I. Introduction

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Cell adhesion is critical for the genesis and maintenance of both three-dimensional structure and normal function in tissues. The biochemical entities mediating cell adhesion are multiprotein complexes comprising three broad classes of macromolecules: the adhesion receptors, the extracellular matrix molecules, and the adhesion plaque proteins (Gumbiner, 1996). Cell adhesion receptors are typically transmembrane glycoproteins that mediate binding to extracellular matrix (ECM)^b molecules or to counter-receptors on other cells; these molecules determine the specificity of cell-cell or cell-ECM interaction. The ECM proteins are usually fibrillar in nature and provide a complex structural and functional network that can interact simultaneously with multiple cell surface receptors. The intracellular plaque proteins (or peripheral membrane proteins) provide structural and functional linkages between adhesion receptors and the actin microfilaments, microtubules, and intermediate filaments of the cytoskeleton. An exciting concept that has emerged from recent cell biological research is that cell adhesion complexes are not simply static architectural entities. Rather, they are dynamic units that are capable of capturing and integrating signals from the extracellular environment (Rosales et al., 1995). Moreover, the functions of cell adhesion complexes are regulated precisely by biochemical events within the cell. Thus cell adhesion receptors are at a nexus of two-way signaling between the cell and its external environment. This review will examine current knowledge concerning signal transduction events initiated or modulated by cell adhesion receptors. The emphasis will be on the integrin family of receptors because their signaling functions have been studied more extensively than those of other adhesion receptor families. However, we also will examine signaling events involving the cadherin, immunoglobulin-cell adhesion molecule (Ig-CAM), and selectin families of adhesion receptors. Adhesion-mediated signaling influences several critical cellular processes including gene expression, cell cycle, and programmed cell death; these aspects will be explored. However, the major emphasis of this review will be on mechanisms of adhesion receptor signaling.

	II. Families of Cell Adhesion Receptors

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This section will review some of the key features of the integrin, cadherin, Ig-CAM, and selectin families of cell adhesion receptors. Because there are thousands of publications on these topics (Edelman, 1993), only a few research articles and reviews will be used here to introduce some key ideas about these molecules. A diagram of the structures of several types of adhesion receptors, their counter-receptors, and some of their associated cytoskeletal components is presented in fig. 1.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Families of cell adhesion receptors. This diagram presents basic information about the integrin, cadherin, Ig-CAM, and selectin families of cell adhesion receptors. It illustrates their approximate structures, their ligands or counter-receptors, and some of their associated cytoskeletal proteins when they are known. A more detailed description is given in the text. The molecules illustrated here are not drawn to scale.

The integrins are a family of cell-surface glycoproteins that act as receptors for ECM proteins, or for membrane-bound counter-receptors on other cells. Integrin-mediated cell-ECM adhesion sites are complex specialized structures termed focal contacts or focal adhesions (Jockush et al., 1995) and are discussed in more detail in Section III. Each integrin is a heterodimer that contains an  and a  subunit with each subunit having a large extracellular domain, a single membrane-spanning region, and in most cases (other than 4), a short cytoplasmic domain (Hynes, 1992; Rosales et al., 1992; Ruoslahti, 1991). The integrin receptor family of vertebrates includes at least 16 distinct  subunits and 8 or more  subunits which can associate to form more than 20 distinct integrins (Hynes, 1992; Rosales et al., 1995). The / pairings specify the ligand-binding abilities of the integrin heterodimers. Although the ligands for integrins are often large ECM proteins such as collagen, laminin, vitronectin, or fibronectin, some integrins recognize rather short peptide sequences within the larger protein, for example, the RGD (Arg-Gly-Asp) sequence found in fibronectin and vitronectin. Because of this, there has been considerable interest in the pharmaceutical industry in developing short peptides or peptidomimetics that can interdict integrin functions in a variety of disease processes including coagulation disorders, inflammation, and cancer (Arap and Pasqualini, 1998; Ruoslahti, 1996). In other cases, however, integrin-ligand recognition depends on the overall conformation of the ligand protein (Kuhn and Eble, 1994). For example, some integrins interact with members of other adhesion receptor families, including Ig-CAMs and cadherins, in a manner that does not involve RGD motifs (Felding-Habermann et al., 1997; Higgins et al., 1998; Piali et al., 1995). Some integrins, such as 51, the "classic" fibronectin receptor, bind to a single ECM protein. More generally, an individual integrin will recognize several distinct proteins (Hynes, 1992; Rosales and Juliano, 1995); for example, the v3 integrin has been reported to bind collagen VI, laminin, fibronectin, vitronectin, thrombospondin, Von Willebrand factor, and fibrinogen (Kuhn and Eble, 1994). Cells often display multiple integrins capable of interacting with a particular ECM protein, thus integrin expression is often apparently redundant, at least in terms of simple cell adhesion. Some integrin subunits undergo alternative splicing of their cytoplasmic domain regions in a tissue-type specific and developmentally regulated manner; this suggests that there are discrete intracellular functions for individual integrins, as we will discuss in much more detail below.

