The unique disulfide bond-stabilized W1 β4-β1 loop in the α4 β-propeller domain regulates integrin α4β7 affinity and signaling

Abstract

Integrin α4β7 mediates rolling and firm adhesion of lymphocytes pre- and post-activation, which is distinct from most integrins only mediating firm cell adhesion upon activation. This two-phase cell adhesion suggests a unique molecular basis for the dynamic interaction of α4β7 with its ligand, mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Here we report that a disulfide bond-stabilized W1 β4-β1 loop in α4 β-propeller domain plays critical roles in regulating integrin α4β7 affinity and signaling. Either breaking the disulfide bond or deleting the disulfide bond-occluded segment in the W1 β4-β1 loop inhibited rolling cell adhesion supported by the low-affinity interaction between MAdCAM-1 and inactive α4β7 but negligibly affected firm cell adhesion supported by the high-affinity interaction between MAdCAM-1 and Mn(2+)-activated α4β7. Additionally, disrupting the disulfide bond or deleting the disulfide bond-occluded segment not only blocked the conformational change and activation of α4β7 triggered by talin or phorbol-12-myristate-13-acetate via inside-out signaling but also disrupted integrin-mediated outside-in signaling and impaired phosphorylation of focal adhesion kinase and paxillin. Thus, these findings reveal a particular molecular basis for α4β7-mediated rolling cell adhesion and a novel regulatory element of integrin affinity and signaling.

Keywords: Affinity Regulation; Cell Adhesion; Cell Signaling; Cell Surface Receptor; Integrin; Integrin α4β7; Ligand Binding Protein; W1 β4-β1 Loop.

FIGURE 1.

The disulfide bond-stabilized α₄ β-propeller W1 β4-β1 loop in α₄β₇. A, crystal structure of the α₄β₇ headpiece (PDB code 3V4P). The β-propeller domain and thigh domain in the α₄ subunit are shown in cyan and magenta, respectively. The disulfide bond-occluded segment in the W1 β4-β1 loop is highlighted in red. The β₇ I domain and hybrid domain are shown in blue and brown, respectively. B, the three amino acid residues occluded by the disulfide bond in the W1 β4-β1 loop are shown in detail. Cys-81 and Cys-85 are shown in green. The disulfide bond formed between Cys-81 and Cys-85 is shown in yellow. Gly-82, Lys-83, and Thr-84 are shown in red. C, sequence alignment of human integrin α subunits near the W1 β4-β1 loop in the α₄ subunit. Residues of the disulfide bond-occluded segment in the β-propeller W1 β4-β1 loop in α₄ and α₉ subunits are highlighted in red.

FIGURE 2.

Effect of the W1 β4-β1 loop mutations on the adhesive modality of α₄β₇ in shear flow. A–D, rolling and firm adhesions of 293T transient transfectants on immobilized MAdCAM-1 substrates (10 μg/ml) in 1 mm Ca²⁺/Mg²⁺ (A and C) or in 0.5 mm Mn²⁺ (B and D). The number of rolling and firmly adherent WT and mutant α₄β₇ transfectants was measured in the indicated divalent cations at a wall shear stress of 2 dynes/cm². Cells preincubated with the α₄β₇ blocking antibody Act-1 (2 μg/ml) for 5 min at 37 °C or treated with 5 mm EDTA were used as controls. Data are mean ± S.E. (n = 3). p values were calculated by one-way ANOVA with Dunnett post-tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

Effect of the W1 β4-β1 loop mutations on the resistance to detachment and rolling velocity of α₄β₇ transfectants in shear flow. A–D, resistance of WT and mutant α₄β₇ 293T transient transfectants to detachment at increasing wall shear stress in 1 mm Ca²⁺/Mg²⁺ (A and C) or in 0.5 mm Mn²⁺ (B and D). The total number of cells remaining bound at each indicated wall shear stress was determined as a percent of adherent cells at 1 dyne/cm². E and F, average rolling velocity of WT and mutant α₄β₇ 293T transient transfectants that adhered to MAdCAM-1 substrates at indicated wall shear stress in 1 mm Ca²⁺/Mg²⁺. All experiments were performed on a surface coated with purified h-MAdCAM-1/Fc (10 μg/ml). Data are mean ± S.E. (n = 3).

FIGURE 4.

