Hostname: page-component-7479d7b7d-jwnkl Total loading time: 0 Render date: 2024-07-14T17:50:52.013Z Has data issue: false hasContentIssue false
  • English
  • Français

Connexin 36 in bovine retina: Lack of phosphorylation but evidence for association with phosphorylated proteins

Published online by Cambridge University Press:  18 November 2003

ARI SITARAMAYYA ,
JOHN W. CRABB ,
DIANE F. MATESIC ,
ALEXANDER MARGULIS ,
VINITA SINGH ,
SADHONA PULUKURI  and
LOAN DANG
ARI SITARAMAYYA
Affiliation:
Eye Research Institute, Oakland University, Rochester
JOHN W. CRABB
Affiliation:
Cole Eye Institute, Cleveland Clinic Foundation, Cleveland
DIANE F. MATESIC
Affiliation:
Department of Pharmaceutical Sciences, Mercer University, Atlanta
ALEXANDER MARGULIS
Affiliation:
Eye Research Institute, Oakland University, Rochester
VINITA SINGH
Affiliation:
Eye Research Institute, Oakland University, Rochester
SADHONA PULUKURI
Affiliation:
Eye Research Institute, Oakland University, Rochester
LOAN DANG
Affiliation:
Eye Research Institute, Oakland University, Rochester
Get access Rights & Permissions [Opens in a new window]

Abstract

In vertebrate retina interneuronal communication through gap junctions is involved in light adaptation and in the transfer of visual information from the rod pathway to the cone pathway. Reports over the last two decades have indicated that these gap junctions are regulated by cyclic nucleotide-dependent protein kinases suggesting that the gap junction proteins, connexins, are phosphorylated. Though all the connexins involved in light adaptation and information transfer from rod to cone pathway are not yet known, connexin 36 has been shown to be definitively involved in the latter process. We have therefore attempted to investigate the cyclic nucleotide-dependent phosphorylation of this connexin in bovine retina. We found several soluble and membrane proteins in bovine retina whose phosphorylation was regulated by cyclic nucleotides. However, no protein of about 36 kDa with cyclic nucleotide-regulated phosphorylation was found in gap junction-enriched membrane preparations. A 36-kDa phosphorylated protein was found in gap junction-enriched membranes phosphorylated in the presence of calcium. However, this protein was not immunoprecipitated by anti-connexin 36 antibodies indicating that it was not connexin 36 in spite of its similarity in molecular weight. Immunoprecipitation did reveal phosphorylated proteins coimmunoprecipitated with connexin 36. Two of these proteins were identified as beta and alpha tubulin subunits. Though cyclic GMP and calcium did not greatly influence the association of these proteins with connexin 36, the results suggest the possibility of connexin 36 associating with other proteins. Together, these observations indicate that interneuronal communication at gap junctions made by connexin 36 may not be regulated by direct phosphorylation of connexin 36, but possibly by phosphorylation of associated proteins.

