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First published online 20 May 2003
doi: 10.1242/jcs.00482

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The type 3 inositol 1,4,5-trisphosphate receptor is concentrated at the tight junction level in polarized MDCK cells

Pascal Colosetti^*,,1, Richard E. A. Tunwell^,2, Caroline Cruttwell¹, Jean-Pierre Arsanto³, Jean-Pierre Mauger^1, and Doris Cassio¹

¹ INSERM U-442, Signalisation cellulaire et calcium, Bât 443, Université Paris-Sud, 91405 Orsay Cedex, France
² Department of Anatomy & Developmental Biology, University College London, Gower Street Campus, London WC1E 6BT, UK
³ Laboratoire de Neurogenése et Morphogenése dans le Développement et chez l'Adulte (NMDA, Unité Mixte de Recherche 6156), Institut de Biologie du Développement de Marseille, Faculté des Sciences de Luminy, Université de la Méditerranée, case 907, 13288 Marseille Cedex 09, France

Author for correspondence (e-mail: jean-pierre.mauger{at}ibaic.u-psud.fr)

Accepted 14 March 2003

The subcellular localization of inositol 1,4,5-trisphosphate (InsP₃)-induced Ca²⁺ signals is important for the activation of many physiological functions. In epithelial cells the spatial distribution of InsP₃ receptor is restricted to specific areas, but little is known about the relationship between the receptor's distribution and cell polarity. To investigate this relationship, the best known polarized cell model, MDCK, was examined. This cell line is characterized by a strong expression of the type 3 InsP₃ receptor and the subcellular localization of this receptor was followed during cell polarization using immunofluorescence and confocal analysis. In non-polarized cells, including ras transformed f3 MDCK cells, the type 3 InsP₃ receptor was found to co-localize with markers of the endoplasmic reticulum in the cytoplasm. In contrast, in polarized cells, this receptor was mostly distributed at the apex of the lateral plasma membrane with the markers of tight junctions, ZO-1 and occludin. The localization of the type 3 InsP₃ receptor in the vicinity of tight junctions was confirmed by immunogold electron microscopy. The culture of MDCK cells in calcium-deprived medium, led to disruption of cell polarity and receptor redistribution in the cytoplasm. Addition of calcium to these deprived cells induced the restoration of polarity and the relocalization of the receptor to the plasma membrane. MDCK cells were stably transfected with a plasmid coding the full-length mouse type 1 InsP₃ receptor tagged with EGFP at the C-terminus. The EGFP-tagged type 1 receptor and the endogenous type 3 co-localized in the cytoplasm of non-polarized cells and at the tight junction level of polarized cells. Thus, the localization of InsP₃ receptor in MDCK depends on polarity.

Key words: Inositol 1,4,5-trisphosphate receptor, Tight junction, Polarity, Calcium, Endoplasmic reticulum, InsP₃R-1-EGFP

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