The relationships between integrin structure and the various functions of integrins are being actively investigated. In terms of ligand binding, it seems clear that both  and  subunit extracellular domains contribute to the formation of the binding site. The ligand binding regions of integrins have been explored with chemical cross-linking of ligands, monoclonal antibodies, mutation, and most recently, molecular modeling and X-ray crystallography (Loftus et al., 1994; Qu and Leahy, 1995; Springer, 1997). Three regions apparently are particularly important: (a) a series of seven repeats of approximately 60 amino acids in the N-terminal portion of the  chain, each containing a putative Ca²⁺ binding site; (b) an inserted domain (I-domain) of approximately 200 amino acids found in several  chains and containing a nucleotide binding fold and a divalent cation coordination site; (c) an I-domain like region of approximately 250 amino acids found in the N-terminal region of the  subunit (Loftus and Liddington, 1997). The seven-repeat sequences of the  chain have been predicted by molecular modeling to form a -propeller structure with the upper face of the propeller being the ligand binding region and the Ca²⁺ coordination motifs lying on the lower face (but probably essential to maintain the structure) (Loftus and Liddington, 1997; Springer, 1997). Propeller structures of this type have been found in a variety of other proteins, including the  subunit of trimeric G-proteins (Sondek et al., 1996), and usually are involved in protein-protein interactions. The structure of the I-domain region has been determined by X-ray crystallography (Lee et al., 1995; Qu and Leahy, 1995); the divalent ion binding motif has been designated the "metal ion-dependent adhesion site" and seems to play a critical role in ligand coordination for integrins with I-domains. Less information is available about the I-domain-like region in the  subunit; however, molecular modeling and mutagenesis studies support its ligand binding role (Loftus and Liddington, 1997). Integrins likely undergo dynamic structural changes as part of the ligand binding process including relative movements of subunits and of domains, and conformational changes within domains (Loftus and Liddington, 1997). As we will discuss in more detail below, integrins can exist in various affinity states for their ligands; structural changes that occur in moving from low- to high-affinity states (or vice versa) can be detected by certain monoclonal antibodies (Hughes et al., 1997).

Integrin cytoplasmic domains are a key nexus of interaction between the extracellular environment and intracellular structures and signaling cascades. Both the  and  subunit cytoplasmic domains make important contributions to various aspects of overall integrin function including cytoskeletal organization, cell motility, signal transduction, and modulation of integrin affinity for ligands ("activation"). Several cytoplasmic proteins including talin, -actinin, and possibly focal adhesion kinase (FAK) bind directly to the 1 cytoplasmic domain and contribute to integrin-cytoskeletal interactions (Burridge and Chrzanowska-Wodnicka, 1996; Yamada and Geiger, 1997). Studies using truncation, mutation, or "domain swaps" of cytoplasmic domains have delineated several important structure-function relationships for this region of integrins. The  cytoplasmic domain is critical for recruitment of integrins to focal contacts because its truncation/mutation impairs this process; in fact, the  cytoplasmic domain alone, expressed as a fusion chimera with another membrane protein, seems sufficient for focal contact localization (LaFlamme et al., 1992; Reszka et al., 1992; Rosales et al., 1992). A widely accepted current model is that the  subunit cytoplasmic domain inhibits certain functions of the  cytoplasmic domain (e.g., focal contact recruitment) but that binding of a ligand to the integrin relieves this inhibition, possibly by allowing the subunits to swing apart like a hinge (Burridge and Chrzanowska-Wodnicka, 1996; Hughes et al., 1996). The  cytoplasmic domain is also important in signal transduction, particularly integrin activation of FAK (discussed in detail below), whereas truncation/mutation of the  cytoplasmic domain has little effect on this process (Akiyama et al., 1994; Bauer et al., 1993; Tahiliani et al., 1997).  cytoplasmic domains have played a critical role in a variety of cellular processes including endocytosis (Van Nhieu et al., 1996), "cross-talk" between different integrins (Blystone et al., 1995), assembly of fibronectin fibrils (Wu et al., 1995), and cell motility (Pasqualini and Hemler, 1994), whereas  cytoplasmic domains also can strongly influence cell motility (Bauer et al., 1993; Chan et al., 1992)_.

