Interaction and functional association of protein disulfide isomerase with α_Vβ₃ integrin on endothelial cells

Abstract

Adhesive properties of endothelial cells are influenced by the thioldisulfide balance. However, the molecular mechanism of this effect is unclear, although recent observations indicate that integrin receptors may be direct targets for redox modulation. The purpose of this study was to examine whether protein disulfide isomerase (PDI) is directly involved in this process. As manganese ions are known to affect the thioldisulfide balance and activate integrins to maximal affinity, we searched for PDI interactions with integrins, particularly with αVβ3, in Mn2+‐treated endothelial cells. By employing confocal microscopy, flow cytometry and coimmunoprecipitation experiments, we showed that exposure of endothelial cells to Mn2+ resulted in: (a) the appearance of surface protein thiol groups, which can be found in PDI and αVβ3, and both proteins colocalizing on the cellular surface; and (b) the formation of the PDI–αVβ3 complex, which dissociates upon reduction. In addition, PDI in a complex with αVβ3 induces conversion of the integrin to the ligand‐competent high‐affinity state, as evidenced by increased binding of vitronectin. The membrane‐impermeable sulfhydryl blockers 3‐N‐maleimidylpropionyl biocytin 3‐N‐maleimidylpropionyl biocytin and p‐chloromercuriphenyl sulfonate, as well as the PDI inhibitors bacitracin, MA3 018, and MA3 019, abolished the binding of vitronectin and LM609 to endothelial cells that is activated by Mn2+. Consistently, LM609 almost completely blocked binding of vitronectin to such cells. The formation of the PDI–αVβ3 stoichiometric complex was further demonstrated by surface plasmon resonance analysis, which showed that the initial reversible binding of PDI becomes irreversible in the presence of Mn2+, probably mediated by disulfide bonds. Thus, we show that Mn2+ simultaneously modulates the thiol isomerase activity of PDI that is bound to αVβ3 and induces its transition to the ligand‐competent state, suggesting an alternative mechanism of integrin regulation.

Ero1α Is Expressed on Blood Platelets in Association with Protein-disulfide Isomerase and Contributes to Redox-controlled Remodeling of αIIbβ3

“…By using either (a) inherent enzymatic PDI activity of ␣ IIb ␤ 3 , it has been shown that the ␤ 3 integrin subunit possesses active site motifs CGXC and expresses endogenous thiol isomerase activity, but it is not known if this activity is sufficient to promote the conformational change in either direction (14), or (b) isomerization of selected disulfide bonds in ␣ IIb ␤ 3 is catalyzed by PDI, although to date, the only physical association on the platelet surface that has been shown for PDI is with glycoprotein Ib␣ (27). However, it is noteworthy that we described such direct interaction of PDI with ␣ V ␤ 3 integrins in endothelial cells (22), which recently was confirmed by in vivo studies using ␤ 3 -null mice and multichannel fluorescence intravital microscopy (28). It was shown that accumulation of PDI observed near the site of vessel injury was almost abolished in ␤ 3 -null mice compared with wild type mice.…”

Peri/Epicellular Protein Disulfide Isomerase Sustains Vascular Lumen Caliber Through an Anticonstrictive Remodeling Effect

“…Importantly, integrins are redox sensitive, and distinct integrin subunits can be (re)activated by reducing agents. 27 Accordingly, as PDI has been reported to interact with integrin at the cell surface and also to exhibit reductase activity 12,26 (and present observations), a potential upstream mechanism regulated by pecPDI during vascular repair is the maintenance of sustained integrin signaling by PDI. This is indeed supported by our findings of lower levels of reduced β1-integrin at the cell surface after pecPDI neutralization ( Figure 6C).…”

“…18,21,26 Thiol reductants generally mediate integrin activation. 13,21,27 We addressed whether β1-integrin is a pecPDI target in our model.…”

Protein Disulfide Isomerase Directly Interacts with β-Actin Cys374 and Regulates Cytoskeleton Reorganization

“…The crucial role for ␥-actin is control of cell motility (23). Our previous work showed that PDI is able to interact with integrin (16). It is possible that PDI is recruited to the adhesion area by active form of integrin ␣IIb␤3, therefore interacting with ␤-actin in these compartments during cell adhesion and spreading.…”