Integrin α_IIbβ₃ Inside-out Activation

Background: The transmembrane domain of integrins plays a critical role in mediating receptor inside-out activation.

Results: Inside-out activation triggers the repartitioning of the intracellular border of α_IIb but not the β₃ transmembrane domain into the lipid bilayer in living cells.

Conclusion: Complex conformational changes occur in the transmembrane domain upon integrin α_IIbβ₃ inside-out activation.

Significance: Our findings represent a new mechanism for integrin inside-out activation.

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_IIbβ₃ 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 β₃ 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_IIbβ₃ 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 Lys⁹⁹⁴ and β₃ TM was at Phe⁷²⁷ respectively; in the active state, the border of α_IIb TM shifted to Pro⁹⁹⁸, whereas the border of β₃ TM remained unchanged, suggesting that complex conformational changes occurred in the TMs upon α_IIbβ₃ inside-out activation. On the basis of the results, we propose a new inside-out activation mechanism for integrin α_IIbβ₃ and by inference, all of the integrins in their native cellular environment.

Introduction

Integrins are a large family of adhesion receptors on the cell surfaces that mediate cell adhesion, migration, and extracellular matrix assembly (1, 2). 18 integrin α subunits and 8 β subunits form 24 heterodimers, of which each subunit consists of a large extracellular domain, a single transmembrane domain (TM),³ and a short unstructured cytoplasmic tail. Integrins exist in a default low affinity state on the cell surfaces. Upon stimulation by intracellular signals, integrins convert to the active state that permits extracellular ligands binding (inside-out activation), which in turn promotes interactions of intracellular proteins with the cytoplasmic tails (outside-in signaling) (3, 4). In this way, integrins transmit signals bi-directionally across plasma membranes.

Biochemical analyses (5, 6), electron microscopy (7, 8), and fluorescent energy transfer studies (9) have established that a constraint is present in the intracellular membrane-proximal (MP) region that restrains integrin α_IIbβ₃ in the inactive state. Intracellular signals interact with the β₃ cytoplasmic tail that breaks the constraint to initiate a conformational change that traverses the TMs, which ultimately triggers activation of the extracellular domain. Two intracellular proteins, talin and kindlin, are reported to interact with the β₃ cytoplasmic tail and work synergistically to activate α_IIbβ₃ (10–12). The extracellular domain of integrin α_IIbβ₃ was crystallized (13), and a low resolution electron cryomicroscopy structure (8) of the full heterodimer was also reported; however, attempts to obtain a crystal structure of the full-length receptor were not successful.

Two stretches of highly conserved amino acids in the intracellular MP region of integrin α_IIbβ₃ play pivotal roles in controlling the receptor inside-out activation (⁹⁹¹GFFKR⁹⁹⁵ in α_IIb; ⁷¹⁷LLITIHD⁷²³ in β₃) (5, 6, 14) (Fig. 1A). A salt bridge between α_IIb Arg⁹⁹⁵ and β₃ Asp⁷²³ was proposed to form a clasp constraining the receptor in the inactive state (15). Intense efforts including site mutagenesis scanning (15, 16), transmission electron microscopy (7), nuclear magnetic resonance (NMR) (17–20), chimeric swapping (21), and computational modeling (16, 22) have been invested to determine how the intracellular clasp is formed, which gave conflicting results. Recent studies using NMR on the structure of associated α_IIbβ₃ TM peptides in the lipid bicelles (20), α_IIbβ₃ TM and cytoplasmic tail complex in CD₃CN/H₂O (1:1) mixture (17), and by using disulfide constraints coupled with structural modeling (16) have yielded three different views on the intracellular clasp of α_IIbβ₃: the first proposed that the clasp is formed by the interaction between residues α_IIb Phe⁹⁹²/Phe⁹⁹³ and β₃ Tyr⁷¹⁵, the second proposed that the clasp is formed by an inhibitory ligand binding in the MP region, and the third proposed that the clasp is formed between β₃ Lys⁷¹⁶ side chain and the peptide backbone of α_IIb ⁹⁹¹GFFKR⁹⁹⁵ motif.

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.

All of the aforementioned studies have greatly improved our knowledge on the conformation of the TMs and the intracellular clasp in the MP region of integrin α_IIbβ₃; however, questions remain. It is known that the regulated association of α_IIbβ₃ TMs is driven by domains outside of the plasma membranes, whether the NMR structures of fragmented α_IIbβ₃ TM peptides correspond to any physiological states of the receptor is obscure. Questions such as how the TMs of integrin α_IIbβ₃ undergo spatial and conformational changes upon activation and how intracellular proteins interact with the cytoplasmic tails of α_IIbβ₃ to initiate inside-out activation can only be genuinely answered by the analyses of intact integrin protein in the cellular environment.

Here, we present a successful analysis of the cellular location of integrin α_IIbβ₃ TM intracellular borders in the receptor inactive and active states in the living cells by using the substituted cysteine scanning accessibility method (SCSAM). Our data revealed a new mechanism for integrin α_IIbβ₃ inside-out activation in cell membranes.