Like other receptors, integrins can exist in different affinity states with respect to their ligands. Integrins can enter a high-affinity ("activated") state in response to certain agents that bind the extracellular domain and influence its conformation (divalent cations, antibodies); they also can respond to signals generated within the cell that presumably have an impact on the cytoplasmic domain ("inside-out signaling") (Humphries, 1996; Keely et al., 1998; O'Toole et al., 1994). The activation status of a particular integrin is cell-type dependent and usually critically depends on cellular energy metabolism. The roles of the cytoplasmic domains in integrin activation have been studied extensively, and the findings are quite complex. For example, partial truncation of 4, 2, or 2 cytoplasmic domains prevents integrin activation, whereas truncation of the IIb subunit activates the fibrinogen-binding integrin IIb3 (O'Toole et al., 1994). Recently, a coherent model of how cytoplasmic domain structure relates to integrin activation has been presented (Hughes et al., 1996). The membrane proximal regions of all  and  subunits are highly conserved, whereas the remainder of the subunits are quite divergent. The conserved  sequence is GFFKR, whereas the conserved  sequence is LLv-iHDR. Deletion of either of these conserved sequences activates the integrin, essentially "locking" it in a high-affinity conformation independent of cellular energy metabolism, whereas mutation at sites C-terminal to these conserved sequences can affect energy-dependent activation (Hughes et al., 1996; O'Toole et al., 1994). The conserved membrane proximal sequences may interact through salt bridges between the subunits, normally keeping the integrin in a low-affinity state. Cytoplasmic events, that is "inside-out signaling," can disrupt the / association allowing the "hinge" to swing and opening up the extracellular ligand binding site (see fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Activation of integrins. Both high-affinity and low-affinity binding states exist for integrins. This may be because of conformational changes that occur when regulatory proteins bind to integrin cytoplasmic tails. The conformational changes are depicted here as integrin subunits swinging on a "hinge" and exposing ligand binding sites (I, I and propeller "prop"). The activating process is regulated by G-protein-coupled receptors and by small GTPases, where R-Ras activates and H-Ras inhibits high-affinity binding. Integrin ligands bind to the  and  subunits with the I domain and I-like domain being particularly important. Adapted from Loftus and Liddington (1997).

Recently, some of the biochemical pathways underlying "inside-out signaling" have begun to come to light. As first elucidated in platelets with regard to the IIb3 integrin (Smyth et al., 1993), several agonists (usually acting through heterotrimeric G-protein-coupled receptors) can activate integrins in various cell types. It also seems clear that signaling pathways coupled to Ras-related guanosine 5'-triphosphatases (GTPases) impinge on integrin activation and function. For example, chemokines such as formyl peptide or interleukin (IL)-8 which enhance 1 and 2 integrin-mediated adhesion in lymphoid cells seem to act via the Rho GTPase (Laudanna et al., 1996); in this case, it is not entirely clear whether this represents is a change in integrin affinity (interaction of a single ligand with its receptor) or in avidity (concerted interaction of several receptors with a polyvalent ligand). Transfection of myeloid cells with a constitutively activated form of the R-Ras GTPase clearly causes an increase in the affinity of several integrins, including v3, 41, and 51, whereas coexpressed IIb3 can be activated by R-Ras in Chinese hamster ovary (CHO) cells (Zhang et al., 1996). By contrast, transfection of activated H-Ras, or of its downstream kinase Raf-1, inhibited the activation of coexpressed IIb3 (Hughes et al., 1997). Thus, different Ras-related small GTPases and their downstream effectors can either positively or negatively modulate the ligand binding affinity of integrins. Presumably integrin activation through inside-out signaling ultimately involves intracellular proteins that directly interact with integrin cytoplasmic domains; a discussion of proteins that interact directly with integrins is presented further on in this review (see Section III.).

In summary, integrins are a particularly complex family of cell adhesion receptors. These heterodimeric cell membrane glycoproteins can bind to a variety of ECM ligands or cellular counter-receptors. They engage with elements of the cytoskeleton and can thus influence cell shape, intracellular architecture, and cell motility. Integrins exist in either high- or low-affinity states with respect to their ligands, and they can engage in both "inside-out" and "outside-in" signal transduction via pathways that just now are being elucidated.

The cadherins comprise a family of transmembrane proteins that share an extracellular domain consisting of multiple repeats of a cadherin-specific motif (Suzuki, 1996) (see fig. 1). Members of the "classic" cadherin subfamily are calcium-dependent homotypic cell-cell adhesion molecules. This subfamily includes the N-, P-, R-, B-, and E-cadherins as well approximately 10 other members (Takeichi, 1995). These molecules localize in specialized sites of cell-to-cell adhesion that are termed adherence junctions; at these sites cadherins can establish linkages with the actin-containing cytoskeleton. The classic cadherins play a key role in developmental processes. For example, "knockout" of the gene for E-cadherin (which mediates epithelial interactions) results in an embryonic lethal that arrests before blastocyst formation (Takeichi, 1995). Another important subfamily of cadherins involved in adhesion is represented by the desmogleins and desmocollins, a group of desmosome-associated cadherins that form intracellular linkages to intermediate filaments rather than actin filaments (Cowin and Burke, 1996).

