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

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Abstract

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, βTC-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.

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References

  • Balda MS, Matter K (2000) The tight junction protein ZO-1 and an interacting transcription factor regulate ErbB-2 expression. EMBO J 19:2024–2033

    Article  CAS  PubMed  Google Scholar 

  • Balda MS, Anderson JM, Matter K (1996) The SH3 domain of the tight junction protein ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this domain. FEBS Lett 399:326–332

    Article  CAS  PubMed  Google Scholar 

  • Becamel C, Gavarini S, Chanrion B, Alonso G, Galeotti N, Dumuis A, Bockaert J, Marin P (2004) The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins. J Biol Chem 279:20257–20266

    Article  CAS  PubMed  Google Scholar 

  • Bischof LJ, Martin CC, Svitek CA, Stadelmaier BT, Hornbuckle LA, Goldman JK, Oeser JK, Hutton JC, O’Brien RM (2001) Characterization of the mouse islet-specific glucose-6-phosphatase catalytic subunit-related protein gene promoter by in situ footprinting: correlation with fusion gene expression in the islet-derived βTC-3 and hamster insulinoma tumor cell lines. Diabetes 50:502–514

    CAS  PubMed  Google Scholar 

  • Calabrese A, Güldenagel M, Charollais A, Mas C, Caton D, Bauquis J, Serre-Beinier V, Caille D, Söhl G, Teubner B, Le Gurun S, Trovato-Salinaro A, Condorelli DF, Haefliger JA, Willecke K, Meda P (2001) Cx36 and the function of endocrine pancreas. Cell Commun Adhes 8:387–391

    CAS  PubMed  Google Scholar 

  • Calabrese A, Zhang M, Serre-Beinier V, Caton D, Mas C, Satin LS, Meda P (2003) Connexin36 controls synchronization of Ca2+ oscillations and insulin secretion in MIN6 cells. Diabetes 52:417–424

    CAS  PubMed  Google Scholar 

  • Calabrese A, Caton D, Meda P (2004) Differentiating the effects of Cx36 and E-cadherin for proper insulin secretion of MIN6 cells. Exp Cell Res 294:379–391

    Article  CAS  PubMed  Google Scholar 

  • Chen ML, Pothoulakis C, LaMont JT (2002) Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A. J Biol Chem 277:4247–4254

    Article  CAS  PubMed  Google Scholar 

  • Condorelli DF, Parenti R, Spinella F, Trovato-Salinare A, Belluardo N, Cardile V, Cicirata F (1998) Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Eur J Neurosci 10:1202–1208

    Google Scholar 

  • Condorelli DF, Belluardo N, Trovato-Salinaro A, Mudo G (2000) Expression of Cx36 in mammalian neurons. Brain Res Brain Res Rev 32:72–85

    CAS  PubMed  Google Scholar 

  • Deans MR, Gibson JR, Sellitto C, Connors BW, Paul DL (2001) Synchronous activity of inhibitory networks in neocortex requires electrical synapses containing connexin36. Neuron 16:477–485

    Article  Google Scholar 

  • Denker BM, Saha C, Khawaja S, Nigam SK (1996) Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis. J Biol Chem 271:25750–25753

    Article  CAS  PubMed  Google Scholar 

  • Dodane V, Kachar B (1996) Identification of isoforms of G proteins and PKC that colocalize with tight junctions. J Membr Biol 149:199–209

    Article  CAS  PubMed  Google Scholar 

  • Doyle JP, Stempak JG, Cowin P, Colman DR, D’Urso D (1995) Protein zero, a nervous system adhesion molecule, triggers epithelial reversion in host carcinoma cells. J Cell Biol 131:465–482

    Article  CAS  PubMed  Google Scholar 

  • Giepmans BN, Moolenaar WH (1998) The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr Biol 8:931–934

    CAS  PubMed  Google Scholar 

  • Gonzalez-Mariscal L, Betanzos A, Avila-Flores A (2000) MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol 11:315–324

    Article  PubMed  Google Scholar 

  • Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE (2003) Tight junction proteins. Prog Biophys Mol Biol 81:1–44

    Article  PubMed  Google Scholar 

  • Goodenough DA, Goliger JA, Paul DL (1996) Connexins, connexons, and intercellular communication. Annu Rev Biochem 65:475–502

    Article  CAS  PubMed  Google Scholar 

  • Grynszpan-Wynograd O, Nicolas G (1980) Intercellular junctions in the adrenal medulla: a comparative freeze-fracture study. Tissue Cell 12:661–672

