Control of integrin affinity for ligands (integrin activation) is essential for normal cell adhesion, migration, and assembly of an extracellular matrix. Integrin activation is usually mediated through the integrin beta subunit cytoplasmic tail and can be regulated by many different biochemical signaling pathways. We report that specific binding of the cytoskeletal protein talin to integrin beta subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity. Thus, regulated binding of talin to integrin beta tails is a final common element of cellular signaling cascades that control integrin activation.

Cell-directed changes in the ligand-binding affinity (‘activation’) of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin’s affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins.

Abstract. We analyzed the binding of fibronectin to integrin a5/31 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd ,~ 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of c~3 joined to the cytoplasmic domains of otsBl. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PACl. The cytoplasmic domains of 0~5/~ conferred an energy-dependent high affinity state on otnbB3 in CliO but not K562 cells. Three additional tx cytoplasmic domains (ix2, otv~, ot~B) conferred PAC1 binding in CHO cells, while three others (otM, OiL, av) did not. In the high affinity ot chimeras, cotransfection with a truncated (~3A724) or mutated (~3(S752"-~P)) 83 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved o~ subunit GFFICR motif, resulted in high affinity binding of ligands to Otnb/~3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the ~ subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, ' the highly conserved GFFKR motif of the c~ Subunit cytoplasmic domain maintains the default low affinity state.

Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin avb3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear-induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of¯ow. The results therefore de®ne the role of integrins and Rho in a pathway leading to endothelial cell adaptation to¯ow.

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Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin αIIbβ3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in αIIbβ3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with αIIbβ3. However, fibrinogen binding caused Csk to dissociate from αIIbβ3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with αIIbβ3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to αIIbβ3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.

The major platelet integrin, ␣IIb␤3, is required for platelet interactions with proteins in plasma and the extracellular matrices (ECMs) that are essential for platelet adhesion and aggregation during hemostasis and arterial thrombosis. Ligand binding to ␣IIb␤3 is controlled by insideout signals that modulate receptor conformation and clustering. In turn, ligand binding triggers outside-in signals through ␣IIb␤3 that, when disrupted, can cause a bleeding diathesis. In the past 5 years there has been an explosion of knowledge about the structure and function of ␣IIb␤3 and the related integrin, ␣V␤3. These developments are discussed here, and current models of bidirectional ␣IIb␤3 signaling are presented as frameworks for future investigations. An understanding that ␣IIb␤3 functions as a dynamic molecular scaffold for extracellular and intracellular proteins has translated into diagnostic and therapeutic insights relevant to hematology and cardiovascular medicine, and further advances can be anticipated. ( IntroductionThe platelet is a tightly regulated adhesion machine. Restrained in its functions while in the bloodstream, its adhesive, hemostatic, and proinflammatory capabilities are unleashed at sites of vessel injury to generate the primary hemostatic plug, catalyze fibrin formation, and supply soluble and membrane-bound factors that promote wound healing. 1 While platelets can adhere to damaged endothelial cells, 2 their principle adhesive surface is the extracellular matrix (ECM), which becomes exposed in injured vessels and offers a panoply of ligands for platelet adhesion receptors. 3 Within this context, integrin adhesion receptors, and ␣IIb␤3 in particular, play critical roles in platelet function.Integrins are heterodimeric (␣␤) type I transmembrane receptors, each subunit typically containing a relatively large extracellular domain, a single-pass transmembrane domain, and a short cytoplasmic tail composed of 20 to 60 amino acids. 4 Platelets express several integrins (␣IIb␤3, also called glycoprotein IIb-IIIa [GPIIb-IIIa]; ␣V␤3; ␣2␤1; ␣5␤1; ␣6␤1). Integrins are, in effect, "2-faced" receptors, one face oriented to the extracellular space and interactive with cognate ECM ligands and the other oriented to the cell interior and interactive with cytoplasmic proteins. Ligand binding to either face can trigger information transfer, or signaling, across the plasma membrane to "activate" cellular functions at the other face. Figure 1 illustrates this bidirectional signaling using ␣IIb␤3 as an example.Basic research conducted in the past 3 decades on many facets of ␣IIb␤3 structure and function has led to remarkable breakthroughs culminating in the development of a chimeric anti-␣IIb␤3 monoclonal antibody and small-molecule receptor antagonists now used parenterally to limit the formation of occlusive platelet thrombi in acute cardiovascular indications. 5,6 On the other hand, clinical trials of oral ␣IIb␤3 antagonists have been disappointing and suggest that long-term extracellular blockade of ligand binding ...

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