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 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 NH(2)-terminal fragment of cingulin (1-378) interacts in vitro with ZO-1 (K(d) approximately 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 NH(2)-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.
Cordenonsi
M
M
Department of Biology, University of Padova, 35121 Padova, Italy.
D'Atri
F
F
Hammar
E
E
Parry
D A
DA
Kendrick-Jones
J
J
Shore
D
D
Citi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D016547:Kinesins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D014162:Transfection; D029867:Xenopus Proteins; D014982:Xenopus laevis; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
C489228:CGN protein, Xenopus; D002352:Carrier Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C569045:TJP1 protein, Xenopus; D029867:Xenopus Proteins; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D009218:Myosins; D016547:Kinesins