Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src (original) (raw)
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Journal of Biological Chemistry, 2001
Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK ؉ ) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK ؉ and, to a lesser extent, with wild-type c-Src, but not with kinasedead c-Src. Mutation of residue Cx43 Tyr 265 (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK ؉ in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43⌬263) that lacks residue Tyr 265 . Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr 265 , resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.
American Journal of Physiology-Cell Physiology, 2002
Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAP...
Relationship between G proteins coupled receptors and tight junctions
Tissue Barriers, 2018
Tight junctions (TJs) are sites of cell-cell adhesion, constituted by a cytoplasmic plaque of molecules linked to integral proteins that form a network of strands around epithelial and endothelial cells at the uppermost portion of the lateral membrane. TJs maintain plasma membrane polarity and form channels and barriers that regulate the transit of ions and molecules through the paracellular pathway. This structure that regulates traffic between the external milieu and the organism is affected in numerous pathological conditions and constitutes an important target for therapeutic intervention. Here, we describe how a wide array of G protein-coupled receptors that are activated by diverse stimuli including light, ions, hormones, peptides, lipids, nucleotides and proteases, signal through heterotrimeric G proteins, arrestins and kinases to regulate TJs present in the blood-brain barrier, the blood-retinal barrier, renal tubular cells, keratinocytes, lung and colon, and the slit diaphragm of the glomerulus.
Interaction of c-Src with Gap Junction Protein Connexin-43
Journal of Biological Chemistry, 2000
Cell-cell communication via connexin-43 (Cx43)-based gap junctions is transiently inhibited by certain mitogens, but the underlying regulatory mechanisms are incompletely understood. Our previous studies have implicated the c-Src tyrosine kinase in mediating transient closure of Cx43-based gap junctions in normal fibroblasts. Here we show that activated c-Src (c-SrcK ؉) phosphorylates the COOH-terminal tail of Cx43, both in vitro and in intact cells. Coimmunoprecipitation experiments reveal that Cx43 associates with c-SrcK ؉ and, to a lesser extent, with wild-type c-Src, but not with kinasedead c-Src. Mutation of residue Cx43 Tyr 265 (Cx43-Y265F mutant) abolishes both tyrosine phosphorylation of Cx43 and its coprecipitation with c-Src. Expression of c-SrcK ؉ in Rat-1 cells disrupts gap junctional communication. Strikingly, the communication-defective phenotype is bypassed after coexpression of the Cx43-Y265F mutant or a COOH-terminally truncated version of Cx43 (Cx43⌬263) that lacks residue Tyr 265. Our results support a model in which activated c-Src phosphorylates the COOH-terminal tail of Cx43 on residue Tyr 265 , resulting in a stable interaction between both proteins leading to inhibition of gap junctional communication.
The Journal of cell biology, 2000
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 stimula...
Close the Gap : a study on the regulation of Connexin43 gap junctional communication
Gap junctions are groups of transmembrane channels that connect the cytoplasms of adjacent cells to mediate the diffusion of small molecules, such as ions, metabolites, second messengers and small peptides. The building blocks of gap junctions are connexin proteins. The most ubiquitous and best studied connexin is connexin43 (Cx43). Cx43 is also known as the heart connexin. In the heart, gap junctional communication between cardiomyocytes ensures efficient electrical coupling and hence the synchronous propagation of action potentials. Misregulation of Cx43 expression, localisation and channel gating may lead to severe cardiac dysfunction. Cx43-based cell-cell coupling is rapidly disrupted following stimulation of certain G protein-coupled receptors (GPCRs). The studies presented in this thesis focus on the quest to unravel the signalling pathways leading to the transient inhibition of Cx43-based communication by GPCR activation. Our results suggest a model in which GPCR agonists inh...
Cellular Signalling, 2012
Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1-ZO-2-occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners.
Role of protein kinase C in the regulation of gap junctional communication
Teratogenesis, Carcinogenesis, and Mutagenesis, 1994
We have examined the effect of protein kinase (PKC) depletion in SV40-transformed Djungarian hamster fibroblasts (DMI 5 cells) on the level of gap junction permeability, Cx43 electrophoretic mobility, and cell sensitivity to different uncoupling stimuli. After 24 hr exposure to 12-0-tetradecanoyl-phorbol-13-acetate (TPA), the total PKC activity in DMl5 cells was reduced to 20-25% in comparison with intact cells. In PKC-depleted cells the level of dye coupling was 3 M O % higher than in the same untreated cultures. Western blot analysis revealed multiple forms of the gap junction protein connexin 43, which correspond to known phosphorylated and dephosphorylated forms of this protein. No decrease in the level of connexin 43 phosphorylation after PKC depletion was observed. TPA (10-7 g/ml), mezerein (10-7 g/ml), teleocidin (1&8 g/ml), Ca-ionophore A23 187 (10-6 gjml), insecticide 1,1,1-trichloro-2,2-bis-(p-chlorphenyl)-ethane (DDT) (10-4 g/ml), and nigericin (10-5 M in hydrolysate lactalbumin solution, pH 6.3) induced a four-to six-fold decrease in the number of recipient cells in the dye-coupling assay. PKC-depleted cells became almost completely resistant to the uncoupling effect of mezerein, teleocidin, and A23187, as well as to new exposure to TPA, and became partially resistant to the effect of DDT. Nigericin inhibited intercellular communication between PKC-depleted cells to the same extent as between control cells. Thus, in the cell system studied, PKC plays a certain role in maintaining the basal level of gdp junction permeability and has an important significance as a mediator of the uncoupling effects of such substances as TPA, mezerein, teleocidin, and Ca2+.