Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule.
Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.
Balzar
M
M
Department of Pathology, Leiden University Medical Centre, Leiden, The Netherlands.
Bakker
H A
HA
Briaire-de-Bruijn
I H
IH
Fleuren
G J
GJ
Warnaar
S O
SO
Litvinov
S V
SV
D000185:Actinin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D002461:Cell Line, Transformed; D015638:Cytochalasin D / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D000071858:Epithelial Cell Adhesion Molecule; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D013347:Subcellular Fractions
D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D002457:Cell Extracts; D003902:Detergents; D000071858:Epithelial Cell Adhesion Molecule; D019384:Nucleic Acid Synthesis Inhibitors; D000185:Actinin; D015638:Cytochalasin D; D017830:Octoxynol