Integrins Nucleate the Formation of Multi Protein Complexes

A central function of the integrins is to mediate a structural linkage between the dynamic intracellular cytoskeleton and the ECM. More that 50 proteins have been identified either as direct integrin-binding proteins or as proteins that localize to adhesion complexes (e.g., focal adhesions) [4]. The proteins found in adhesion complexes fall into two broad categories: those that serve a structural role to anchor and regulate the actin cytoskeleton and those that are responsible for integrin-mediated signaling and the remodeling of adhesion complexes.

Handbook of Cell Signaling, Volume 1

The association of talin, a-actinin and vinculin with inte-grin receptors serves to illustrate how integrins are linked to actin filaments and the cytoplasm (Fig. 1). Talin is a major structural component of focal adhesions [3]. Talin binds directly to the tails of pi, P2, and P3 integrins. In addition, talin contains binding sites for actin, vinculin, focal adhesion kinase (FAK), and phospholipids. Cells deficient for the expression of talin exhibit significant increases in membrane blebbing, defects in cell adhesion and spreading, and a failure to assemble focal adhesions and stress fibers, underscoring the role of talin in the organization of adhesion structures [5]. a-Actinin is an actin-binding protein that binds the cytoplasmic tails of Pi, P2, and P3 integrins [3] as well as several additional focal adhesion proteins, including vinculin and zyxin. Localization of a-actinin to adhesion complexes occurs by a direct interaction with P-integrin cytoplasmic tails. Vinculin is one of the most abundant focal adhesion proteins, although it does not bind integrins directly [3]. Vinculin binds F-actin and the adhesion-associated proteins paxillin and VASP and is recruited to focal adhesions indirectly via an interaction with an integrin cytoplasmic tail binding protein (e.g., talin or a-actinin). Vinculin functions as a molecular bridge to stabilize integrin-F-actin linkages. Vinculin-deficient cells exhibit decreased mechanical stiffness and increased cell motility [3,5].

Integrins also serve to recruit proteins that are directly involved in regulating the formation and turnover of adhesion complexes and the promotion of intracellular signals (Fig. 1). FAK is a focal-adhesion-associated, nonreceptor protein tyrosine kinase. FAK binds in vitro to the cytoplasmic tails of P1 and P3 integrins, although to date this interaction

Figure 1 Integrin connections. Schematic diagram of protein interactions initiated by integrin receptors, as described in the text. The following abbreviations are used: ARF (ADP-ribosylation factor), Arp2/3 (actin-related protein 2/3 complex), ASAP (ARF-GAP containing SH3, ankyrin repeats, and PH domain), C3G (Crk SH3-binding GNRP), Cas (Crk-associated substrate), Crk (CT10 regulator of kinase), FAK (focal adhesion kinase), GRAF (GTPase regulator associated with FAK), ILK (integrin-linked kinase), MAPK (mitogen-activated protein kinase), myosin PTPases (myosin phosphatase), p190RhoGAP (p190 Rho GTPase-activating protein), PAK (p21-activated kinase), PI3K (phos-phatidylinositol 3-kinase), PIX/COOL (Pak-interacting exchange factor/cloned out of library), PKL/GIT1 (paxillin-kinase linker/G-protein-coupled receptor kinase-interacting protein 1), PTEN (phosphatase and tensin homolog deleted on chromosome ten), ROCK (Rho kinase), RTKs (receptor tyrosine kinases), SOS (Son of Sevenless), VASP (vasodilator-stimulated phosphoprotein), and WASP (Wiskott-Aldrich syndrome protein).

Figure 1 Integrin connections. Schematic diagram of protein interactions initiated by integrin receptors, as described in the text. The following abbreviations are used: ARF (ADP-ribosylation factor), Arp2/3 (actin-related protein 2/3 complex), ASAP (ARF-GAP containing SH3, ankyrin repeats, and PH domain), C3G (Crk SH3-binding GNRP), Cas (Crk-associated substrate), Crk (CT10 regulator of kinase), FAK (focal adhesion kinase), GRAF (GTPase regulator associated with FAK), ILK (integrin-linked kinase), MAPK (mitogen-activated protein kinase), myosin PTPases (myosin phosphatase), p190RhoGAP (p190 Rho GTPase-activating protein), PAK (p21-activated kinase), PI3K (phos-phatidylinositol 3-kinase), PIX/COOL (Pak-interacting exchange factor/cloned out of library), PKL/GIT1 (paxillin-kinase linker/G-protein-coupled receptor kinase-interacting protein 1), PTEN (phosphatase and tensin homolog deleted on chromosome ten), ROCK (Rho kinase), RTKs (receptor tyrosine kinases), SOS (Son of Sevenless), VASP (vasodilator-stimulated phosphoprotein), and WASP (Wiskott-Aldrich syndrome protein).

