Disruption of disulfide-restriction at integrin knees induces activation and ligand-independent signaling of α4β7

Abstract

SummaryControl of integrin activation and signaling plays crucial roles in cell adhesion, spreading and migration. Here, we report that selective breakage of two conserved disulfide bonds located at the knees of integrin a 4 C589-C594 and b 7 C494-C526 activated a 4 b 7 . This activated integrin had a unique structure that was different from the typical extended conformation of active integrin. In addition, these activated a 4 b 7 integrins spontaneously clustered on the cell membrane and triggered integrin downstream signaling independent of ligand binding. Although these disulfide bonds were not broken during a 4 b 7 activation by inside-out signaling or Mn 2+ , they could be specifically reduced by 0.1 mM dithiothreitol, a reducing strength that could be produced in vivo under certain conditions. Our findings reveal a novel mechanism of integrin activation under specific reducing conditions by which integrin can signal and promote cell spreading in the absence of ligand.

Extracellular Redox Regulation of α7β Integrin-Mediated Cell Migration Is Signaled via a Dominant Thiol-Switch

“…Several other disulfide bonds within integrin ectodomains are in discussion to fulfill similar redox-regulatory roles, such as cysteine pairs within the β3 subunit located within the I-domain [27], within the EGF-domains of the stalk region [24,[28][29][30], or between the PSI-and EGF1-domain as a long-range cysteine bond [31]. Within the integrin α subunits, cysteine bridges within the propeller domain and in the hinge region of integrin subunit α4 were delineated for their reducibility [24][25][26]. However, the integrin α4 subunit stands apart from other integrins because of its specific proteolytic processing likely within the thigh domain close to the hinge region and because of its easy dissociation into a heavy and light chain [47].…”

“…Along with reversible disulfide bond formation, this cysteine pair changes the protein conformation and activity. Previous studies reported that specific cysteine residues within integrins, located within [24][25][26] and outside [24,[27][28][29][30][31] of the α-subunit hinge region, are relevant for integrin function. However, most of these studies did not show their redox-dependent functional reversibility.…”

Disulfide Bonds as Regulators of Integrin Function in Thrombosis and Hemostasis

“…This bond is the 1-5 disulfide bond in the b2 EGF-3 domain and is homologous to the Cys523-Cys544 bond in b3 EGF-3, suggesting that this integrin unique bond is functional in both b3 and b2 integrins. A recent study showed that disruption of the b7 Cys494-Cys526 disulfide bond as well as the a4 Cys589-Cys594 disulfide bond resulted in increased ligand-binding affinity of the a4b7 integrin and outside-in signaling of the integrin independent of ligand binding (150). These bonds were also shown to be specifically reduced by low concentrations of DTT, giving rise to a unique non-extended active conformation of the FIG.…”

“…A model structure of the a4b7 integrin showed that similar to the homologous cysteines in aIIbb3 and avb3, the b7 Cys494-Cys526 and the a4 Cys589-Cys594 disulfide bonds are located in the integrin knee, implying that the knee region and disulfide bonds in it are important for the regulation of integrin activation. The authors suggested a novel mechanism for integrin affinity and signaling regulation through reduction of the two disulfide bonds at the knees of integrin (150). Cysteine substitutions that elevate ligand binding affinity were also identified in the homologous b subunit from Drosophila (bPS), and were located in the EGF-1, EGF-3, PSI, and hybrid domain of bPS subunit (66).…”

“…Abnormalities in integrins stand at the basis of many diseases and constitute targets for therapy of thrombosis and inflammation. Some of the disulfide bonds in integrins have been shown to have a functional role in regulating integrin function (88,89,150). Integrin aIIbb3 is the most abundant integrin in platelets and exerts an essential role in thrombus formation by mediating platelet aggregation (105,115).…”