Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of neurodegenerative diseases and stroke. However, the mechanism of MMP activation remains unclear. We report that MMP activation involves S-nitrosylation. During cerebral ischemia in vivo, MMP-9 colocalized with neuronal nitric oxide synthase. S-Nitrosylation activated MMP-9 in vitro and induced neuronal apoptosis. Mass spectrometry identified the active derivative of MMP-9, both in vitro and in vivo, as a stable sulfinic or sulfonic acid, whose formation was triggered by S-nitrosylation. These findings suggest a potential extracellular proteolysis pathway to neuronal cell death in which S-nitrosylation activates MMPs, and further oxidation results in a stable posttranslational modification with pathological activity.

Cellular regulation of the ligand binding affinity of integrin adhesion receptors (integrin activation) depends on the integrin ␤ cytoplasmic domains (tails). The head domain of talin binds to several integrin ␤ tails and activates integrins. This head domain contains a predicted FERM domain composed of three subdomains (F1, F2, and F3). An integrin-activating talin fragment was predicted to contain the F2 and F3 subdomains. Both isolated subdomains bound specifically to the integrin ␤ 3 tail. However, talin F3 bound the ␤ 3 tail with a 4-fold higher affinity than talin F2. Furthermore, expression of talin F3 (but not F2) in cells led to activation of integrin ␣ IIb ␤ 3 . A molecular model of talin F3 indicated that it resembles a phosphotyrosine-binding (PTB) domain. PTB domains recognize peptide ligands containing ␤ turns, often formed by NPXY motifs. NPX(Y/F) motifs are highly conserved in integrin ␤ tails, and mutations that disrupt this motif interfere with both integrin activation and talin binding. Thus, integrin binding to talin resembles the interactions of PTB domains with peptide ligands. These resemblances suggest that the activation of integrins requires the presence of a ␤ turn at NPX(Y/F) motifs conserved in integrin ␤ cytoplasmic domains.Integrin adhesion receptors are essential for the development and survival of multicellular animals. Normal functioning of the Ͼ20 human integrins often requires dynamic cellular regulation of integrin ligand binding affinity (integrin activation). Activation of integrins is important in many biological processes, including cell migration, hemostasis, extracellular matrix assembly, tumor metastasis, and the immune response (1, 2). Integrin ␣␤ heterodimers generally possess two short cytoplasmic domains (tails). The integrin ␤ tail plays a central role in the activation process, probably by undergoing regulated interactions with certain cytoplasmic proteins (1, 3).Talin is an abundant and widely expressed 250-kDa integrin ␤ tail-binding protein implicated in integrin activation (4). Talin is composed of a 50-kDa head and 205-kDa rod domain. The head domain contains a major integrin-binding site (5-7), and expression of a 1071-residue fragment of talin containing the head domain in cells leads to activation of integrin ␣ IIb ␤ 3 (5). The capacity of this fragment to activate integrin ␣ IIb ␤ 3 is lost when the head domain is deleted from it or when the ␤ 3 cytoplasmic domain is truncated (5). Talin binding to integrin ␤ tails can be regulated via calpain proteolysis (6) or through the binding of phosphoinositides (8). Furthermore, the phosphorylation of either talin and/or integrin (9, 10) could provide additional mechanisms for regulation of integrin-talin interactions. Thus, the talin head domain is implicated in integrin activation, and modulation of its binding to integrins is likely to contribute to the regulation of integrin activation.The talin head domain contains a predicted FERM domain (band four-point-one/ezrin/radixin/moesin homology domain) (11). FERM ...

Talin links integrin ␤ cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin ␤ cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the ␤ 3 integrin cytoplasmic domain. Here we report that the N-terminal ϳ47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the ␤ 3 tail. The affinity difference is mainly due to a difference in k on . Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K a and a 100-fold increase in apparent k on . The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the ␤ 3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band fourpoint-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.Integrins play important roles in the development and functioning of all multicellular animals (1). Integrins are non-covalent heterodimers of type I transmembrane protein subunits termed ␣ and ␤. Each subunit has a large (Ͼ700 residue) N-terminal extracellular domain. A single membrane-spanning domain links this extracellular domain to a generally (with the exception of ␤4) short (13-70 residues) cytoplasmic domain (2). Integrins bind to insoluble ligands (e.g. collagen fibrils) and link them to the intracellular cytoskeleton. In addition to forming these physical linkages, integrins regulate cell growth, survival, and differentiation (1). These signaling activities and cytoskeletal linkages depend on the integrin cytoplasmic domains (3). Conversely, integrin-mediated adhesion is rapidly and precisely regulated by changes in integrin affinity for ligand (activation) (4, 5) and by affinity-independent mechanisms such as changes in integrin clustering (6). Integrin linkages with the cytoskeleton can affect both integrin activation (7, 8) and affinity-independent (7, 9) regulation of integrinmediated cell adhesion. Thus, integrin-cytoskeletal linkages play a pivotal role in their regulation and in their signaling properties.Talin is an actin-binding protein that links integrins to the actin cytoskeleton (10). Talin co-localizes with clustered integrins in all known species that possess these receptors. Furthermore, genetic and cell biological analyses show that talin is required for integrin clustering into focal adhesions (11). Talinintegrin interactions can also regulate integrin activation (12). Talin consists of an N-terminal ϳ47-kDa globular head domain (talin-H) 1 and a ϳ190-kDa C-terminal rod (talin-R) domain (13...