    Article  CAS  PubMed  Google Scholar 

  • Howarth AG, Hughes MR, Stevenson BR (1992) Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am J Physiol 262:C461–C469

    CAS  PubMed  Google Scholar 

  • In’t Veld PA, Pipeleers DG, Gepts W (1984) Evidence against the presence of tight junctions in normal endocrine pancreas. Diabetes 33:101–104

    PubMed  Google Scholar 

  • Itoh M, Nagafuchi A, Yonemura S, Kitani-Yasuda T, Tsukita S, Tsukita S (1993) The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol 121:491–502

    Article  CAS  PubMed  Google Scholar 

  • Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S (1999) Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2 and ZO-3, with the COOH termini of claudins. J Cell Biol 147:1351–1363

    Article  CAS  PubMed  Google Scholar 

  • Kausalya PJ, Reichert M, Hunziker W (2001) Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells. FEBS Lett 505:92–96

    Article  CAS  PubMed  Google Scholar 

  • Kleeff J, Shi X, Bode HP, Hoover K, Shrikhande S, Bryant PJ, Korc M, Buchler MW, Friess H (2001) Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer. Pancreas 23:259–265

    Article  CAS  PubMed  Google Scholar 

  • Kojima T, Kokai Y, Chiba H, Yamamoto M, Mochizuki Y, Sawada N (2001) Cx32 but not Cx26 is associated with tight junctions in primary cultures of rat hepatocytes. Exp Cell Res 263:193–201

    Article  CAS  PubMed  Google Scholar 

  • Kumar NM, Gilula NB (1996) The gap junction communication channel. Cell 84:381–388

    Article  CAS  PubMed  Google Scholar 

  • Laing JG, Manley-Markowski RN, Koval M, Civitelli R, Steinberg TH (2001a) Connexin45 interacts with zonula occludens-1 and connexin43 in osteoblastic cells. J Biol Chem 276:23051–23055

    Article  CAS  PubMed  Google Scholar 

  • Laing JG, Manley-Markowski RN, Koval M, Civitelli R, Steinberg TH (2001b) Connexin45 interacts with zonula occludens-1 in osteoblastic cells. Cell Commun Adhes 8:209–212

    CAS  PubMed  Google Scholar 

  • Le Gurun S, Martin D, Formenton A, Maeschler P, Caille D, Waeber G, Meda P, Haefliger JA (2003) Connexin36 contributes to control function of insulin-producing cells. J Biol Chem 278:37690–37697

    Article  PubMed  Google Scholar 

  • Li X, Lynn BD, Olson C, Meier C, Davidson KG, Yasumura T, Rash JE, Nagy JI (2002) Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve. Eur J Neurosci 16:795–806

    Article  PubMed  Google Scholar 

  • Li X, Ionescu AV, Lynn BD, Lu S, Kamasawa N, Morita M, Davidson KGV, Yasumura T, Rash JE, Nagy JI (2004a) Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and Cx47 association with zonula occludens-1 (ZO-1) in mouse brain. Neuroscience 126:611–630

    Article  CAS  PubMed  Google Scholar 

  • Li X, Olson C, Lu S, Kamasawa N, Yasumura T, Rash JE, Nagy JI (2004b) Neuronal connexin36 association with zonula occludens-1 protein (ZO-1) in mouse brain and interaction with the first PDZ domain of ZO-1. Eur J Neurosci 19:2132–2146

    Article  PubMed  Google Scholar 

  • Lynn BD, Li X, Cattini PA, Turley EA, Nagy JI (2001) Identification of sequence, protein isoforms, and distribution of the hyaluronan-binding protein RHAMM in adult and developing rat brain. J Comp Neurol 439:315–330

    Article  CAS  PubMed  Google Scholar 

  • Martin AQ, Mathieu MN, Chevillard C, Guerineau NC (2001) Gap junctions mediate electrical signaling and ensuing cytosolic Ca2+ increases between chromaffin cells in adrenal slices: a role in catecholamine release. J Neurosci 21:5397–5405

    CAS  PubMed  Google Scholar 

  • Martin AQ, Mathieu MN, Guerineau NC (2003) Evidence for long-lasting cholinergic control of gap junctional communication between adrenal chromaffin cells. J Neurosci 23:3669–3678

    CAS  PubMed  Google Scholar 

  • Matter K, Balda MS (2003) Signalling to and from tight junctions. Nat Rev Mol Cell Biol 4:225–236

    Article  CAS  PubMed  Google Scholar 

  • Meda P, Pepper MS, Traub O, Willecke K, Gros D, Beyer E, Nicholson B, Paul D, Orci L (1993) Differential expression of gap junction connexins in endocrine and exocrine glands. Endocrinology 133:2371–2378

