The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter platebinding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility.
INTRODUCTIONCell shape and movement form the basis for several biological phenomena such as morphogenesis, invasion, and metastasis. The actin cytoskeleton is a major determinant of changes in cell shape, and interactions between actin filaments and the cell membrane are essential for cell adhesion, spreading, and migration, as well as for signal transduction (Hall, 1998;Schoenwaelder and Burridge, 1999). Several molecules have been recognized as linkers between the actin cytoskeleton and the cell membrane. For example, members of the band 4.1/ERM superfamily (band 4.1, talin, ezrin, radixin, moesin, and merlin, the product of the neurofibromatosis type 2 tumor suppressor gene) are characterized by the presence of a conserved N-terminal membrane-binding domain, which is able to bind to the cytoplasmic region of several transmembrane receptors, including CD44, CD43, ICAM-2, and ICAM-3 (Sainio et al., 1997;Heiska et al., 1998;Yonemura et al., 1998). The C-terminal regions of ERM proteins are thought to bind to actin filaments, thereby linking these transmembrane receptors to the cytoskeleton (reviewed by Mangeat et al., 1999).Layilin is a recently cloned ϳ55-kDa membrane-binding partner for talin (Borowsky and Hynes, 1998) that is widely expressed in different cell types and tissue extracts. It is found in peripheral ruffles of spreading cells and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments. Layilin colocalizes with talin in ruffles and binds to talin's ϳ50-kDa head domain (amino acids 280 -435)...