intact chromaffin granules or inhibit their V-ATPase activity, but do bind to osmotically disrupted granule membrane. This suggests that ductin has an orientation (N-terminus pointing away from the cytoplasm) in the vacuolar membrane opposite to that in the gap junction membrane.     

Prevalence of temperature sensitive folding mutations in the parallel beta coil domain of the phage P22 tailspike endorhamnosidase

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.     

Dynamic recycling of ERGIC53 between the endoplasmic reticulum and the Golgi complex is disrupted by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, has recently been demonstrated to block protein transport from the endoplasmic reticulum (ER) to the Golgi complex. The ER to Golgi transport is primarily operated by the ER-Golgi intermediate compartment (ERGIC). We examined the effect of NDGA on the ERGIC, focusing on the distribution of its marker ERGIC53. In control cells ERGIC53 was distributed to vesicular tubular structures corresponding to the ERGIC as well as to the ER and the cis-Golgi, reflecting its cycling between these compartments. Upon treatment of cells with NDGA, ERGIC53 was rapidly accumulated in the Golgi and undetectable in the ER and the ERGIC. Prolonged incubation of cells with the drug, however, caused redistribution of ERGIC53 and resident Golgi proteins to the ER. Thus, it is likely that NDGA has dual effects on ERGIC53 cycling; the initial accumulation in the Golgi may be caused by blocking its retrieval from the cis-Golgi to the ER/ERGIC, while the delayed redistribution to the ER may occur through a pathway induced by the drug that is different from the COPI-dependent pathway.     

Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy

OBJECTIVE: Experimental brain tumor gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) gene has demonstrated that not only HSV-tk transduced but surrounding non-HSV-tk transduced cells are killed when given ganciclovir. This so-called bystander effect has recently been shown to be dependent on connexin-mediated intercellular communication. To assess potential susceptibility to the bystander effect, we examined levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Connexin-26 expression has not previously been studied in primary brain tumors and connexin-43 expression has not been studied in nonastrocytic primary brain tumors. We also attempted to enhance the bystander effect in vitro by overexpressing connexin in tumor cells with high basal levels of connexin expression. METHODS: Western blot analysis and immunohistochemistry were used to determine levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Wild-type 9L gliosarcoma cells were transfected in vitro with the connexin-43 gene and the HSV-tk gene or the HSV-tk gene alone. The bystander effect of each transfectant was then assessed and compared. RESULTS: Most of the primary brain tumors tested, including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, gangliogliomas, meningiomas, and medulloblastomas, showed connexin-26 and connexin-43 expression. Bystander experiments revealed a significant enhancement of the bystander effect in the gliosarcoma cells transfected with connexin-43 and HSV-tk, as compared with gliosarcoma cells transfected with HSV-tk alone. CONCLUSION: Most primary brain tumors express connexin-26 and connexin-43. This suggests that most primary brain tumors may be susceptible to the bystander effect of HSV-tk gene therapy. The bystander effect can be enhanced in vitro by overexpression of connexin-43 in a cell line with a high basal level of connexin-43 expression.     

Analysis of the thrombin inhibitor DuP 714 by an enzyme-linked immunosorbent assay

Resident proteins of the exocytic pathway contain at least two types of information in their primary sequence for determining their subcellular location. The first type of information is found at the carboxyl terminus of soluble proteins of the endoplasmic reticulum (ER) and in the cytoplasmic domain of some ER and Golgi membrane proteins. It acts as a retrieval signal, returning proteins that have left the compartment in which they reside. The second type of information has been found in the membrane-spanning domain of several ER and Golgi proteins and, though the mechanism by which it operates is still unclear, it acts as a retention signal, keeping the protein at a particular location within the organelle. The presence of both a retrieval signal and a retention signal in a trans-Golgi network resident protein suggests that more than one mechanism operates to ensure correct localization of resident proteins along the exocytic pathway.     

Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase

We have studied the localization of mutant cystic fibrosis transmembrane regulator delta F508CFTR in pancreatic adenocarcinoma cells (CFPAC), which naturally express the mutant protein. Our goal was to investigate whether delta F508CFTR is strictly retained in the endoplasmic reticulum (ER) or alternatively whether it can be transported beyond the ER and reach the endoplasmic reticulum-Golgi intermediate compartment (ERGIC). This compartment, defined by the presence of the 53-kDa protein ERGIC-53, was identified by subcellular fractionation and by immunofluorescence. Part of the delta F508CFTR population and ERGIC-53 showed similar distributions in membrane fractions analyzed on Nycodenz density gradients. Both proteins were present in density fractions distinct from the ones containing the ER marker proteins calnexin and Sec61. Immunofluorescence microscopy of CFPAC cells revealed some colocalization of delta F508CFTR with ERGIC-53. Following incubation of CFPAC cells at 15 degrees C, a condition known to block ER to Golgi transport, both ERGIC-53 and delta F508CFTR subcellular localizations were altered. By contrast, this temperature shift had no effect on the localization of the ER marker Sec61. Our observations indicate that the abnormal protein delta F508CFTR can leak out of the ER and reach the ERGIC. These results support the idea that this intermediate compartment plays a role in the trafficking events leading to retention and finally degradation of the misfolded delta F508CFTR protein.