has not been demonstrated in vivo [6]. FAK contains an approximately 100-amino-acid domain that is both necessary and sufficient to target FAK to focal adhesions [7]. In addition, FAK also contains sequences that mediate its association with focal adhesion proteins paxillin and talin, as well as to the cytoskeletal adaptor protein Cas and GTPase-activating proteins (GAPs) for Rho (GRAF [8]) and ARF1 (ASAP1 [9]) (Fig. 2). Paxillin is a multidomain protein that not only binds to FAK, but also serves as a scaffold to recruit and organize a number of additional signaling molecules at the sites of adhesion. Paxillin binds Src, Crk, vinculin, actopaxin, and the serine/threonine kinase ILK, as well as the ARF GAPs PKL and GIT1 [10,11]. In addition, paxillin binds directly to the cytoplasmic tails of a4 integrins [12]. Like FAK-deficient cells, paxillin-null cells exhibit defects in cell spreading and cell migration, as well as decreased tyrosine phosphorylation of FAK and Cas [13,14]. Cas is another adaptor protein that binds to both FAK and Src and serves to recruit additional signaling molecules to focal adhesions. Cas associates with the guanine nucleotide exchange factor C3G, protein phosphatases, and adaptor proteins Crk and Nck [15]. Coupling between FAK, Src, and Cas appears to be important for FAK-stimulated cell migration [16].

A number of other proteins and kinases have been classified as integrin binding or integrin-associated proteins, including adaptor proteins and kinases (for example, RACK1, Shc, Grb2, and ILK); growth factor receptors (EGF receptor, ErbB2, PDGF receptor-P, insulin receptor, VEGF receptor); cytoplasmic, chaperone, calcium-binding proteins (calnexin, calreticulin, CIB, endonexin); and membrane-associated proteins (tetraspanins, Ig superfamily proteins, GPI-linked receptors, transmembrane proteins, and ion channels). The functional and structural diversity amongst these integrin-associated proteins underscores the importance of integrins as initiators of many intracellular signaling pathways. How integrins function in a structural versus a signaling role and how such complexes are organized temporally and spatially within the cell remain important and unanswered questions.

Cell Migration: A Paradigm for Studying Integrin Signaling

Cell migration provides an exceptionally relevant model to study integrin signaling. Migration is a complex cellular process that involves the extension of lamellipodia, adhesion

Figure 2 Focal adhesion kinase and its binding partners. FAK consists of a centrally located kinase domain flanked by an amino-terminal FERM domain and a carboxy-terminal region containing proline-rich sequences (site I and site II), as well as the focal adhesion targeting (FAT) domain. FAK autophosphorylation at Y397 in the amino-terminal domain creates a binding site for Src as well as the p85 subunit of PI3K. The carboxy-terminal domain of FAK binds the adaptor protein Cas, the GAP proteins GRAF and ASAP, and focal adhesion proteins talin and paxillin. Phosphorylation of FAK on Y925 creates a binding site for Grb2, potentially leading to the activation of the Ras-MAPK pathway.

Figure 2 Focal adhesion kinase and its binding partners. FAK consists of a centrally located kinase domain flanked by an amino-terminal FERM domain and a carboxy-terminal region containing proline-rich sequences (site I and site II), as well as the focal adhesion targeting (FAT) domain. FAK autophosphorylation at Y397 in the amino-terminal domain creates a binding site for Src as well as the p85 subunit of PI3K. The carboxy-terminal domain of FAK binds the adaptor protein Cas, the GAP proteins GRAF and ASAP, and focal adhesion proteins talin and paxillin. Phosphorylation of FAK on Y925 creates a binding site for Grb2, potentially leading to the activation of the Ras-MAPK pathway.

at sites within newly formed lamella, organization of forcegenerating adhesions, contraction and cell-body displacement, and detachment of the cell rear. These events require the coordination of multiple signaling pathways.

Lamellipodia Extension and Formation of New Adhesions

The initial steps in cell migration require the formation of protrusive structures (lamellipodia) at the leading edge of the cell and the stabilization of the protrusion by newly formed adhesion complexes. Cell protrusions are regulated by the activity of surface receptors and Rho family GTPases Cdc42 and Rac [17]. Actin polymerization at the cell front is regulated by Cdc42 and Rac via their interaction with members of the Wiskott-Aldrich syndrome protein (WASP)/Scar1 superfamily [18]. Binding of Cdc42/Rac to WASP/Scar proteins activates the Arp2/3 complex [19], triggering its binding to the sides of preexisting actin filaments and stimulating new filament formation, which results in branched actin networks [20]. The formation of the branched actin network serves to drive the forward extension of the cell membrane, leading to the formation of lamellipodia [20,21].

Formation of new adhesions within lamellipodia involves integrin-induced assembly of FAK/Src complexes and the recruitment of two adaptor proteins, Cas and paxillin. Formation of this signaling complex is likely important to sustain activation of Rac and activation of serine/threonine kinase PAK (p21-activated kinase). FAK/Src-mediated phosphorylation of Cas or paxillin creates binding sites for the adaptor protein Crk. Cas/Crk complexes mediate Rac activation by binding DOCK180 [22,23], the human counterpart of the Drosophila and Caenorhabditis elegans genes mbc and ced-5, respectively [24,25]. While paxillin binds Crk following FAK/Src-mediated phosphorylation and signals to Rac, FAK mutants deficient in binding to paxillin efficiently restore migration of FAK null cells to a wild-type level [26]. Thus, in this setting, signaling to Cas appears to be sufficient to mediate adhesion formation.

