Cingulin contains globular and coiled-coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin

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.