The structure of a typical classic cadherin consists of an amino-terminal external domain having five tandem repeats, a single transmembrane segment, and a cytoplasmic carboxy-terminal domain of approximately 150 amino acids (fig. 1). The binding functions of the cadherin are localized in the amino-terminal tandem repeat, whereas the other repeats are bridged by calcium binding sites that impart rigidity to the molecule (Aberle et al., 1996). A recent model based on the X-ray structure of N-cadherin (Shapiro et al., 1995) suggests that cell-cell adhesion mediated by cadherins involves a "zipper" type of organization. Cadherins on one cell surface form a series of rigid dimers that presents the first (N-terminal) cadherin repeat to equivalent dimers on the opposing cells (Aberle et al., 1996; Gumbiner, 1996). Lateral motion of these complexes allows the cell junction site to "zip up."

The cytoplasmic domains of cadherins interact strongly with a group of intracellular proteins known as catenins; these proteins are essential for cadherin function, because truncation of the cadherin cytoplasmic domain to delete catenin binding sites leads to a loss of cadherin-mediated adhesion (Gumbiner, 1996; Takeichi, 1995). Because there is considerable homology among their cytoplasmic domains, different classic cadherins can compete for the same pool of catenins (Kinter, 1992). The catenins were described initially as a set of three proteins, -, -, and -catenin (also termed plakoglobin). -Catenin binds directly to the cadherin cytoplasmic domain; subsequently, -catenin binds to -catenin and links the complex to the actin cytoskeleton by direct interaction with actin and by binding -actinin, an actin-bundling protein (Cowin and Burke, 1996). The structure of -catenin shows substantial homology to the protein vinculin, which binds -actinin and talin and is critical for cytoskeletal assembly at integrin-mediated focal adhesion sites (see Section III. below) (Aberle et al., 1996). In some cases plakoglobin replaces -catenin in mediating cadherin-cytoskeletal complexes.

The cadherins play a critical role in the development of tissue organization during ontogeny and in maintenance of normal tissue structures in adult organisms (Huber et al., 1996). For example, a variety of cadherins have been implicated in generating the complex architecture of the brain (Redies and Takeichi, 1996). In adult organisms, loss of cadherin expression or function can lead to serious pathophysiological consequences. Thus progression toward an invasive, malignant phenotype in epithelial tumors is associated with loss or mutation of E-cadherin, or with the disruption of cadherin-catenin complexes. Phosphorylation of -catenin by v-Src or other tyrosine kinases can lead to a breakdown of cadherin-cytoskeleton associations and loss of the adherence junctions that maintain normal epithelial organization (Behrens and Birchmeier, 1994; Birchmeier, 1995).

Recent evidence has indicated that cadherins and catenins also play an important role in signal transduction. As discussed in more detail below (Section VIII.), -catenin not only interacts with cadherins but also with components of the wingless/Wnt signaling pathway (Peifer, 1996). Thus the cell-cell adherence junction, maintained by cadherins and their associated catenins, is a key element in both tissue organization and in regulation of signaling cascades.

A diverse array of cell adhesion receptors are included in the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs). Proteins of this family are defined by the presence of one or more copies of the Ig fold, a compact structure with two cysteine residues separated by 55 to 75 amino acids arranged as two antiparallel  sheets (Vaughn and Bjorkman, 1996). In many (but not all) cases, CAMs in the Ig superfamily also contain one or more copies of a fibronectin type III repeat domain. Ig family adhesion receptors typically have a large amino-terminal extracellular domain, a single transmembrane helical segment, and a cytoplasmic tail (fig. 1). Members of the Ig-CAM family function in a wide variety of cell types and are involved in many different biological processes.

One of the most important contexts for Ig-CAMs is the developing nervous system, where many different members of this superfamily are involved in axon guidance and in the establishment and maintenance of neural connections (Baldwin et al., 1996; Tessier-Lavigne and Goodman, 1996). The "classic" example of an Ig superfamily adhesion receptor is NCAM, which contains five Ig folds in its extracellular portion; there are three forms of NCAM, two with transmembrane domains and one having a glycosylphosphatidylinositol (GPI) link to the membrane (Edelman and Crossin, 1991). NCAM functions as a homotypic calcium-independent adhesion receptor and seems to be almost universally present in the nervous system. Adhesive interactions mediated by NCAMs are known to be regulated by both the abundance of receptor and its degree of polysialyation (Edelman and Crossin, 1991; Tessier-Lavigne and Goodman, 1996). Several other homotypic neural cell adhesion molecules belong to the Ig superfamily, including L1, TAG1, contactin, and Drosophila fasiculin II (Baldwin et al., 1996; Tessier-Lavigne and Goodman, 1996). Another group of Ig-CAMs important in neural development are the netrin receptors. Netrins are ECM matrix proteins, distantly related to laminins, that provide guidance cues to migrating axons (Tessier-Lavigne and Goodman, 1996). Vertebrate netrin receptors belong to the Ig family and include DCC, a protein originally identified as the product of a key tumor suppressor gene for colon cancer (Keino-Masu et al., 1996). Yet another group of key adhesion receptors involved in neural development is the dozen or so members of the Eph subfamily of transmembrane tyrosine kinases; these proteins contain an Ig fold in their extracellular domains and are thus Ig-CAMs (Tessier-Lavigne and Goodman, 1996). The Eph kinases and their cell-bound ligands seem to be involved primarily in axon-target cell interactions. Thus neural Ig-CAMs can be involved in either homotypic (NCAM) or heterotypic (DCC, Eph kinases) adhesive interactions. There is relatively little known about the interactions of neural Ig-CAMs with cytoskeletal proteins. One report has suggested an association between the 180 kDa form of NCAM and spectrin (which can link to the actin cytoskeleton) (Pollerberg et al., 1997). However, it seems likely that other interactions exist and contribute to the functions of neural Ig-CAMs. Presumably many of the interactions between neural adhesion molecules result in signal generation, as well as in establishment of adhesive contacts between cells; however, with a few exceptions (see Section VIII.), the mechanistic details of the signaling pathways are poorly understood at present.