Paxillin is an important regulator of Cdc42 and Rac through its binding to PKL and the subsequent interaction of PKL with two members of the Cdc42/Rac GEF family PIX/COOL [27-29]. The PIX/COOL family of proteins was originally reported to exhibit GEF activity for Rac and Cdc42 [30], although recently this property has been questioned, raising speculation that PIX/COOL proteins might activate PAK by binding the GTP form of Cdc42/Rac rather than directly activating the GTPases [27]. While the formation of the paxillin/PKL/PIX complex has been reported to activate PAK, recent data indicate that the interaction of paxillin with Rac leads to the downregulation of Rac activity [31], providing a possible mechanism for Rac turnover/ downregulation. A suggested pathway (Fig. 1) may be inte-grin recruitment and activation of FAK/Src, binding and phosphorylation of Cas, and activation of Rac via Cas/Crk/ Dock180 complexes. GTP-Rac may then bind to PIX/ COOL proteins complexed with paxillin/PKL, resulting in PAK activation and Rac downregulation.

Maturation of Newly Formed Adhesions

Activation of Rho is required for the organization of F-actin into stress fibers and the formation of focal adhesions [32]. Both functions are regulated by the ability of Rho to promote the generation of directional forces, via its regulation of Rho kinase and myosin phosphatase and the subsequent regulation of myosin light chain (MLC) phosphorylation. Rho activation of Rho kinase inhibits myosin phosphatase, thereby maintaining MLCs in a highly phosphorylated (contractile) state. The resultant contractile forces are essential for the organization of actin filaments and adhesion complexes [32].

During cell migration, Cdc42/Rac and Rho signaling are regulated in a reciprocal fashion, leading to the breakdown of stress fibers and focal adhesions (due to the downregulation of Rho) and the commensurate reorganization of cortical actin networks at the leading edge of the cells [33-35]. Plating cells on ECM stimulates a transient decrease in Rho activity, which is necessary for cell spreading [36,37]. FAK appears to contribute to the transient decrease in Rho activity, as such changes in Rho activity are not observed in cells deficient for FAK expression [38]. The mechanism by which FAK regulates the initial decrease in Rho activity may involve its interaction with the Rho GTPase-activating protein GRAF [8] or its ability to activate Src, which has been shown to phosphorylate p190RhoGAP, resulting in decreased Rho activity upon integrin engagement [36,39]. A subsequent increase in Rho activity is necessary to restore contractile forces, leading to strengthening of attachment sites, stress fiber formation, and generation of the forces necessary for continued cell movement [32].

Detachment and Release of Adhesions

In addition to stabilizing lamellipodia formation at the front of the cell, detachment at the rear of the cell requires sustained contraction and disassembly of integrin complexes. Mitogen-activated protein kinase (MAPK), like Rho kinase, phosphorylates MLCK, stimulating MLC phosphorylation and cell contraction [40]. In addition, phosphorylation of focal-adhesion-localized calpain by active MAPK [41] stimulates calpain-mediated cleavage of adhesion proteins and cell detachment [42,43]. Integrins activate MAPK through three different Ras-dependent pathways. Integrin-mediated activation of FAK and recruitment of Src results in phosphorylation of FAK on Tyr925 [44,45]. Phosphorylation on Tyr925 creates a binding site for Grb2 [44], an SH2/SH3 adaptor protein that links growth factor receptor tyrosine kinases to the Ras/MEK/MAPK pathway through the Ras guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange protein SOS (Fig. 1). Integrins also activate SOS through caveolin-1-mediated recruitment of Shc to integrins and subsequent phosphorylation by Fyn [46] (Fig. 1). Finally, integrin engagement results in phosphory-lation and activation of the epidermal growth factor (EGF) receptor in the absence of EGF stimulation [47]. Activated EGF receptor recruits Shc to the receptor, where phospho-rylation creates a binding site for Grb2/SOS [47] (Fig. 1). Indeed, in this setting, ECM-mediated phosphorylation of Shc and activation of MAPK is blocked by inhibitors of EGF receptor tyrosine kinase activity. Integrin-stimulated migration is inhibited by MAPK inhibitors and stimulated by expression of active MEK [40]. Interestingly, dominantnegative Ras expression has little effect on ECM-stimulated migration [48,49], indicating the existence of Ras-independent mechanisms of MAPK activation.

Several studies show that Rac synergizes with Raf to stimulate MAPK-dependent migration in response to EGF [50]. Rac-dependent activation of PAK stimulates phospho-rylation of MEK, resulting in an increased affinity of MEK for Raf [51]. An important consequence of Rac activation may be the enhancement of Raf-MEK interaction, leading to maximum MAPK activity in a setting of only basal Ras and Raf stimulation. This may provide a mechanism by which integrins potentiate signals from growth factor receptors, allowing cells to respond to low levels (gradients) of chemo-tactic signals in the environment.

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