EXPERIMENTAL PROCEDURES

Materials

Site-directed mutagenesis kits were from Strategene. Biotin maleimide, DMEM, and all cell culture reagents were from Invitrogen. Anti-integrin α_IIb or β₃ rabbit or mouse antibodies were from Santa Cruz Biotechnology. Protein A- and G-Sepharose, streptavidin/biotinylated-horseradish peroxidase complex (streptavidin-HRP), and goat anti-rabbit IgG-conjugated horseradish peroxidase were from GE Healthcare. Igepal was from Sigma. PVDF membrane was from Millipore.

Site-directed Mutagenesis

A wild-type human α_IIb and a β₃ cDNA were used as the templates for site-directed mutagenesis. Amino acids at the position of Leu⁹⁸⁵ to Glu¹⁰⁰⁸ in α_IIb and Leu⁷¹² to Thr⁷⁶² in β₃ were individually replaced with cysteines. Mutagenesis was performed using the Strategene site-directed mutagenesis kit following the manufacturer's instructions. The complete cDNA sequence of each mutant was verified by DNA sequencing.

Protein Expression

Cysteine-substituted α_IIbβ₃ was transiently expressed in the human embryonic kidney 293 cells (HEK 293) by using Lipofectamine 2000 (from Invitrogen) transfection following the manufacturer's instructions. Chinese hamster ovary (CHO) cells were also used in some experiments. Cells were grown at 37 °C in a 5% CO₂ atmosphere and harvested 48 h after transfection.

Flow Cytometry

Flow cytometry assays for assessing the effects of cysteine substitutions on α_IIbβ₃ activation were performed as described previously (23). In brief, cysteine-substituted α_IIb or β₃ constructs were co-expressed with the wild-type partners in the CHO cells. 24 h after transfection, cells were stained with antibody D57, which measures receptor surface expression, and PAC1, which detects the active state of α_IIbβ₃, in the presence and absence of Ro43-5054 or anti-LIBS6, and then subjected to FACS scan. Ro43-5054 is a competitive antagonist of α_IIbβ₃, which was used to estimate nonspecific PAC1 binding (F₀), and anti-LIBS6 is an α_IIbβ₃-activating antibody that was used to estimate maximal PAC1 binding (F_max). The activation index was calculated as 100 × (F − F₀)/(F_max − F₀), where F = mean fluorescence intensity of PAC1 staining under the test condition. For detailed methods, see Ref. 23.

Biotin Maleimide Labeling and Immunoprecipitation

Whole cell labeling with BM was performed as described previously (24). Briefly, transfected HEK 293 cells were collected and resuspended in PBSCM (PBS containing 0.1 mm CaCl₂ and 1 mm MgCl₂, pH 7.0) solution and subsequently labeled with BM (0.2 mm final) at room temperature for 20 min. Reactions were stopped by adding 5-fold glutathione in molar ratio. Cells were then washed with PBSCM and lysed in IPB buffer (150 mm NaCl, 1% (v/v) Igepal, 0.5% (w/v) sodium deoxycholate, 10 mm Tris-HCl, pH 7.5) containing 0.2% (w/v) BSA and protease inhibitors (from Roche Applied Science) on ice for 10 min. α_IIbβ₃ proteins were immunoprecipitated by a mouse anti-human α_IIbβ₃ monoclonal antibody (sc-21783 from Santa Cruz Biotechnology) and protein G beads for 4 h at 4 °C.

SDS-PAGE and Immunoblotting

Protein samples were resolved on 7.5% (for β₃ subunit) and 10% (for α_IIb light chain) SDS-polyacrylamide gels, respectively, and transferred to PVDF membranes. Biotinylated proteins were detected by incubation of blots with 1:10,000 diluted streptavidin-biotinylated horseradish peroxidase (GE Healthcare) in TBSTB buffer (TBST buffer (0.1% (v/v) Tween 20, 137 mm NaCl, 20 mm Tris, pH 7.5), containing 0.5% (w/v) BSA). Protein expression levels were determined by probing the blot with rabbit anti-α_IIb or β₃ polyclonal antibodies at 1:3,000 dilutions in TBSTM buffer (TBST buffer containing 5% (w/v) nonfat milk).

Membrane Isolation and Na₂CO₃ Treatment

Membrane treatment with Na₂CO₃ was performed as described previously (25). Briefly, transfected cells (10-cm plate) were collected, washed (TBS buffer: 140 mm NaCl, 10 mm Tris, pH 7.4), and incubated with the homogenization buffer (10 mm Tris, pH 7.4, with Roche protease inhibitors) for 30 min on ice. Cells were then lysed by Dounce homogenization. Cell debris were removed by low speed centrifugation (4,000 × g, 5 min, 4 °C), and the membrane fractions were collected by high speed centrifugation (35,000 × g, 30 min, 4 °C). Membrane pellets were first resuspended in 100 μl of 0.3 m sucrose and then mixed with 2 ml of ice-cold 0.1 m or 3 m Na₂CO₃, pH 11.5, and incubated for 30 min on a rotating shaker at 4 °C. Membranes were then collected by centrifugation, washed with PBSCM, and resuspended in 1.0 ml of PBSCM followed by BM labeling. In some experiments, the protein levels of the stripped and unstripped membranes were determined by the BCA method (from Thermo Scientific). Equal amounts of protein samples were resolved on SDS-PAGE and stained with Coomassie Blue.