Another important biological context for Ig-CAMs lies in the immune system. In fact, integrins, selectins, and Ig-CAMs are all critically involved in multiple aspects of immune function (Dustin and Springer, 1991; Rosales and Juliano, 1995; Springer, 1995). T lymphocytes express several Ig superfamily receptors including CD2, CD4, or CD8, ICAMs 1 and 2, and the T-cell receptor (TCR) itself. These receptors play important roles in antigen recognition, cytotoxic T-cell functions, and lymphocyte recirculation. In contrast to many of the neural Ig-CAMs, which are often homotypic receptors, Ig family proteins involved in the immune system primarily engage in heterotypic interactions. For example, CD2 on T cells interacts with LFA-3 (another Ig-CAM) expressed on target cells, the TCR interacts with major histocompatibility complex (MHC) class II proteins on antigen presenting cells (both Ig superfamily), whereas ICAMs on endothelial cells are recognized by 2 integrins on leukocytes. Other Ig-CAM family adhesion receptors are found on vascular endothelial cells and play an important role in leukocyte trafficking to inflamed tissue sites. For example, vascular cell adhesion molecule-1 (VCAM-1) is an endothelial cell counter-receptor for the integrin 41 found on leukocytes. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an Ig-family cell-cell adhesion molecule that can engage in both homotypic and heterotypic interactions; one of its roles seems to be maintaining tight contacts between adjacent vascular endothelial cells (DeLisser et al., 1994).

Recently, X-ray crystal structure was obtained for the extracellular domains of several Ig-CAMs important in immune function including intercellular adhesion molecule (ICAM)-2, vascular cell adhesion molecule-1 and CD2 (Casanovas et al., 1997). This has allowed a more detailed understanding of how these molecules recognize their ligands and support cell-cell contact. As with the cadherins, the intracellular domains of Ig-CAMs may also be important in regulating the adhesive functions of these receptors (DeLisser et al., 1994; Doherty et al., 1992).

Immune system Ig-CAMs are critically involved in the key signal transduction processes leading to activation of T cells and B cells by antigens (Weiss and Littman, 1994). A detailed account of these complex events is beyond the scope of this review. In essence, however, Ig-CAM and integrin-mediated contacts are established between the antigen-presenting cell and the T cell such that the TCR recognizes antigen bound to an MHC protein on the presenting cell. This triggers the activation of intracellular tyrosine kinases associated with the TCR and with accessory receptors (Crabtree and Clipstone, 1994). In B cells, the B-cell receptor (also an Ig-CAM) can recognize either soluble or particulate antigen, and it also can activate intracellular tyrosine kinases upon ligation. Both the Src family and the spleen tyrosine kinase (SYK)/ZAP-70 family of tyrosine kinases have been implicated in antigen-induced T- and B-lymphocyte signaling (Weiss and Littman, 1994). Further on in this review, we will return to the theme of adhesion receptors activating intracellular tyrosine kinases in great detail as we consider adhesion receptor signaling in fibroblasts, epithelial cells, and tumor cells.

Another important group of signaling proteins that overlaps the Ig-CAM superfamily are the receptor protein tyrosine phosphatases (RPTPs) (Neel and Tonks, 1997). Many of these transmembrane enzymes have extracellular domains that include Ig folds (and often fibronectin type III repeats as well). The RPTPs typically have a large external domain, a single transmembrane helix, and a cytoplasmic domain containing two signature tyrosine phosphatase domains flanked by a variety of noncatalytic sequences. RPTPs seem to function conversely to receptor tyrosine kinases (RTKs); that is, ligand binding results in dimerization of RPTPs but inhibits enzyme activity (rather than activating it as with RTKs). It has become clear recently that several RPTPs can engage in homotypic or heterotypic cell adhesion through their extracellular domains. For example, Drosophila LAR, a Drosophila Ig-CAM-RPTP, plays an important role in axon migration and innervation of muscle targets (Neel and Tonks, 1997).

