Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin). Characterization and extractability of the protein complex from the cell cytostructure.
The transmembrane epithelial cell-cell adhesion protein E-cadherin (uvomorulin) associates via its cytoplasmic domain with three or more proteins whose structure and function are not yet established. The associated proteins, also termed catenins (Ozawa, M., Baribault, H., and Kemler, R. (1989) EMBO J. 8, 1711-1717), are of interest because they may form a link to the cytoskeleton, and/or regulate E-cadherin function. In this report immunoprecipitates of E-cadherin complexes, isolated from Xenopus laevis A6 and Madin-Darby canine kidney cell monolayers, were stringently washed to leave a single very tightly associated protein of approximately 92 kDa. We report on the 92-kDa protein's association with E-cadherin in the presence of various perturbants, its preferential dissociation from the immune complex upon exposure to mixed detergent micelles containing sodium dodecyl sulfate, and its preparative purification to homogeneity. We have additionally found that E-cadherin and its associated proteins may be easily and quantitatively extracted from both subconfluent and fully confluent cells by a variety of mild nonionic detergents made up in isotonic buffers. In contrast, such extractions at short times left most of the cytoskeletal protein fodrin in the insoluble pellet fraction. Western blots of immunoprecipitated E-cadherin complexes failed to detect the presence of fodrin, or that of the cytoskeletal proteins adducin, alpha-actinin, and vinculin. If the E-cadherin-associated protein complex interacts with known proteins of the cell cytoskeleton, such interactions are labile and/or transient.
McCrea
P D
PD
Department of Pharmacology, University of California, San Francisco 94143-0450.
Gumbiner
B M
BM
D000818:Animals; D015820:Cadherins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D003599:Cytoskeleton / Q000378:metabolism*; D004285:Dogs; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism; D008970:Molecular Weight; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014982:Xenopus laevis; D013049:Spectrin
D015820:Cadherins; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D011506:Proteins; D013049:Spectrin