Integrin adhesion receptors contain an on/off switch that regulates ligand binding affinity and cell adhesion. The switch from "off" to "on" is commonly referred to as integrin activation. The objective of this study was to gain insight into the nature of the on/off switch in platelet integrin ␣ IIb ␤ 3 . Here, we show that a select group of the cysteines, located within the extracellular cysteinerich domain of the ␤ subunit, remain unpaired. These unpaired cysteine residues exhibit the properties of a redox site involved in integrin activation. Alterations to the redox site prevent the inter-conversion between resting and active integrin. Altogether, the study establishes integrin as a direct target for redox modulation, revealing an unappreciated link between cell adhesion and redox biology.Integrins are transmembrane receptors that mediate cell adhesion and cell migration (1, 2). The integrin protein family is directly involved in most cell-matrix contacts and cell adhesion events. Many pathologic events, including tumor progression, angiogenesis, and vascular disease, also involve integrins (3-5). In most cases cell adhesion is stringently regulated, both spatially and temporally. Spatial regulation is achieved by the expression patterns of the integrins and their various ligands. Temporal regulation of adhesion is conferred by a process called integrin "activation." Activation unshields the integrin ligand binding site, increasing its ligand binding affinity. The activation, and de-activation, of integrins is crucial to events like morphogenesis, tumor cell invasion, and platelet aggregation (6 -8).The subject of this study is platelet integrin ␣ IIb ␤ 3 , an excellent paradigm of the integrin protein family. Integrin ␣ IIb ␤ 3 is a particularly relevant model for the study of integrin activation because its function on the platelet requires physiologic activation (9). Integrin ␣ IIb ␤ 3 is maintained in a resting state on circulating platelets. However, agonists like ADP or thrombin induce activation of the integrin. This activation facilitates the binding of soluble fibrinogen, leading to the formation of a platelet aggregate, or thrombus, that halts the loss of blood. Consequently, activation of ␣ IIb ␤ 3 is key for normal hemostasis. Importantly, though, improper activation of ␣ IIb ␤ 3 can have lethal consequences. Rupture of atherosclerotic plaques can cause activation of ␣ IIb ␤ 3 on platelets and can lead to myocardial infarct (10).The precise mechanism by which integrins are activated and de-activated is still not completely understood. A significant body of work has focused on how alterations to integrin cytoplasmic domains control activation. In one well supported model, changes in the conformation of the cytoplasmic tails are thought to release a conformational constraint, or open an "integrin hinge" (11). The release of this hinge is believed to translate into conformational rearrangements in the extracellular face of the integrin that expose the ligand binding site. The physical association of...

Integrin alphaIIbbeta3 plays a pivotal role in hemostasis and thrombosis by mediating platelet adhesion and platelet aggregation. Integrin alphaIIbbeta3 contains an on/off switch that regulates its ligand binding affinity. The switch from "off" to "on" is commonly referred to as integrin activation. We recently identified a redox site within the extracellular domain of the platelet integrin alphaIIbbeta3 that exhibits many properties that one might expect of the on/off switch [Yan, B., and Smith, J. W. (2000) J. Biol. Chem. 275, 39964-39972]. Several independent reports show that reducing agents, such as dithiothreitol, can activate integrins. The objective of the present study was to determine if the effects of DTT can be attributed to a perturbation at the integrin redox site. Indeed, we find that DTT reduces two disulfide bonds within the integrin's cysteine-rich domain. Such bond reduction leads to global conformational changes within both alphaIIb and beta3 and the opening of the RGD and fibrinogen binding sites. These findings causally link the reduction of disulfide bonds within the integrin's redox site to transitions in the integrin's activation state.

Integrin ␣ IIb ␤ 3 is the fibrinogen receptor that mediates platelet adhesion and aggregation. The ligand binding function of ␣ IIb ␤ 3 is "activated" on the platelet surface by physiologic stimuli. Two forms of ␣ IIb ␤ 3 can be purified from platelet lysates. These forms are facsimiles of the resting (Activation State-1 or AS-1) and the active (Activation State-2 or AS-2) conformations of the integrin found on the platelet surface. Here, the differences between purified AS-1 and AS-2 were examined to gain insight into the mechanism of activation. Four major findings are put forth. 1) The association rate (k 1 ) between fibrinogen and the integrin is a key difference between AS-1 and AS-2. 2) Although the divalent ion Mn 2؉ enhances the ligand binding function of AS-1, this ion is unable to convert AS-1 to AS-2. Therefore, its effect on integrin is unrelated to activation. 3) Peptide mass fingerprints indicate that the chemical structure of AS-1 and AS-2 are virtually identical, calling into question the idea that post-translational modifications are necessary for activation. 4) The two forms of ␣ IIb ␤ 3 have significant conformational differences at three positions. These include the junction of the heavy and light chain of ␣ IIb , the divalent ion binding sites on ␣ IIb , and at a disulfide-bonded knot linking the amino terminus of ␤ 3 to the cysteine-rich domain. These observations indicate that integrin is activated by a series of specific conformational rearrangements in the ectodomain that increase the rate of ligand association.

No abstract