Thus Ig-CAMs play multiple roles in the developing embryo and in the adult organism. In addition to mediating adhesive contacts that are important in tissue organization, or in cellular trafficking in the immune system, many Ig-CAMs function in key signal transduction processes as well.

The selectins are a small family of lectin-like adhesion receptors composed of three members, L-, E-, and P-selectin (Lasky, 1995; Rosen and Bertozzi, 1994; Tedder et al., 1995). The structure of a selectin includes an amino-terminal domain that is homologous to calcium-dependent animal lectins, followed by an epidermal growth factor (EGF)-type domain, two to nine complement regulatory protein repeats, a transmembrane helical segment, and a short cytoplasmic tail (fig. 1). Selectins mediate heterotypic cell-cell interactions through calcium-dependent recognition of sialyated glycans. The best defined physiological role for selectins concerns leukocyte adherence to endothelial cells and platelets during inflammatory processes (Rosales and Juliano, 1995; Springer, 1995). The expression and function of selectins is tightly regulated so as to come into play only when leukocytes need to stick to the vessel wall as part of normal immune system cellular trafficking or during inflammation. Thus P-selectin is present in latent form in endothelial cells and platelets and it is rapidly translocated from secretory granules to the cell surface upon cell activation by thrombin or other agonists. E-selectin is synthesized and expressed on endothelial cells in response to inflammatory cytokines such as tumor necrosis factor (TNF) or IL-1. L-selectin is expressed constitutively on leukocytes, but its presentation at the cell surface may be regulated.

The precise identities of the ligands for the three currently known selectins are being pursued actively and are a matter of some controversy (Varki, 1997). For all three selectins, a key component of the binding ligand is tetrasaccharide residues of the sialyl-Lewis^X type (previously defined as a blood group antigen); such motifs appear on glycolipids as well as on glycoproteins. However, the binding affinities of selectins for isolated sialyl-Lewis^X saccharides are very poor, although selectins clearly are responsible for high-affinity cell-cell binding. Thus physiological high-affinity ligands for selectins likely must include sialyl-Lewis^X saccharides in the context of a macromolecular scaffold. The best documented high-affinity counter-receptor for a selectin is P-selectin glycoprotein ligand-1 (PSGL-1), an O-glycosylated mucin-like transmembrane glycoprotein found on leukocytes and lymphoid cells (Norman et al., 1995). Glycoproteins of this type likely have a rigid, rod-like structure and may be able to present multiple copies of sialyl-Lewis^X to endothelial cell selectins. Two potential counter-receptors for L-selectin have been identified; GlyCAM-1 which is a small, secreted mucin-like glycoprotein, and CD34 a transmembrane mucin-like protein found on endothelial cells. A putative counter-receptor for E-selectin has been termed ESL-1 and is a glycoprotein bearing N-linked carbohydrate residues. However, the physiological importance of GlyCAM-1, CD34, and ESL-1 in leukocyte to endothelial adherence and leukocyte trafficking is still unclear (Varki, 1997).

There is much less known about selectin-mediated signal transduction than is the case for integrins, cadherins, and Ig-CAMs. However, it is rapidly becoming clear that selectins play an important role in signaling processes that regulate leukocyte-endothelial cell interactions (Zimmerman et al., 1996b).

	III. The Components of Adhesive Junctions

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Signal transduction by cell adhesion receptors primarily occurs in the context of highly organized supramolecular complexes that are assembled at sites of cell-to-cell or cell-to-ECM adhesion. There are several distinct types of adhesive junctions. However, our present understanding of the role of adhesive junctions in signaling extends primarily to one type of junction, the integrin-containing focal contact (also termed focal adhesion) and thus we will discuss these structures in some detail. Another growing body of information concerns cadherin-containing adherence junctions, whereas less is known about the signaling properties of other types of adhesive junctions.

Focal contacts are specialized sites where cells attach to the ECM (Burridge and Chrzanowska-Wodnicka, 1996; Jockush et al., 1995). At focal adhesions, clusters of integrins bind externally to ECM proteins and internally to several specialized cytoplasmic proteins that in turn bind to actin filaments. It is becoming clear that focal adhesions are dynamic structures that change in size (and likely in composition) as the cell adhesion process progresses. Initially, rather small punctate structures are formed at sites of cell-to-substratum contact. As the focal contact matures, the actin filaments extend and bundle to form prominent structures termed stress fibers. Integrins can interact with numerous proteins at focal contacts and at other sites in the cell. In addition, there is a complex network of interactions among the specialized cytoplasmic focal contact proteins. In this section of the review we will introduce some of the key components of focal contacts. We also will discuss transmembrane proteins that directly interact with integrins. Finally, we will introduce several newly discovered cytoplasmic proteins that bind directly to integrin  or  subunit cytoplasmic tails and which may play important roles in integrin signaling. The known interactions of most of the proteins to be discussed below are depicted in fig. 3.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Proteins associated with integrin-containing focal contacts. (A) Focal contact proteins and their interactions. This diagram illustrates the patterns of binding interactions of key structural proteins of the focal contact including talin (Tal), vinculin (Vinc), -actinin (-act), tensin (Tens), and paxillin (Pax). See text for additional details. (B) Proteins interacting directly with integrins. This diagram recapitulates current information concerning proteins that bind directly to integrins. In many cases it is clear that the interaction occurs with a particular  or  subunit or with an individual integrin heterodimer, whereas in other cases the interaction may involve binding to many different integrins. Clearly only some of these interactions will take place in any given type of cell, with the pattern of interaction determined by the expression of the various binding proteins, of various integrins, and by the physiological status of the cell.

