Connexin45 Interacts with Zonula Occludens-1 in Osteoblastic Cells

Connexin43(Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Connexin45 Interacts with Zonula Occludens-1 in Osteoblastic Cells

Connexin43(Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells

The relative expression of connexin43 and connexin45 modulates gap junctional communication and production of bone matrix proteins in osteoblastic cells. It is likely that changes in gap junction permeability are determined by the interaction between these two proteins. Cx43 interacts with ZO-1, which may be involved in trafficking of Cx43 or facilitating interactions between Cx43 and other proteins. In this study we sought to identify proteins that associate with Cx45 by coprecipitation in non-denaturing conditions. Cx45 was isolated with a 220-kDa protein that we identified as ZO-1. Under the same conditions, Cx43 also was isolated with anti-Cx45 antiserum from Cx45-transfected ROS cells (ROS/Cx45 cells). Cx43 antiserum could also coprecipitate ZO-1 in the transfected and untransfected ROS cells. Double label immunofluorescence studies showed that ZO-1, Cx43, and Cx45 colocalized at appositional membranes in ROS/Cx45 cells suggesting that all three proteins are normally associated in the cells. Additionally, we found that in vitro translated ZO-1 binds to the carboxyl-terminal of Cx45 indicating that there is a direct interaction between the carboxyl-terminal of Cx45 and ZO-1. These studies demonstrate that ZO-1 interacts with Cx45 as well as with Cx43, and suggest that the interaction of connexins with ZO-1 may play a role in regulating the composition of the gap junction and may modulate connexin-connexin interactions.     

Association with ZO-1 Correlates with Plasma Membrane Partitioning in Truncated Connexin45 Mutants

Zonula occludens-1 (ZO-1), the most abundant known connexin-interacting protein in osteoblastic cells, associates with the carboxyl termini of both Cx43 and Cx45. To learn more about the role of the cormexin-ZO-1 interaction, we analyzed connexin trafficking and function in ROS 17/2.8 cells that were stably transfected either with full length Cx45 or with Cx45 lacking 34 or 37 amino acids on the carboxyl terminus (Cx45t34 or Cx45t37). All three proteins were transported to appositional membranes in the transfected cells: Cx45 and Cx45t34 displayed a punctate appositional membrane-staining pattern, while Cx45t37 staining at appositional membranes was more linear. Expression of Cx45 decreased gap junction communication as assayed by dye transfer, while expression of Cx45t34 or Cx45t37 increased the amount of dye transfer seen in these cells. We found that Cx43, Cx45 and Cx45t34 co-precipitated with ZO-1 in these cells, while Cx45t37 did not. We also found that Cx45t37 was much more soluble in 1% Triton X-100 than the other connexins examined. In addition, Cx45t37 migrated to a fraction of lighter buoyant density on sucrose flotation gradients than Cx43, Cx45, ZO-1 and Cx45t34. As ZO-1 is an actin-binding protein, this suggested that the differences in Cx45t37 solubility might be due to a difference between the interaction of gap junctions and the actin cytoskeleton in the ROS/Cx45t37 and in the other transfected ROS cells. To examine this possibility, the transfected ROS cells were stained with fluorescently labeled phalloidin and demonstrated that there was a notable loss of actin stress fibers in the ROS/Cx45t37 cells. These findings suggest that association with ZO-1 alters the plasma membrane localization of Cx45 by removing it from a lipid raft compartment and rendering it Triton-insoluble, presumably by promoting an interaction with the actin cytoskeleton; they also suggest that Cx45 has a complex binding interaction with ZO-1 that involves either an extended carboxyl terminal domain or two distinct binding sites.     

Transfected connexin45 alters gap junction permeability in cells expressing endogenous connexin43

Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.     

Connexin43 PDZ2 binding domain mutants create functional gap junctions and exhibit altered phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43deltaI382) or the last five amino acids (Cx43delta378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43deltaI382 and Cx43 delta378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage-specific manner

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry (MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 (Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 (ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein "pull down," co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.     

Connexin43 PDZ2 Binding Domain Mutants Create Functional Gap Junctions and Exhibit Altered Phosphorylation

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43ΔI382) or the last five amino acids (Cx43Δ378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43ΔI382 and Cx43Δ378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.     

Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells.