Effect of the W1 β4-β1 loop mutations on the activation of α₄β₇ by inside-out signaling. A, flow cytometry analysis of α₄β₇ and mCherry-talin head domain expression in 293T cells cotransfected with integrin α₄β₇ and the mCherry-talin head domain. The expression levels of integrin α₄β₇ were determined by staining cells with anti-β₇ mAb FIB504 followed by Alexa Fluor 488-conjugated mAb goat anti-rat IgG. One representative of three independent experiments is shown as a histogram. Numbers show the specific mean fluorescence intensity of integrin α₄β₇ (top panels) and mCherry-talin (bottom panels). Results are the mean ± S.E. of three independent experiments. Open histogram, control; filled histogram, integrin α₄β₇ (top panels) and mCherry-talin (bottom panels). B and C, the number of rolling and firmly adherent WT and mutant α₄β₇ 293T transient transfectants on immobilized MAdCAM-1 substrates (10 μg/ml) at a wall shear stress of 2 dynes/cm² in 1 mm Ca²⁺/Mg²⁺ before and after overexpression of mCherry-talin (B) or pre- and post-stimulation by 0.1 μm PMA (C). Data are mean ± S.E. (n = 3). p values were calculated by two-way ANOVA with Bonferroni post-tests. *, p < 0.05; **, p < 0.01.

FIGURE 5.

Influence of the W1 β4-β1 loop mutations on integrin conformation. A–C, FRET between the β₇ I domain and the plasma membrane. The FRET efficiency of WT and mutant α₄β₇ 293T transient transfectants under the indicated conditions: 1 mm Ca²⁺/Mg²⁺ and 0.5 mm Mn²⁺ (A), 1 mm Ca²⁺/Mg²⁺ and 1 mm Ca²⁺/Mg²⁺ with expression of mCherry-talin (B), and 1 mm Ca²⁺/Mg²⁺ and 1 mm Ca²⁺/Mg²⁺ with stimulation by 0.1 μm PMA (C). Data are mean ± S.E. (n = 20). p values were calculated by two-way ANOVA with Bonferroni post-tests. ***, p < 0.001.

FIGURE 6.

Influence of the W1 β4-β1 loop mutations on α₄β₇-mediated outside-in signaling and cell spreading. A and B, rolling and firm adhesions of CHO-K1 stable transfectants on immobilized MAdCAM-1 substrates (10 μg/ml) in 1 mm Ca²⁺/Mg²⁺ (A) or in 0.5 mm Mn²⁺ (B). The number of rolling and firmly adherent WT and mutant α₄β₇ transfectants was measured in the indicated divalent cations at a wall shear stress of 2 dynes/cm^2. Cells treated with 5 mm EDTA were used as a control. Data are mean ± S.E. (n = 3). p values were calculated by one-way ANOVA with Dunnett post-tests. ***, p < 0.001. C and D, differential interference contrast and interference reflection microscopy images of CHO-K1 stable transfectants that adhered to immobilized MAdCAM-1 in 1 mm Ca²⁺/Mg²⁺ (C) or 0.5 mm Mn²⁺ (D). The images are representatives from one of three independent experiments. Scale bars = 50 μm. E, quantification of cell spreading area (projection on substrates) of α₄β₇ CHO-K1 transfectants on the basis of differential interference contrast images. Data are mean ± S.E. (n = 50). p values were calculated by two-way ANOVA with Bonferroni post-tests. ***, p < 0.001. F, CHO-K1 cells stably expressing WT or mutant α₄β₇ were plated on poly-L-lysine in serum-free Ham's F12 medium or on MAdCAM-1 in Ham's F12 medium containing 1 mm Ca²⁺/Mg²⁺ or 0.5 mm Mn²⁺ for 2 h, lysed, and blotted for indicated molecules. Phosphorylated Y397-FAK and Y118-paxillin were blotted as indications of FAK and paxillin activation, respectively. A representative result of three independent experiments is shown.

FIGURE 7.

Effect of the W1 β4-β1 loop mutations on the adhesive modality of integrin α₄β₇, α₄β₁, and α₉β₁ on VCAM-1 substrates in shear flow. A, rolling and firm adhesions of WT and mutant α₄β₇ 293T transient transfectants on immobilized VCAM-1 substrates (10 μg/ml) in 1 mm Ca²⁺/Mg²⁺ or 0.5 mm Mn²⁺. B, rolling and firm adhesions of WT and mutant α₄β₁ 293T transient transfectants on immobilized VCAM-1 substrates (3.5 μg/ml) in 1 mm Ca²⁺/Mg²⁺ or 0.5 mm Mn²⁺. C, rolling and firm adhesions of WT and mutant α₉β₁ 293T transient transfectants on immobilized VCAM-1 substrates (20 μg/ml) in 1 mm Ca²⁺/Mg²⁺ or 0.5 mm Mn²⁺. The number of rolling and firmly adherent WT and mutant transfectants was measured in the indicated divalent cations at a wall shear stress of 2 dynes/cm². Cells treated with 5 mm EDTA were used as a control. Data are mean ± S.E. (n = 3). p values were calculated by one-way ANOVA with Dunnett post-tests. **, p < 0.01; ***, p < 0.001.