1. Cytoskeletal proteins The cytoplasmic structural proteins of the focal contact that directly bind to integrins include talin and -actinin, which in turn bind to other structural proteins including vinculin, paxillin, and tensin, ultimately leading to the recruitment of actin filaments. Integrin-mediated phosphorylation of several cytoskeletal proteins seems to have a role in the organization of stress fibers at focal adhesions. It has been shown that several kinases, including FAK, play a role in the organization of cytoskeleton (see Section IV. below). The structure and function of several of the key structural proteins of the focal contact is described below (fig. 3A).

b. -ACTININ. -Actinin is an actin-bundling protein that comprises two identical polypeptides, each of which is approximately 104 kDa in size. The protein can be divided into three regions: the N-terminal actin binding site, a central region with four -helical motifs, and a carboxy-terminal region (Jockush et al., 1995

d. VINCULIN. Vinculin is a 116 kDa polypeptide enriched in focal contacts. Vinculin binds to actin, -actinin, talin, and paxillin (reviewed in Jockush et al., 1995

2. Transmembrane proteins: tetraspanners, integrin-associated protein 50, caveolin As discussed above, integrins interact via their cytoplasmic tails with an assemblage of cytoplasmic proteins. However, the external and transmembrane domains of integrins also can participate in protein-protein interactions; in some cases these interactions may occur at sites away from focal contacts (fig. 3B).

3. Cytoplasmic integrin-binding proteins The cytoplasmic domains of integrins have been implicated in the bi-directional transfer of information across the plasma membrane. Integrin cytoplasmic tails interact with several proteins that may participate in integrin-mediated functions including signal transduction. Novel proteins have been identified that interact with either the  or  cytoplasmic domains of integrins (fig. 3B).

a. SUBUNIT-ASSOCIATED PROTEINS.

i. 3 endonexin. A novel 111 amino acid protein, 3 endonexin, was cloned by using the integrin 3 cytoplasmic tail as bait in the yeast two-hybrid system (Shattil et al., 1995). This interaction is very specific to 3, because a mutation of serine to proline at position 752 in the 3 cytoplasmic tail reduced the binding by 64%; this mutation is critical for IIb3 integrin-mediated function (Chen et al., 1994b). Because this 3 mutation abolishes binding to fibrinogen (Chen et al., 1992b) and cell spreading (Chen et al., 1994b), it suggests that 3 endonexin is involved in the selective modulation of 3 integrin function. In addition, it has been proposed that 3 endonexin has a function independent of integrins because endonexin is expressed in a wide variety of tissues. An NITY motif at positions 756 to 759 of 3 is critical for the interaction with endonexin (Eigenthaler et al., 1997); this motif is highly conserved in 3 integrins of different species. Another study from the same group (Kashiwagi et al., 1997) demonstrated that 3 endonexin bound to IIb3 in mammalian cells and was able to modulate the affinity state of IIb3, leading to fibrinogen-dependent aggregation. Only a weak staining of 3 endonexin was observed in focal adhesions, suggesting that 3 endonexin interacts with integrins transiently, so that other interactions with the integrin tails can follow after endonexin dissociation (Kashiwagi et al., 1997). A role of 3 endonexin in outside-in signaling events has yet to be demonstrated.

ii. Integrin-linked kinase. A yeast two-hybrid approach also was used to identify another  subunit-binding protein termed integrin-linked kinase (ILK) (Hannigan et al., 1996). ILK is a 59 kDa serine-threonine kinase that can associate with 1, 2, and 3 cytoplasmic domains and has been shown to phosphorylate a 1 integrin cytoplasmic domain peptide in vitro (Dedhar and Hannigan, 1996). Cell attachment to fibronectin reduced ILK kinase activity, whereas overexpression of ILK induced anchorage-independent growth and reduced cell adhesion to laminin, fibronectin, and vitronectin (Hannigan et al., 1996). ILK and 1 integrins colocalize in focal adhesions, suggesting a possible role in cytoskeletal assembly. Recently, it has been shown that ILK overexpression induces anchorage-independent cell cycle progression (Radeva et al., 1997). As discussed in detail in Section X. below, upon integrin-mediated cell adhesion, levels of cyclins D1 and A are up-regulated and cyclin-dependent kinase (CDK) inhibitor levels are down-regulated. Normal cells placed in suspension have down-regulated expression of cyclin D1 and cyclin A and hypophosphorylated retinoblastoma (Rb), whereas suspension cells overexpressing ILK have higher expression of cyclin D1 and cyclin A and hyperphosphorylated Rb (Radeva et al., 1997). Thus ILK may be a key link between integrins and downstream signaling events that influence the cell cycle. In addition, very recent evidence suggests that ILK is involved in epithelial cell differentiation and gene expression (see Section XI.).