We examined the expression and function of gap junctions in two rat osteoblastic cell lines, ROS 17/2.8 and UMR 106-01. The pattern of expression of gap junction proteins in these two cell lines was distinct: ROS cells expressed only connexin43 on their cell surface, while UMR expressed predominantly connexin45. Immunoprecipitation and RNA blot analysis confirmed the relative quantitation of these connexins. Microinjected ROS cells passed Lucifer yellow to many neighboring cells, but UMR cells were poorly coupled by this criterion. Nevertheless, both UMR and ROS cells were electrically coupled, as characterized by the double whole cell patch-clamp technique. These studies suggested that Cx43 in ROS cells mediated cell-cell coupling for both small ions and larger molecules, but Cx45 in UMR cells allowed passage only of small ions. To demonstrate that the expression of different connexins alone accounted for the lack of dye coupling in UMR cells, we assessed dye coupling in UMR cells transfected with either Cx43 or Cx45. The UMR/Cx43 transfectants were highly dye coupled compared with the untransfected UMR cells, but the UMR/Cx45 transfectants demonstrated no increase in dye transfer. These data demonstrate that different gap junction proteins create channels with different molecular permeabilities; they suggest that different connexins permit different types of signalling between cells.     

A proposed role for ZO-1 in targeting connexin 43 gap junctions to the endocytic pathway

Gap junctions are intercellular channels organized in plaque that directly link adjacent cells. Connexins (Cx), the constitutive proteins of gap junctions are associated with several partner proteins (cytoskeletal, anchoring) which could participate in plaque formation and degradation. Coimmunoprecipitation and indirect immunofluorescence analyses showed that ZO-1, a tight junction-associated protein, was linked to Cx43 in the testis. By using gamma-hexachlorocyclohexane (HCH), known to induce gap junction endocytosis, we demonstrated that endocytosis increased Cx43/ZO-1 association within the cytoplasm of treated Sertoli cells. In control cells, the two proteins were present, as expected, at the plasma membrane level, but poorly colocalized. The increased intracytoplasmic Cx43/ZO-1 complex was associated with a shift towards increased levels of Cx43 P1 and P2 isoforms. The HCH induced Cx43 hyperphosphorylation was abolished by the ERK inhibitor PD98059 suggesting that this effect could be mediated through activation of the ERK pathway. These data strongly support a novel role for ZO-1 in the turnover of Cx43 during gap junction plaque endocytosis.     

Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

We tested the hypothesis that fluidshear stress () modifies the expression, function, and distributionof junctional proteins [connexin (Cx)43, Cx45, and zona occludens(ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to-values of 5 or 20 dyn/cm² for 1-3 h.Immunostaining indicated that at 5 dyn/cm², thedistribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cellmembranes; at 20 dyn/cm², disruption was more severe.Intercellular coupling was significantly decreased at both shear stresslevels. Western blots showed the downregulation of membrane-bound Cx43and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at differentlevels of shear stress. Similarly, Northern blots revealed thatexpression of Cx43, Cx45, and ZO-1 was selectively up- anddownregulated in response to different shear stress levels. Theseresults indicate that in cultured bone cells, fluid shear stressdisrupts junctional communication, rearranges junctional proteins, anddetermines de novo synthesis of specific connexins to an extent thatdepends on the magnitude of the shear stress. Such disconnection fromthe bone cell network may provide part of the signal whereby thedisconnected cells or the remaining network initiate focal bone remodeling.

     

Association of connexin36 with zonula occludens-1 in HeLa cells, betaTC-3 cells, pancreas, and adrenal gland

The PDZ domain-containing protein zonula occludens-1 (ZO-1), a well-established component of tight junctions, has recently been shown to interact with various connexin proteins that form gap junctions. We investigated the association of connexin36 (Cx36) with ZO-1 in various cultured cells and tissues. Punctate immunofluorescence labeling for Cx36 was detected in Cx36-transfected HeLa cells, betaTC-3 cells, pancreatic islets, and adrenal medulla. Immunofluorescence for ZO-1 was also punctate in cells and tissues, and was colocalized with Cx36 at points of cell-cell contact. Immunoprecipitation of either Cx36 or ZO-1 from cell lysates and tissue homogenates resulted in immunoblot detection of ZO-1 or Cx36, respectively, in immunoprecipitates. A 14-amino acid peptide corresponding to the carboxy-terminus of Cx36 showed binding capacity to the PDZ1 domain of ZO-1, which was eliminated after removal of the last 4 carboxy-terminus amino acids. Low micromolar concentrations of the 14-amino acid peptide produced up to 85% inhibition of Cx36 interaction with the PDZ1 domain of ZO-1. These results provide evidence for molecular interaction between Cx36 and ZO-1 in vitro, and in vivo, and suggest that the interference with Cx36/ZO-1 interaction by short carboxy-terminus peptides of Cx36 may be of value for functional studies of this interaction.     