    Article  CAS  PubMed  Google Scholar 

  • Meyer TN, Schwesinger C, Denker BM (2002) Zonula occludens-1 is a scaffolding protein for signaling molecules. Galpha(12) directly binds to the Src homology 3 domain and regulates paracellular permeability in epithelial cells. J Biol Chem 277:24855–24858

    Article  CAS  PubMed  Google Scholar 

  • Mitic LL, Anderson JM (1998) Molecular architecture of tight junctions. Annu Rev Physiol 60:121–142

    Article  CAS  PubMed  Google Scholar 

  • Moser T (1998) Low-conductance intercellular coupling between mouse chromaffin cells in situ. J Physiol 506:195–205

    Article  CAS  PubMed  Google Scholar 

  • Murray SA, Davis K, Gay V (2003) ACTH and adrenocortical gap junctions. Microsc Res Tech 61:240–246

    Article  CAS  PubMed  Google Scholar 

  • Nagy JI, Li X, Rempel J, Stelmack G, Patel D, Staines WA, Yasumura T, Rash JE (2001) Connexin26 in adult rodent central nervous system: demonstration at astrocytic gap junctions and co-localization with connexin30 and connexin43. J Comp Neurol 441:302–323

    Article  CAS  PubMed  Google Scholar 

  • Nielsen PA, Baruch A, Giepmans BN, Kumar NM (2001) Characterization of the association of connexins and ZO-1 in the lens. Cell Commun Adhes 8:213–217

    CAS  PubMed  Google Scholar 

  • Nielsen PA, Beahm DL, Giepmans BN, Baruch A, Hall JE, Kumar NM (2002) Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1. J Biol Chem 277:38272–38283

    Article  CAS  PubMed  Google Scholar 

  • Nielsen PA, Baruch A, Shestopalov VI, Giepmans BN, Benedetti EI, Kumar NM (2003) Lens connexins Cx46 and CxCHCHx50 interact with zonula occludens protein-1 (ZO-1). Mol Biol Cell 14:2470–2481

    Article  CAS  PubMed  Google Scholar 

  • Orci L (1974) A portrait of the pancreatic B-cell. Diabetologia 10:163–187

    CAS  PubMed  Google Scholar 

  • Orci L, Malaisse-Lagae F, Amherdt M, Ravazzola M, Weisswange A, Dobbs R, Perrelet A, Unger R (1975a) Cell contacts in human islets of Langerhans. J Clin Endocrinol Metab 41:841–844

    CAS  PubMed  Google Scholar 

  • Orci L, Malaisse-Lagae F, Ravazzola M, Rouiller D, Renold AE, Perrelet A, Unger R (1975b) A morphological basis for intercellular communication between alpha- and beta-cells in the endocrine pancreas. J Clin Invest 56:1066–1070

    CAS  PubMed  Google Scholar 

  • Rash JE, Staines WA, Yasumura T, Patel D, Furman CS, Stelmack GL, Nagy JI (2000) Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43. Proc Natl Acad Sci U S A 97:7573–7578

    Article  CAS  PubMed  Google Scholar 

  • Rash JE, Yasumura T, Dudek FE, Nagy JI (2001) Cell-specific expression of connexins and evidence of restricted gap junctional coupling between glial cells and between neurons. J Neurosci 15:1983–2000

    Google Scholar 

  • Scadding JW (1981) Development of ongoing activity, mechanosensitivity, and adrenaline sensitivity in severed peripheral nerve axons. Exp Neurol 73:345–364

    Article  CAS  PubMed  Google Scholar 

  • Schnabel E, Anderson JM, Farquhar MG (1990) The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium. J Cell Biol 111:1255–1263

    Article  CAS  PubMed  Google Scholar 

  • Serre-Beinier V, Le Gurun S, Belluardo N, Trovato-Salinaro A, Charollais A, Haefliger JA, Condorelli DF, Meda P (2000) Cx36 preferentially connects beta-cells within pancreatic islets. Diabetes 49:727–734

    CAS  PubMed  Google Scholar 

  • Serre-Beinier V, Mas C, Calabrese A, Caton D, Bauquis J, Caille D, Charollais A, Cirulli V, Meda P (2002) Connexins and secretion. Biol Cell 94:477–492

    Article  CAS  PubMed  Google Scholar 

  • Sheng M, Sala C (2001) PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24:1–29

    Article  CAS  PubMed  Google Scholar 

  • Söhl G, Degen J, Teubner B, Willecke K (1998) The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development. FEBS Lett 428:27–31