iii. Cytohesin 1. Using the 2 cytoplasmic domain as bait in the yeast two-hybrid system, Kolanus et al. (1996) identified a protein they termed cytohesin-1. In lymphoid cells cytohesin-1 binds to 2; further, there seems to be a significant role for cytohesin-1 in the regulation of integrin activation, as measured by L2 adhesion to ICAM1. Cytohesin-1 has a plekstrin homology (PH) domain and a domain that resembles the yeast SEC7 protein. The SEC7 domain alone induces L2 binding to ICAM1, whereas the PH domain inhibits the process, seemingly acting in a dominant negative role. It has been postulated that PH domains function like SH2 and SH3 domains, mediating protein-protein associations in signal transduction pathways and/or localizing proteins to the plasma membrane (Pawson, 1995). Cytohesin-1 recently has been shown to function as a guanine nucleotide exchange factor (GNEF) probably via its SEC7 domain (Meacci et al., 1997). Moreover, cytohesin-1 also binds via its PH domain to phosphoinositol 3,4,5-triphosphate, suggesting a link with PI-3K pathways (Klarlund et al., 1997). Thus cytohesin-1 may play several important roles in integrin activation and in intracellular signaling.

iv. Integrin cytoplasmic domain-associated protein 1. Recently, another novel 1 integrin cytoplasmic domain-associated protein (ICAP1) was isolated using a yeast two-hybrid approach. Two isoforms of ICAP1 that are derived from alternatively spliced mRNA have been reported; the longer isoform has 200 amino acids (ICAP1) and the shorter isoform has 150 amino acids (ICAP1). The interaction of ICAP1 with 1 integrins has been shown in vitro and in vivo. ICAP is a phosphoprotein, and its phosphorylation is regulated during cell-matrix interactions. Cell attachment to fibronectin resulted in enhancement of ICAP1 phosphorylation, whereas expression of Rho A that disrupts cell-matrix interactions resulted in dephosphorylation of ICAP1. These data indicate that ICAP1 has a role in integrin-mediated cell adhesion and spreading (Chang et al., 1997).

b. SUBUNIT INTERACTING PROTEINS.

i. Calreticulin. A conserved stretch of seven amino acids (KXGFFKR) exists in all integrins and subunits. To understand the role of such a highly conserved region, investigators tried to identify proteins that interact with this region. Rojiani et al. (1991) reported a 60 kDa protein that specifically binds to the KXGFFKR sequence. This protein had significant homologies to rabbit calreticulin (Fliegel et al., 1989) and the Ro/SS-A antigen, its human equivalent (McCauliffe et al., 1990). An in vivo interaction of calreticulin with the active form of the 21 integrin has been demonstrated (Coppolino et al., 1995). Further, inhibition of expression of calreticulin by antisense oligonucleotides resulted in inhibition of cell adhesion and less cell spreading, indicating a role for calreticulin in cell-ECM interactions (Leung-Hagesteijn et al., 1994). Recently, a double knockout of calreticulin in embryonic stem cells suggested that calreticulin is essential for the function of integrins including "outside-in" signaling (Coppolino et al., 1997).

ii. Calcium- and integrin-binding protein. A novel calcium- and integrin-binding protein (CIB) of 25 kDa that interacts with IIb was identified using the yeast two-hybrid system. This protein specifically binds to IIb and has similarities to the calcium-binding proteins calmodulin and calcineurin. CIB interacts with IIb3 in vitro and is expressed in platelets, which suggests that it may function as a regulatory molecule for IIb3 (Naik et al., 1997).

iii. Integrin-binding protein 1. Using a yeast two-hybrid approach, we identified a novel protein that interacts with the 5 subunit cytoplasmic tail (Alahari and Juliano, unpublished results). This protein does not have significant homologies to known proteins, and henceforth was tentatively named integrin-binding protein 1 (IBP1). IBP1 binds to human 51 in vitro, but its physiological role is currently unknown.

a. INSULIN RECEPTOR SUBSTRATE 1 AND 190 KDA. Two cytoplasmic proteins have been shown to associate with v3 integrin in response to growth factor stimulation. A 190 kDa tyrosine-phosphorylated protein associates with v3 in PDGF-stimulated fibroblasts (Bartfeld et al., 1993