Identification of Connexin-43 Interacting Proteins

Connexin-43 (Cx43), the most ubiquitously expressed vertebrate gap junction protein, has been shown to interact directly with Zonula Occludens-1 (ZO-1). Although several potential functions have been proposed for the ZO-1/Cx43 interaction, the role that ZO-1 and other Cx43-interacting partners play in the regulation of Cx43 trafficking, assembly, gating and turnover are not well understood. We believed a thorough analysis and classification of other Cx43-interacting proteins might help us to understand and better test these roles. We approached this question by utilizing Tandem Mass Spectrometry (MS/MS) analysis to identify proteins from normal rat kidney whole cell lysates that could interact with the C-terminal region of Cx43. Comparison against protein sequence databases identified 19 probable protein matches, including kinases, phosphatases, membrane receptors, cell signaling molecules and scaffolding proteins. We have further characterized some of these interacting proteins, including Zonula Occludens-2 (ZO-2), via western blotting and “pull down” experiments. Further in vitro/in vivoanalysis of these interacting proteins will help in our understanding of the global role of connexins in regulating development, cell metabolism and growth.     

Identification of connexin-43 interacting proteins

Connexin-43 (Cx43), the most ubiquitously expressed vertebrate gap junction protein, has been shown to interact directly with Zonula Occludens-1 (ZO-1). Although several potential functions have been proposed for the ZO-1/Cx43 interaction, the role that ZO-1 and other Cx43-interacting partners play in the regulation of Cx43 trafficking, assembly, gating and turnover are not well understood. We believed a thorough analysis and classification of other Cx43-interacting proteins might help us to understand and better test these roles. We approached this question by utilizing Tandem Mass Spectrometry (MS/MS) analysis to identify proteins from normal rat kidney whole cell lysates that could interact with the C-terminal region of Cx43. Comparison against protein sequence databases identified 19 probable protein matches, including kinases, phosphatases, membrane receptors, cell signaling molecules and scaffolding proteins. We have further characterized some of these interacting proteins, including Zonula Occludens-2 (ZO-2), via western blotting and "pull down" experiments. Further in vitro/in vivo analysis of these interacting proteins will help in our understanding of the global role of connexins in regulating development, cell metabolism and growth.     

Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P₁ phosphorylated and P₀ unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.     

Lens connexins alpha3Cx46 and alpha8Cx50 interact with zonula occludens protein-1 (ZO-1)

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.     

Association of connexin36 with zonula occludens-1 in HeLa cells, βTC-3 cells, pancreas, and adrenal gland

The PDZ domain-containing protein zonula occludens-1 (ZO-1), a well-established component of tight junctions, has recently been shown to interact with various connexin proteins that form gap junctions. We investigated the association of connexin36 (Cx36) with ZO-1 in various cultured cells and tissues. Punctate immunofluorescence labeling for Cx36 was detected in Cx36-transfected HeLa cells, TC-3 cells, pancreatic islets, and adrenal medulla. Immunofluorescence for ZO-1 was also punctate in cells and tissues, and was colocalized with Cx36 at points of cell–cell contact. Immunoprecipitation of either Cx36 or ZO-1 from cell lysates and tissue homogenates resulted in immunoblot detection of ZO-1 or Cx36, respectively, in immunoprecipitates. A 14-amino acid peptide corresponding to the carboxy-terminus of Cx36 showed binding capacity to the PDZ1 domain of ZO-1, which was eliminated after removal of the last 4 carboxy-terminus amino acids. Low micromolar concentrations of the 14-amino acid peptide produced up to 85% inhibition of Cx36 interaction with the PDZ1 domain of ZO-1. These results provide evidence for molecular interaction between Cx36 and ZO-1 in vitro, and in vivo, and suggest that the interference with Cx36/ZO-1 interaction by short carboxy-terminus peptides of Cx36 may be of value for functional studies of this interaction.*co-1^st authors