    Article  PubMed  Google Scholar 

  • Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC (1997) Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275:73–77

    Article  CAS  PubMed  Google Scholar 

  • Spiryda LB, Colman DR (1998) Protein zero, a myelin IgCAM, induces physiologically operative tight junctions in nonadhesive carcinoma cells. J Neurosci Res 54:282–288

    Article  CAS  PubMed  Google Scholar 

  • Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA (1986) Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 103:755–766

    Article  CAS  PubMed  Google Scholar 

  • Stuart RQ, Nigam SK (1995) Regulated assembly of tight junctions by protein kinase C. Proc Natl Acad Sci U S A 20:6072–6076

    Google Scholar 

  • Theis M, Mas C, Doring B, Degen J, Brink C, Caille D, Charollais A, Krüger O, Plum A, Nepote V, Herrera P, Meda P, Willecke K (2004) Replacement by a lacZ reporter gene assigns mouse connexin36, 45 and 43 to distinct cell types in pancreatic islets. Exp Cell Res 294:18–29

    Article  CAS  PubMed  Google Scholar 

  • Thomas MA, Huang S, Cokoja A, Riccio O, Staub O, Suter S, Chanson M (2002) Interaction of connexins with protein partners in the control of channel turnover and gating. Biol Cell 94:445–456

    Article  CAS  PubMed  Google Scholar 

  • Toyofuku T, Yabuki M, Otsu K, Kuzuya T, Hori M, Tada M (1998) Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes. J Biol Chem 273:12725–12731

    Article  CAS  PubMed  Google Scholar 

  • Unsicker K, Seidl K, Hofmann HD (1989) The neuro-endocrine ambiguity of sympathoadrenal cells. Int J Dev Neurosci 7:413–417

    Article  CAS  PubMed  Google Scholar 

  • White TW, Bruzzone R (1996) Multiple connexin proteins in single intercellular channels: connexin compatibility and functional consequences. J Bioenerg Biomembr 28:339–350

    CAS  PubMed  Google Scholar 

  • White TW, Paul DL (1999) Genetic diseases and gene knockouts reveal diverse connexin functions. Annu Rev Physiol 61:283–310

    Article  CAS  PubMed  Google Scholar 

  • Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Güldenagel M, Deutsch U, Söhl G (2002) Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem 383:725–737

    Article  CAS  PubMed  Google Scholar 

  • Yamagami K, Moritoyo T, Wakamori M, Sorimachi M (2002) Limited intercellular spread of spontaneous Ca2+ signals via gap junctions between mouse chromaffin cells in situ. Neurosci Lett 323:97–100

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto T, Hertzberg EL, Nagy JI (1995) Differential distribution of connexin43-immunoreactive gap junctions in the rat adrenal cortex. In: Kanno Y (ed) Progress in cell research, vol 4. Elsevier, Amsterdam, pp 289–292

  • Yokoyama S, Tachibana K, Nakanishi H , Yamamoto Y, Irie K, Mandai K, Nagafuchi A, Monden M, Takai Y (2001) Alpha-catenin-independent recruitment of ZO-1 to nectin-based cell-cell adhesion sites through afadin. Mol Biol Cell 12:1595–1609

    CAS  PubMed  Google Scholar 

  • Zhurinsky J, Shtutman M, Ben-Ze’ev A (2000) Differential mechanisms of LEF/TCF family-dependent transcriptional activation by beta-catenin and plakoglobin. Mol Cell Biol 20:4238–4252

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Brett McLean and Nora Nolette for excellent technical assistance. We also thank Dr. David Paul (Department of Neuroscience, Harvard University) for providing heterozygous Cx36/LacZ mice, and Dr. Christopher McIntosh (Department of Physiology, University of British Columbia) for providing βTC-3 cells. This work was supported by grants from the Canadian Institutes of Health Research to J.I.N.

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  1. Shijun Lu

    Present address: Department of Pathology, Weifang Medical College, Peoples’ Republic of China

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  1. Department of Physiology, Faculty of Medicine, University of Manitoba, 730 William Avenue, Winnipeg, Manitoba, R3E 3J7, Canada

    Xinbo Li*, Carl Olson*, Shijun Lu & James I. Nagy

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Correspondence to James I. Nagy.

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Li*, X., Olson*, C., Lu, S. et al. Association of connexin36 with zonula occludens-1 in HeLa cells, βTC-3 cells, pancreas, and adrenal gland. Histochem Cell Biol 122, 485–498 (2000). https://doi.org/10.1007/s00418-004-0718-5

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