Alexey M. Belkin, S. Francesco Retta. Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill, North Carolina 27599.
Expression of muscle-specific b 1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, b 1 integrin-minus cells leads to their phenotypic conversion.B 1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their b 1A-expressing counterparts. The transfected b 1D is targeted to focal adhesions and efficiently displaces the endogenous b 1A and a v b 3 integrins from the sites of cell–matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in b 1D transfectants. Whereas a significant part of cellular b 1A integrin is extractable in digitonin, the majority of the transfected b 1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton.
Increased interaction of b 1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, b 1A interacts more strongly with a-actinin, than b 1D. Inside-out driven activation of the b 1D ectodomain increases ligand binding and fibronectin matrix assembly by b 1D transfectants. Phenotypic effects of b 1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of b 1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton–matrix link in muscle cells. This reflects the major role for b 1D integrin in muscle, where extremely stable association is required for contraction.
Integrins are a large family of transmembrane heterodimeric receptors that play a key role in cell adhesion to extracellular matrix (Hynes, 1992). Integrin receptors serve a dual purpose, linking extracellular matrix to the actin cytoskeleton and providing bidirectional transmission of signals between the extracellular matrix and the cytoplasm (Schwartz et al., 1995; Yamada and Miyamoto, 1995; Burridge and Chrzanowska-Wodnicka, 1996). At least two major actin-binding proteins, talin and a -actinin, are thought to interact directly with the cytoplasmic domain of several b subunits, providing a link to the actin cytoskeleton (Horwitz et al., 1986; Otey et al., 1990; Hemler et al., 1994). Among integrins, b 1 is typically the most abundant and ubiquitously expressed subunit associated with a number of subunits to form distinct heterodimers. These interact with a variety of extracellular matrix and cell adhesion molecules (Hynes, 1992). The entire structure of the b 1 integrin cytoplasmic domain is critical for integrin– cytoskeleton interaction (Hayashi et al., 1990; LaFlamme et al., 1992; Reszka et al., 1992; Ylanne et al., 1993; Lewis and Schwartz, 1995).