Interaction of ct-Actinin with the Cadherin / Catenin Cell-Cell Adhesion Complex via oL-Catenin

Cadherins are CaZ+-dependent, cell surface glycoproteins involved in cell--cell adhesion. Extracellularly, transmembrane cadherins such as E-, P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intraceUular proteins termed catenins, including a-catenin, B-catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts a-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that a-actinin, but not vinculin, co-immunoprecipitates specifically with aand ~3-catenin from Nand E-cadherin--expressing cells, but only if a-catenin is present. Moreover, we show that a-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin-independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between a-actinin and a-catenin. T HE cadherins form a family of cell surface glycoproteins that function in promoting Ca-dependent cellcell adhesion and serve as the transmembrane components of cell--cell adherens junctions (Takeichi, 1988; Geiger, 1989; Geiger et al., 1990; Takeichi, 1991; Geiger and Ayalon, 1992; Grunwald, 1993). Adherens junctions are found between many cell types, including epithelial cells, cardiac myocytes, and fibroblasts (Volk and Geiger, 1984; Geiger et al., 1987, 1990; Geiger and Ayalon, 1992; Heaysman and Pegrum, 1973). Extracellularly the individual cadherins self-associate to promote specific cell-cell interactions (Nose et al., 1988, 1990; Friedlander et al., 1989), while intracellularly they interact with a group of proteins, collectively termed catenins (Ozawa and Kemler, 1992; Ozawa et al., 1989; Magee and Buxton, 1991). The catenins (e~, [~, and 7) are thought to link cadhefins to the actin-based cytoskeleton, although the mechanism is not understood. Both the cadherin cytoplasmic domain and the associated catenins have been shown to be required for full cadherin activity (Nagafuchi and Takeichi, 1988, 1989; Ozawa et al., 1989, 1990; Jaffe et al., 1990; Tsukita et al., 1992; Hirano et al., 1992). c~-Catenin is a 102-kD protein with homology to vinculin (Nagafuchi et al., 1991; Herrenknecht et al., 1991). [3-cateAddress all correspondense to Dr. Margaret J. Wheelock, Department of Biology, University of Toledo, Toledo, OH 43606. Tel. : (419) 537-4919. FAX: (419) 537-7737. K. J. Johnson and M. J. Wheelock contributed equally to this study. Scientific correspondence may be directed to any of the authors. nin is a 92-kD protein related to both the protein product of armadillo, a Drosophila segment polarity gene, and plakoglobin (Peifer and Wieschaus, 1990; McCrea et al., 1991), a vertebrate protein found at desmosomes and cellcell adherens junctions (Cowin et al., 1986). Plakoglobin also associates with cadherins and is thought to be the same as 7-catenin (Peifer et al., 1992; Knudsen and Wheelock, 1992). A variety of cell types express cadherins that have been shown to promote specific cell-ceU adhesion. E-cadherin is the major cadherin expressed by polarized epithelial cells, whereas both Eand P-cadherin are expressed by squamous epithelial cells (reviewed by Takeichi, 1988). N-cadherin is expressed by developing and mature cardiomyocytes (Volk and Geiger, 1984) and has been shown to play an important role in promoting both myocyte interaction and myofibrillogenesis (Peralta Soler and Knudsen, 1994). N-cadherin, along with other adhesion molecules, functions as a cell-cell adhesion molecule in nerve (Matsunaga et al., 1988) and in developing skeletal muscle (Knudsen et al., 1990). N-cadherin has been shown to be expressed by primary chicken fibroblasts and P-cadherin by a normal rat fibroblast cell line (Hirano et al., 1987; Geiger et al., 1990; Itoh et al., 1991). Cadherins localize to the cell--cell adherens junctions in epithelial cells and cardiomyocytes. At the electron microscopic level, these junctions are characterized by close plasma membrane apposition between interacting cells and by the presence of electron dense material at the intracellular face of the plasma membrane. The dense, submembranous material, sometimes referred to as the plas© The Rockefeller University Press, 0021-9525195/07167111 $2.00 The Journal of Cell Biology, Volume 130, Number 1, July 1995 67-77 67 on A uust 3, 2017 jcb.rress. org D ow nladed fom malemmal undercoat, contains a number of cytoplasmic proteins, many of which are well characterized (Tsukita et al., 1989, 1992; Tsukita et al., 1993). Examples include a-catenin, 13-catenin, plakoglobin, vinculin, a-actinin, radixin, the 220-kD protein (ZO-1), zyxin, spectrin, and actin. Exactly how these various proteins interact with one another is an area of on-going investigation. Some interactions among these cytoplasmic proteins have been described, whereas others are still a matter of speculation. For example, vinculin and zyxin have both been shown to bind to a-actinin (Belkin and Koteliansky, 1987: Wachsstock et al., 1987; Crawford et al., 1992; McGregor et al., 1994). Vinculin (Menkel et al., 1994) and a-actinin (Burridge and Feramisco, 1982; Blanchard et al., 1989) both interact with actin. Radixin caps the barbed end of actin filaments (Tsukita et al., 1989) and the 220-kD protein may interact with spectrin (Itoh et al., 1991). Due to homology between a-catenin and a domain in vinculin thought to be involved in vinculin self-association, a-catenin has been proposed to self-associate and thereby consolidate cadherins in the adherens junction (Nagafuchi et al., 1991; Herrenknecht et al., 1991). Alternatively, c~-catenin has been proposed to bind to vinculin and thereby link the cadherirdcatenin complex to the cytoskeleton. However, no evidence has been presented to date to support either of these possibilities. In this paper we show that a-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex expressed by fibroblasts. This is in contrast to epithelial cells where both a-actinin and vinculin colocalize with the E-cadherin/catenin complex. We present evidence that a-actinin, but not vinculin, coimmunoprecipitates specifically with c~and 13-catenin from Nand E-cadherin--expressing cells, but only if a-catenin is present. In addition, we present evidence that a-actinin associates with the N-cadherin/catenin complex in a manner not dependent upon the presence of actin. We therefore propose that cadherin/ catenin complexes are linked to the actin cytoskeleton via a direct interaction between c~-catenin and ~-actinin. Materials and Methods