A Redox Site Involved in Integrin Activation

Abstract

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...

Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH2), a beta integrin antagonist, in patients with solid tumours

“…Thus, ATN-161 may inhibit the function of several integrins implicated in tumour angiogenesis and metastasis. Disulphide interchange has been proposed to mediate integrin activation (Yan and Smith, 2000); we hypothesise that the free cysteine thiol in ATN-161 blocks this interchange by forming a disulphide with the integrin target, thereby suppressing integrin function.…”

Modulation of CD11b/CD18 Adhesive Activity by Its Extracellular, Membrane-Proximal Regions

“…Given the similarity between the three mutant receptors and the activated CD11b/CD18 receptor on peripheral leukocytes in their reactivity toward mAb MEM148, the three active CD11b/CD18 mutants may exhibit certain intermediate conformations that exist temporarily along the physiological activation pathway of the CD11b/ CD18 receptor. Moreover, several other mechanisms could also play important roles in integrin activation, such as disulfide bond exchange within the cysteine-rich region of the ␤ subunit (47), association between the ␣ subunit Calf-2 and the ␤ subunit ␤TD domains (48), the hinge model (16), etc. As the inserted sequence (GGGGSGGGGS) in our study exists in a random flexible conformation in solution (28), we also speculated that the rigidity of the extracellular membrane-proximal regions of the CD11b/CD18 receptor is critical to modulation of integrin activity by inside-out signaling.…”

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

“…This does not exclude other mechanisms of cGMP-independent action such as inhibition of intracellular calcium entry [37] or direct nitrosation of α IIb β 3 [33,38].…”

“…Free thiols become available on the platelet surface upon activation [10] and those present on integrin α IIb β 3 provide a direct target for redox modification [33]. Thiol-disulphide exchange within α IIb β 3 , catalysed by an endogenous isomerase activity [34], by PDI [11,35], or by ERP5 [36] is necessary for transition to its active conformation.…”