Integrin cytoplasmic domain-binding proteins

Shouchun Liu 0 David A. Calderwood 0 Mark H. Ginsberg ) 0 0 Department of Vascular Biology, VB-2, The Scripps Research Institute , 10550 N. Torrey Pines Rd, La Jolla, CA 92037 , USA SUMMARY Integrins are a large family of cell surface receptors that mediate cell adhesion and influence migration, signal transduction, and gene expression. The cytoplasmic domains of integrins play a pivotal role in these integrinmediated cellular functions. Through interaction with the cytoskeleton, signaling molecules, and other cellular proteins, integrin cytoplasmic domains transduce signals from both the outside and inside of the cell and regulate integrin-mediated biological functions. Identification and functional analyses of integrin cytoplasmic domain-binding proteins have been pursued intensively. In recent years, more cellular proteins have been reported to directly Integrin adhesion receptors are heterodimers of a and b subunits that contain a large extracellular domain responsible for ligand binding, a single transmembrane domain and a cytoplasmic domain that in most cases consists of 20-70 amino acid residues (Hynes, 1992; Sastry and Horwitz, 1993). Integrins play central roles in cell adhesion, cell migration and control of cell differentiation, proliferation and programmed cell death. They mediate signal transduction through the cell membrane in both directions: binding of ligands to integrins transmits signals into the cell and results in cytoskeletal re-organization, gene expression and cellular differentiation (outside-in signaling); on the other side, signals from within the cell can also propagate through integrins and regulate integrin ligand-binding affinity and cell adhesion (inside-out signaling; Hynes, 1992; Schwartz et al., 1995). The cytoplasmic domains of integrins play a pivotal role in these bi-directional signaling processes and intensive efforts have focused on identifying cellular proteins that can directly interact with integrin cytoplasmic domains in order to elucidate molecular mechanisms by which integrin mediate bi-directional signal transduction (Dedhar and Hannigan, 1996; Hemler, 1998; Hughes and Pfaff, 1998). Here, we focus on the most recent advances in this field. interact with integrin cytoplasmic domains and some of these interactions may play important roles in integrinmediated biological responses. Integrin b chains, for example, interact with actin-binding proteins (e.g. talin and filamin), which form mechanical links to the cytoskeleton. These and other proteins (e.g. FAK, ILK and novel proteins such as TAP20) might also link integrins to signaling mechanisms and, in some cases (e.g. JAB1) mediate integrin-dependent gene regulation. Extensive mutational analysis has demonstrated that integrin b cytoplasmic tails play a central role in integrin functions. b 1, b 2 and b 3 integrins lacking b tails fail to localize to focal adhesions, and show reduced ligand-binding activity and impaired activation of downstream signaling molecules (Solowska et al., 1989; Hayashi et al., 1990; Marcantonio et al., 1990; OToole et al., 1994). Furthermore, b 1A, b 1D, b 3, b 5 and b 7 tails expressed in isolation as transmembrane chimeras localize to pre-existing focal adhesions and exhibit a dominant negative effect on the ligand-binding activity of b 1, b 3 and b 5 integrins (Akiyama et al., 1994; Chen et al., 1994; LaFlamme et al., 1992; Lukashev et al., 1994; Zent et al., 2000). Isolated b tails are also sufficient to activate downstream signaling molecules, such as FAK, and can regulate cell cycle progression and actin cytoskeleton assembly (Belkin and Retta, 1998; David et al., 1999; Tahiliani et al., 1997). b tails are thus necessary and sufficient for correct subcellular localization of integrins and for activation of signaling pathways, and regulate the affinity of integrins for their ligands. The mechanisms by which integrin b tails function in both outside-in and inside-out signaling remain to be fully resolved. Nonetheless, these processes are probably mediated mainly through direct associations between integrin b tails and signaling and structural proteins. A complete understanding of the molecular basis of integrin regulation will require identification of these integrin-binding proteins and characterization of their activities. At least 21 proteins are known to bind to one or more integrin b tails (Table 1). This diverse list of proteins includes actin-binding proteins, enzymes, adaptor proteins, a transcriptional co-activator and additional proteins of unknown function. As the list lengthens, the challenge becomes determination of which interactions are significant in vivo and the roles of these interactions in specific cellular activities. Actin-binding proteins Correct localization of integrins, and their role in cell spreading, migration and matrix assembly require connection to the actin cytoskeleton. This connection is formed by the direct or indirect association of actin-binding proteins with integrin b tails (reviewed by Calderwood et al., 2000 and Critchley, 2000). These interactions represent some of the bestcharacterized integrin b tail associations, and their significance has been investigated in a variety of contexts. The first cytoplasmic protein shown to bind to integrins directly was the actin-binding protein talin (Horwitz et al., 1986). Talin colocalizes with integrins at certain sites of cellsubstratum contact, and Horwitz et al., proposed that the talinintegrin interaction provides the link between integrins and the actin cytoskeleton. Subsequent experiments revealed that the b cytoplasmic tail is responsible for binding to talin (Pfaff et al., 1998; Knezevic et al., 1996; Table 1), although one report COIP--Coimmunoprecipitation; PEP--Synthetic/recombinant peptide studies; 2HYB--Yeast two-hybrid screen; INT--Binding to purified integrins; SLS--Static light scattering; EQ--Equilibrium gel filtration. indicates that talin also binds to the a IIb tail (Knezevic et al., 1996). The integrin-binding site has been localized to the head domain of talin, and overexpression of a fragment of talin containing this binding site leads to increased binding of soluble ligand (activation) by a IIbb 3 in CHO cells (Calderwood et al., 1999). These data, together with the observation that reduced expression of talin disrupts cell surface expression of integrins and export from the Golgi, and impairs focal adhesion formation and cell migration (Priddle et al., 1998; Martel et al., 2000), suggest that binding of talin to integrin b tails is important for a variety of integrin functions. However, proof of this hypothesis requires evidence that specific disruption of the talin-integrin interaction alters integrin-dependent functions. To date no point mutants of talin that lack integrin-binding activity have been reported. However, fragments that lack the entire, integrin-binding, head domain cannot activate a IIbb 3 in CHO cells. The exact (...truncated)