M. Cordenonsi et al., Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin, J CELL BIOL, 147(7), 1999, pp. 1569-1581
We characterized the sequence and protein interactions of cingulin, an M-r
140-160-kD phosphoprotein localized on the cytoplasmic surface of epithelia
l tight junctions (TJ). The derived amino acid sequence of a full-length Xe
nopus 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). Seq
uence analysis, electron microscopy, and pull-down assays indicate that the
cingulin rod is responsible for the formation of coiled-coil parallel dime
rs, which can further aggregate through intermolecular interactions. Pull-d
own assays from epithelial, insect cell, and reticulocyte lysates show that
an NH2-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1
(K-d similar to 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 inter
actions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragm
ents, colocalizes with endogenous cingulin in transfected MDCK cells, indic
ating that sequences within both head and rod domains are required for TJ l
ocalization. We propose that cingulin is a functionally important component
of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytos
keleton.