Integrin αIIbβ3 inside-out activation: an in situ conformational analysis reveals a new mechanism

Abstract

Integrins are a family of heterodimeric adhesion receptors that transmit signals bi-directionally across the plasma membranes. The transmembrane domain (TM) of integrin plays a critical role in mediating transition of the receptor from the default inactive to the active state on the cell surfaces. In this study, we successfully applied the substituted cysteine scanning accessibility method to determine the intracellular border of the integrin α(IIb)β(3) TM in the inactive and active states in living cells. We examined the aqueous accessibility of 75 substituted cysteines comprising the C terminus of both α(IIb) and β(3) TMs, the intracellular membrane-proximal regions, and the whole cytoplasmic tails, to the labeling of a membrane-permeable, cysteine-specific chemical biotin maleimide (BM). The active state of integrin α(IIb)β(3) heterodimer was generated by co-expression of activating partners with the cysteine-substituted constructs. Our data revealed that, in the inactive state, the intracellular lipid/aqueous border of α(IIb) TM was at Lys(994) and β(3) TM was at Phe(727) respectively; in the active state, the border of α(IIb) TM shifted to Pro(998), whereas the border of β(3) TM remained unchanged, suggesting that complex conformational changes occurred in the TMs upon α(IIb)β(3) inside-out activation. On the basis of the results, we propose a new inside-out activation mechanism for integrin α(IIb)β(3) and by inference, all of the integrins in their native cellular environment.

FIGURE 1.

A, amino acid sequences of the TMs and the cytoplasmic tails of integrin α_IIbβ₃. The proposed TMs are depicted in a box with broken lines, MP regions are colored in gray, and cysteine-substituted residues are highlighted in bold. The potential ionic interaction between α_IIb Arg⁹⁹⁵ and β₃ Asp⁷²³ is indicated as circled positive and negative symbols. B, BM labeling of wild-type and cysteine-substituted integrin α_IIbβ₃. HEK 293 cells transfected with cloning vector pcDNA3, wild-type α_IIbβ₃, and mutant α_IIb E1008C/β₃ T762C were collected and labeled with BM at room temperature for 20 min. Cells were lysed, and integrin α_IIbβ₃ heterodimer was immunoprecipitated, resolved on 10% SDS-PAGE, and transferred to a PVDF membrane. Incorporated biotin was detected by HRP-streptavidin and ECL. The blot was stripped and probed with an anti-α_IIb light chain and an anti-β₃ antibody simultaneously to detect the amount of protein in each sample.

FIGURE 2.

BM labeling of cysteine-substituted α_IIb co-expressed with wild-type β₃. A, representative results of BM labeling on integrin α_IIb-substituted cysteines. 24 amino acids in the C terminus of α_IIb were individually substituted with cysteines and labeled with BM as described under “Experimental Procedures.” B, summary of BM labeling of integrin α_IIb-substituted cysteines. The level of biotin incorporation into each sample was quantified by densitometry, and the signal was normalized to the amount of integrin α_IIb light chain present in the sample. In each experiment, the level of biotinylation was compared with that of the α_IIb E1008C, whose labeling was set to 100%. Data represent mean of 3–5 experiments ± S.E. (error bars).

FIGURE 3.

BM labeling on integrin β₃-substituted cysteines. Amino acids between Leu⁷¹² and Phe⁷³⁰ in the β₃ subunit were individually substituted with cysteines and labeled with BM. A, BM labeling of cysteine-substituted β₃ co-expressed with wild-type α_IIb. B, BM labeling of cysteine-substituted β₃ co-expressed with mutant α_IIb F992A/F993A. Similar results were obtained with co-expression of α_IIb Gly⁹⁹¹ truncation. C, summary of BM labeling of cysteine-substituted β₃. Biotin incorporation into each sample was quantified as described in Fig. 2 and was compared with β₃-T762C, whose labeling was set to 100%. Data represent mean of 3–6 experiments ± S.E. (error bars).

FIGURE 4.

Effect of cysteine substitutions in α_IIb or β₃ subunit on integrin α_IIbβ₃ activation. Upper, activation index of cysteine-substituted α_IIb co-expressed with wild-type β₃. Lower, activation index of cysteine-substituted β₃ co-expressed with wild-type α_IIb. Cysteine-substituted α_IIb or β₃ were co-expressed with the respective wild-type partners in the CHO cells. 24 h after transfection, cells were stained with PAC1 antibody to measure activation and with D57 antibody to measure surface expression. Data represent mean of 1–3 experiments ± S.E. (error bars). Details are described under “Experimental Procedures.”

FIGURE 5.

BM labeling of cysteine-substituted α_IIb co-expressed with β₃ K716P. A, representative results of BM labeling on integrin α_IIb-substituted cysteines. The experiments were performed as described in the Fig. 3. B, summary of BM labeling of integrin α_IIb-substituted cysteines. Results were analyzed same as described in Fig. 3. Data represent mean of 3–5 experiments ± S.E. (error bars). Similar labeling results were obtained on cysteine-substituted α_IIb co-expressed with β₃ Lys⁷¹⁶ truncation or β₃ G708I mutation.

FIGURE 6.

BM labeling of cysteine-substituted α_IIb co-expressed with β₃-K716P after Na₂CO₃ stripping. A, comparison of protein samples from membranes without and with Na₂CO₃ stripping. Half-fraction of the isolated cell membranes was treated with 0.1 m Na₂CO₃ for 30 min on ice. Membranes were then pelleted by centrifugation and washed once with PBS. Membrane pellets were lysed in IPB buffer, and protein levels were determined by the BCA method. Equal amounts of protein samples were loaded on the 4–20% SDS-polyacrylamide gel. B, representative BM labeling of cysteine-substituted α_IIb Phe⁹⁹³-Pro⁹⁹⁸ co-expressed with β₃ K716P after 0.1 m Na₂CO₃ treatment. Similar labeling results were obtained after 3 m Na₂CO₃ treatment. C, summary of BM labeling. Results were analyzed the same as described in Fig. 3. Data represent mean of 3–6 experiments ± S.E. (error bars).

FIGURE 7.

BM labeling of cysteine-substituted integrin β₃ intracellular tail. Amino acids between Glu⁷³¹ and Thr⁷⁶² in the β₃ subunit were individually substituted with cysteines and labeled with BM. A, representative results of BM labeling on substituted cysteines in integrin β₃ intracellular tail co-expressed with wild-type α_IIb. B, summary of BM labeling of cysteine-substituted β₃ intracellular tail. Results were analyzed the same as described in Fig. 3. Data represent mean of 3 experiments ± S.E. (error bars).

FIGURE 8.

Proposed mechanism of integrin inside-out activation. A, schemes showing that in the inactive state, the α_IIb TM (aqua) associates with β₃ TM (blue) in the lipid bilayer with a clasp formed by GFFKRNR (red line) and Lys⁷¹⁶ (red dot) at the intracellular lipid/aqueous interface. Upon inside-out activation, extension of the α_IIb extracellular domain applies stretching forces on the α_IIb TM, causing its upward shift that results repartition of four amino acids in the MP region (red line) into the lipid bilayer and drawing the negatively charged tail (dashed black lines) close to the inner leaflet of the plasma membrane that may form ionic interactions with the positive head groups of membrane phospholipids. B, detailed positions of the residues in the α_IIb MP region involving lipid bilayer repartitioning upon inside-out activation. The structure of the α_IIb MP region was adapted from Refs. and .