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© The Rockefeller University Press, 0021-9525/1999/12/1569/ $5.00
The Journal of Cell Biology, Volume 147, Number 7, December 27, 1999 1569-1582

Original Article

Cingulin Contains Globular and Coiled-coil Domains and Interacts with ZO-1, ZO-2, ZO-3, and Myosin

Michelangelo Cordenonsia, Fabio D'Atrib,c, Eva Hammarc, David A.D. Parryd, John Kendrick-Jonese, David Shorec, and Sandra Citia,c
a Department of Biology, University of Padova, 35121 Padova, Italy
b Department of Biochemistry, University of Geneva, 1211 Geneva 4, Switzerland
c Department of Molecular Biology, University of Geneva, 1211 Geneva 4, Switzerland
d Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
e Medical Research Council Laboratory of Molecular Biology, Cambridge, CB22QH UK

Correspondence to: Sandra Citi, Department of Molecular Biology, University of Geneva, 30 Quai Ernest Ansermet, 1211 Geneva 4, Switzerland. Tel:41-22-7026182 Fax:41-22-7026868

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central {alpha}-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ~5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

Key Words: cingulin, tight junction, epithelia, MDCK, protein


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