Keywords

Connexin 36Gap junctionsConnexin-associated proteinsPhosphorylationRetina
Type
Research Article
Information
Visual Neuroscience , Volume 20 , Issue 4 , July 2003 , pp. 385 - 395
Copyright
2003 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Baldridge, W.H., Ball, A.K., & Miller, R.G. (1987). Dopaminergic regulation of horizontal cell gap junction particle density in goldfish retina. Journal of Comparative Neurology 265, 428–436.Google Scholar
Baldridge, W.H., Vaney, D.I., & Weiler, R. (1998). The modulation of intercellular coupling in the retina. Seminars in Cell and Developmental Biology 9, 311–318.Google Scholar
Becker, D., Bonness, V., & Mobbs, P. (1998). Cell coupling in the retina: Patterns and purpose. Cell Biology International 22, 781–792.Google Scholar
Belluardo, N., Mudo, G., Trovato-Salinaro, A., Le Gurun, S., Charollais, A., Serre-Beinier, V., Amato, G., Haefliger, J.A., Meda, P., & Condorelli, D.F. (2000). Expression of connexin36 in the adult and developing rat brain. Brain Research 865, 121–138.Google Scholar
Colbran, R.J., Smith, M.K., Schworer, C.M., Fong, Y.L., & Soderling, T.R. (1989). Regulatory domain of calcium/calmodulin-dependent protein kinase II. Mechanism of inhibition and regulation by phosphorylation. Journal of Biological Chemistry 264, 4800–4804.Google Scholar
Condorelli, D.F., Parenti, R., Spinella, F., Trovato, S.A., Belluardo, N., Cardile, V., & Cicirata, F. (1998). Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. European Journal of Neuroscience 10, 1202–1208.Google Scholar
Cook, J.E. & Becker, D.L. (1995). Gap junctions in the vertebrate retina. Microscopy Research and Technique 31, 408–419.Google Scholar
Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei, M., Hasan, A., Yan, L., Rayborn, M.E., Salomon, R.G., & Hollyfield, J.G. (2002). Drusen proteome analysis: An approach to the etiology of age-related macular degeneration. Proceedings of the National Academy of Sciences of the U.S.A. 99, 14682–14687.Google Scholar
Deans, M.R., Volgyi, B., Goodenough, D.A., Bloomfield, S.A., & Paul, D.L. (2002). Connexin36 is essential for transmission of rod-mediated visual signals in the mammalian retina. Neuron 36, 703–712.Google Scholar
DeVries, S.H. & Schwartz, E.A. (1992). Hemi-gap-junction channels in solitary horizontal cells of the catfish retina. Journal of Physiology 445, 201–230.Google Scholar
Falk, M.M. (2000). Cell-free synthesis for analyzing the membrane integration, oligomerization, and assembly characteristics of gap junction connexins. Methods 20, 165–179.Google Scholar
Feigenspan, A., Teubner, B., Willecke, K., & Weiler, R. (2001). Expression of neuronal connexin36 in AII amacrine cells of the mammalian retina. Journal of Neuroscience 21, 230–239.Google Scholar
Giepmans, B.N., Hengeveld, T., Postma, F.R., & Moolenaar, W.H. (2001a). Interaction of c-Src with gap junction protein connexin-43. Role in the regulation of cell–cell communication. Journal of Biological Chemistry 276, 8544–8549.Google Scholar
Giepmans, B.N., Verlaan, I., Hengeveld, T., Janssen, H., Calafat, J., Falk, M.M., & Moolenaar, W.H. (2001b). Gap junction protein connexin-43 interacts directly with microtubules. Current Biology 11, 1364–1368.Google Scholar
Giepmans, B.N., Verlaan, I., & Moolenaar, W.H. (2001c). Connexin-43 interactions with ZO-1 and alpha- and beta-tubulin. Cell Communication and Adhesion 8, 219–223.Google Scholar
Goodenough, D.A., Goliger, J.A., & Paul, D.L. (1996). Connexins, connexons, and intercellular communication. Annual Review of Biochemistry 65, 475–502.Google Scholar
Guldenagel, M., Sohl, G., Plum, A., Traub, O., Teubner, B., Weiler, R., & Willecke, K. (2000). Expression patterns of connexin genes in mouse retina. Journal of Comparative Neurology 425, 193–201.Google Scholar
Guldenagel, M., Ammermuller, J., Feigenspan, A., Teubner, B., Degen, J., Sohl, G., Willecke, K., & Weiler, R. (2001). Visual transmission deficits in mice with targeted disruption of the gap junction gene connexin36. Journal of Neuroscience 21, 6036–6044.Google Scholar
Hampson, E.C., Vaney, D.I., & Weiler, R. (1992). Dopaminergic modulation of gap junction permeability between amacrine cells in mammalian retina. Journal of Neuroscience 12, 4911–4922.Google Scholar
Hertzberg, E.L. & Gilula, N.B. (1979). Isolation and characterization of gap junctions from rat liver. Journal of Biological Chemistry 254, 2138–2147.Google Scholar
Kadle, R., Zhang, J.T., & Nicholson, B.J. (1991). Tissue-specific distribution of differentially phosphorylated forms of Cx43. Molecular and Cellular Biology 11, 363–369.Google Scholar
Kanemitsu, M.Y., Loo, L.W., Simon, S., Lau, A.F., & Eckhart, W. (1997). Tyrosine phosphorylation of connexin 43 by v-Src is mediated by SH2 and SH3 domain interactions. Journal of Biological Chemistry 272, 22824–22831.Google Scholar
Ko, L., Koestner, A., & Wechsler, W. (1980). Morphological characterization of nitrosourea-induced glioma cell lines and clones. Acta Neuropathologica (Berlin) 51, 23–31.Google Scholar
