In epithelial cells, α-, β-, and γ-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. α-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell–matrix and cell–cell contacts, α-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell–cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and α-catenin. We show that α-catenin colocalizes at cell–cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH2-terminal head binds to α-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The K d of the complex was determined to 2–4 × 10−7 M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of α-catenin is involved in this interaction.Complex formation of vinculin and α-catenin was challenged in transfected cells. In PtK2 cells, intact α-catenin and α-catenin1-670, harboring the β-catenin– binding site, were directed to cell–cell contacts. In contrast, α-catenin697–906 fragments were recruited to cell–cell contacts, focal adhesions, and stress fibers. Our results imply that in vivo α-catenin, like vinculin, is tightly regulated in its ligand binding activity.

Abstraet Using blot overlay techniques we have investigated the interaction of vinculm with u-a&tin. We show that an a-actinin binding site is located in the 90 kDa vinculin head and co&m a vinculin binding site in the C-terminal rod of a-actinin, as recently reported by McGregor et al. [(1994) B&hem. J. 310,2252331. The isolated vinculin head binds much more strongly to a-actinm than intact vinculin. Using a proteolytic 81 kDa head fragment, we show that vinculin residues l-107 are required for a-actinin binding. Antibodies directed against vinculin residues 808-850 inhibit the vmculin-u-actinin binding, suggesting that this sequence is directly involved in, or topographically related to, the a-actinin binding site.