Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.

Cadherins are Ca(2+)-dependent cell adhesion molecules found in several kinds of cell-cell contact, including adherens junctions and desmosomes. In the presence of Ca(2+), cells expressing the same type of cadherin form stable contacts with one another, a phenomenon designated homophilic, or homotypic, adhesion. Most cadherins are single-pass transmembrane proteins whose extracellular regions mediate specific cell-cell interactions. The intracellular faces of these contacts are associated with the actin cytoskeleton in adherens junctions or the intermediate-filament system in desmosomes. The close coordination of the transmembrane adhesion molecules with the cytoskeleton is believed to be essential in coordinating morphogenetic movements of tissues during development and in conferring the appropriate mechanical properties to cell-cell contacts. Structural, biochemical, and biophysical analysis of the molecules that comprise these contacts has provided unique mechanistic insights into the specificity of homophilic adhesion, the functional connection to the underlying cytoskeleton, and the dynamics of junction formation.

In adherens junctions, alpha-catenin links the cadherin-beta-catenin complex to the actin-based cytoskeleton. alpha-catenin is a homodimer in solution, but forms a 1:1 heterodimer with beta-catenin. The crystal structure of the alpha-catenin dimerization domain, residues 82-279, shows that alpha-catenin dimerizes through formation of a four-helix bundle in which two antiparallel helices are contributed by each protomer. A slightly larger fragment, comprising residues 57-264, binds to beta-catenin. A chimera consisting of the alpha-catenin-binding region of beta-catenin linked to the amino terminus of alpha-catenin 57-264 behaves as a monomer in solution, as expected, since beta-catenin binding disrupts the alpha-catenin dimer. The crystal structure of this chimera reveals the interaction between alpha- and beta-catenin, and provides a basis for understanding adherens junction assembly.

The cell-adhesion protein E-cadheriduvomorulin exhibits a calcium-dependent homoassociation. The effect of Ca2+ on the extracellular fragment of E-cadherin was studied using the recombinant protein expressed in the baculovirus expression system. The recombinant and native fragment of E-cadherin were found to be similar by many biochemical criteria [Herrenknecht, K. & Kemler, R. (1993) J. Cell Sci. 17, 147-1541. A large and reversible conformational transition was observed upon Ca2+ depletion. A change from a rod-like structure, 22 nm in length, to a more globular assembly of the five subdomains became evident by electron-microscopical analysis. In the presence of Ca", the circular dichroic spectra indicated predominantly p-structure but a more negative ellipticity was observed in the absence of Ca2+. The intrinsic tryptophan fluorescence decreased by 12% upon Ca2+ depletion. Both effects were used for calcium titrations which indicated calcium binding to several sites with average Kd values of 45-150 pM. Cleavage of the protein fragment by trypsin occurred only at low Caz+ concentrations and from the calcium-dependence of cleavage rates, a Kd value of 24 pM was derived. The major site of cleavage was identified by partial sequencing to be located between the two putative calcium-binding sites in the third subdomain from the N-terminus. In agreement with earlier results with the native fragment, the recombinant protein did not associate in the presence or absence of Ca2+. We suggest the calcium-dependent homoassociation therefore depends on additional effects connected with the cell surface association of E-cadherin.

No abstract

αE-catenin has cell–cell contact–dependent and –independent functions in regulating actin and membrane dynamics.

αE-catenin, an essential component of the adherens junction, interacts with the classical cadherin-β-catenin complex and with F-actin, but its precise role is unknown. αE-catenin also binds to the F-actinbinding protein vinculin, which also appears to be important in junction assembly. Vinculin and αE-catenin are homologs that contain a series of helical bundle domains, D1-D5. We mapped the vinculin-binding site to a sequence in D3a comprising the central two helices of a four-helix bundle. The crystal structure of this peptide motif bound to vinculin D1 shows that the two helices adopt a parallel, colinear arrangement suggesting that the αE-catenin D3a bundle must unfold in order to bind vinculin. We show that αE-catenin D3 binds strongly to vinculin, whereas larger fragments and full-length αE-catenin bind approximately 1,000-fold more weakly. Thus, intramolecular interactions within αE-catenin inhibit binding to vinculin. The actin-binding activity of vinculin is inhibited by an intramolecular interaction between the head (D1-D4) and the actin-binding D5 tail. In the absence of F-actin, there is no detectable binding of αE-catenin D3 to full-length vinculin; however, αE-catenin D3 promotes binding of vinculin to F-actin whereas full-length αE-catenin does not. These findings support the combinatorial or "coincidence" model of activation in which binding of high-affinity proteins to the vinculin head and tail is required to shift the conformational equilibrium of vinculin from a closed, autoinhibited state to an open, stable F-actin-binding state. The data also imply that αE-catenin must be activated in order to bind to vinculin.