Kolb, H. & Famiglietti, E.V. (1974). Rod and cone pathways in the inner plexiform layer of cat retina. Science 186, 47–49.Google Scholar
Kolb, H. & Nelson, R. (1983). Rod pathways in the retina of the cat. Vision Research 23, 301–312.Google Scholar
Lampe, P.D. & Lau, A.F. (2000). Regulation of gap junctions by phosphorylation of connexins. Archives of Biochemistry and Biophysics 384, 205–215.Google Scholar
Lasater, E.M. (1987). Retinal horizontal cell gap junctional conductance is modulated by dopamine through a cyclic AMP-dependent protein kinase. Proceedings of the National Academy of Sciences of the U.S.A. 84, 7319–7323.Google Scholar
Lasater, E.M. & Dowling, J.E. (1985). Dopamine decreases conductance of the electrical junctions between cultured retinal horizontal cells. Proceedings of the National Academy of Sciences of the U.S.A. 82, 3025–3029.Google Scholar
Lu, C. & McMahon, D.G. (1997). Modulation of hybrid bass retinal gap junctional channel gating by nitric oxide. Journal of Physiology 499 (Pt 3), 689–699.Google Scholar
Matesic, D.F., Germak, J.A., Dupont, E., & Madhukar, B.V. (1993). Immortalized hypothalamic luteinizing hormone-releasing hormone neurons express a connexin 26-like protein and display functional gap junction coupling assayed by fluorescence recovery after photobleaching. Neuroendocrinology 58, 485–492.Google Scholar
Matesic, D.F., Rupp, H.L., Bonney, W.J., Ruch, R.J., & Trosko, J.E. (1994). Changes in gap-junction permeability, phosphorylation, and number mediated by phorbol ester and non-phorbol-ester tumor promoters in rat liver epithelial cells. Molecular Carcinogenesis 10, 226–236.Google Scholar
Matesic, D.F., Blommel, M.L., Sunman, J.A., Cutler, S.J., & Cutler, H.G. (2001). Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes. Cell Biology and Toxicology 17, 395–408.Google Scholar
Mills, S.L. & Massey, S.C. (1995). Differential properties of two gap junctional pathways made by AII amacrine cells. Nature 377, 734–737.Google Scholar
Mills, S.L. & Massey, S.C. (2000). A series of biotinylated tracers distinguishes three types of gap junction in retina. Journal of Neuroscience 20, 8629–8636.Google Scholar
Mills, S.L., O'Brien, J.J., Li, W., O'Brien, J., & Massey, S.C. (2001). Rod pathways in the mammalian retina use connexin 36. Journal of Comparative Neurology 436, 336–350.Google Scholar
Musil, L.S. & Goodenough, D.A. (1991). Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. Journal of Cell Biology 115, 1357–1374.Google Scholar
O'Brien, J., Al Ubaidi, M.R., & Ripps, H. (1996). Connexin 35: A gap-junctional protein expressed preferentially in the skate retina. Molecular Biology of the Cell 7, 233–243.Google Scholar
O'Brien, J., Bruzzone, R., White, T.W., Al Ubaidi, M.R., & Ripps, H. (1998). Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family. Journal of Neuroscience 18, 7625–7637.Google Scholar
O'Farrell, P.H. (1975). High resolution two-dimensional electrophoresis of proteins. Journal of Biological Chemistry 250, 4007–4021.Google Scholar
Piccolino, M., Neyton, J., & Gerschenfeld, H.M. (1984). Decrease of gap junction permeability induced by dopamine and cyclic adenosine 3′:5′-monophosphate in horizontal cells of turtle retina. Journal of Neuroscience 4, 2477–2488.Google Scholar
Piccolino, M., Witkovsky, P., & Trimarchi, C. (1987). Dopaminergic mechanisms underlying the reduction of electrical coupling between horizontal cells of the turtle retina induced by d-amphetamine, bicuculline, and veratridine. Journal of Neuroscience 7, 2273–2284.Google Scholar
Schubert, A.L., Schubert, W., Spray, D.C., & Lisanti, M.P. (2002). Connexin family members target to lipid raft domains and interact with caveolin-1. Biochemistry 41, 5754–5764.Google Scholar
Sitaramayya, A., Pozdnyakov, N., & Margulis, A. (2000). Nitric oxide influences protein phosphorylation in retina. Investigative Ophthalmology and Visual Science 41, S879.Google Scholar
Sohl, 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 Letters 428, 27–31.Google Scholar
Sohl, G., Guldenagel, M., Traub, O., & Willecke, K. (2000). Connexin expression in the retina. Brain Research Reviews 32, 138–145.Google Scholar
Teubner, B., Odermatt, B., Guldenagel, M., Sohl, G., Degen, J., Bukauskas, F., Kronengold, J., Verselis, V.K., Jung, Y.T., Kozak, C.A., Schilling, K., & Willecke, K. (2001). Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons. Journal of Neuroscience 21, 1117–1126.Google Scholar
West, K.A., Yan, L., Shadrach, K., Sun, J., Hasan, A., Miyagi, M., Crabb, J.S., Hollyfield, J.G., Marmorstein, A.D., & Crabb, J.W. (2003). Protein database: Human retinal pigment epithelium. Molecular Cell Proteomics 2, 37–49.Google Scholar
Xin, D. & Bloomfield, S.A. (1999). Comparison of the responses of AII amacrine cells in the dark- and light-adapted rabbit retina. Visual Neuroscience 16, 653–665.Google Scholar
Zoidl, G., Meier, C., Petrasch-Parwez, E., Zoidl, C., Habbes, H.W., Kremer, M., Srinivas, M., Spray, D.C., & Dermietzel, R. (2002). Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36). Journal of Neuroscience Research 69, 448–465.Google Scholar