Image and Data Analysis

Films from immunoblots and biotinylation blots were scanned with a Hewlett-Packard Scanjet 5590. Scanned images were quantified with UN-SCAN-IT gel^TM version 6.1 software. Biotinylation levels were calculated according to Ref. 24.

Statistical Analysis

Means ± S.E. were calculated with SigmaPlot 10 software. Statistical analysis was performed using SigmaPlot 10 software.

RESULTS

Substituted Cysteine Scanning Accessibility Method for Integrin α_IIbβ₃ Conformational Analysis

SCSAM uses sulfhydryl reactive chemical probes to determine the location of the introduced cysteines in a target membrane protein that is free of endogenous reactive cysteines (26, 27). BM, a membrane-permeable sulfhydryl-specific reagent, has been used successfully for the topological analysis of transmembrane proteins (24, 28). SCSAM is based on the observation that the chemical reaction only occurs in the aqueous environment. Cysteines residing in the aqueous medium can react with BM, whereas cysteines residing in the lipid bilayer, the protein interior, or forming disulfide bonds cannot. BM minimally labels the endoplasmic reticulum-retained membrane proteins (24), which is probably due to the high content of glutathione in the cytosol. Integrin α_IIbβ₃ is a cysteine-rich membrane receptor that contains 20 endogenous cysteines in α_IIb and 56 in the β₃ subunit. The recent crystal structure of the extracellular domain of α_IIbβ₃ showed that all of the cysteines in α_IIbβ₃ form disulfide bonds (13); however, conflicting reports also suggested that the receptor may have endogenous thiol isomerase activity that exposes free cysteines (29). To determine whether free reactive cysteines are present in the wild-type α_IIbβ₃, we tested wild-type integrin α_IIbβ₃ for labeling with BM. Wild-type α_IIbβ₃ and mutant constructs α_IIb-E1008C/β₃-T762C were expressed in the HEK 293 cells and subjected to the whole cell labeling. Fig. 1B shows that the wild-type α_IIb or β₃ has no detectable BM labeling, whereas the light chain of α_IIb with a single cysteine substitution (E1008C) at the C terminus or β₃ with a cysteine substitution at Thr⁷⁶² (residue at the C terminus of β₃) was strongly labeled, suggesting that no free endogenous cysteines are present in wild-type α_IIb or β₃ that are available for BM labeling. Western blots verified that α_IIb (sc-6602, from Santa Cruz Biotechnology) and β₃ (sc-6627, from Santa Cruz Biotechnology) are both well expressed. Based on the results, we individually substituted single amino acids (between Leu⁹⁸⁵ and Glu¹⁰⁰⁸) with cysteines in the C-terminal region of α_IIb that covers the proposed C terminus of the TM, the intracellular MP region, and the whole intracellular tail, and the positions between Leu⁷¹² and Phe⁷³⁰ in β₃ covering the proposed MP region of β₃ TM (Fig. 1A).

Whole Cell Labeling of Cysteine-substituted Integrin α_IIbβ₃ with BM

Integrin α_IIb carrying substituted cysteines was co-expressed with the wild-type β₃ in the HEK 293 cells using Lipofectamine 2000 transfection. 48 h after transfection, cells were collected and subjected to BM labeling. Fig. 2 shows that the positive control, α_IIb E1008C was strongly labeled, whereas the negative control, wild-type α_IIb, had no labeling. Amino acids between Leu⁹⁸⁵ and Phe⁹⁹³ were not labeled, suggesting that they are embedded in the lipid bilayer; amino acids between Asn⁹⁹⁶ and Glu¹⁰⁰⁸ were increasingly labeled, indicating the C-terminal tail is free of any cytosolic protein interactions. Interestingly, K994C was weakly labeled, whereas the adjacent residue, R995C was unlabeled compared with the controls. These data suggest that in the intact α_IIbβ₃ complex in living cells, the intracellular border of α_IIb TM is at Lys⁹⁹⁴, which leaves the proposed salt bridge α_IIb Arg⁹⁹⁵/β₃ Asp⁷²³ outside of the plasma membrane at the lipid/aqueous interface in the cytosol.

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).

We then performed whole cell labeling on the cysteine-substituted integrin β₃ constructs co-expressed with wild-type α_IIb in HEK 293 cells. Fig. 3, A and C, shows that none of the substituted cysteines in the region between Leu⁷¹² and Glu⁷²⁶ was labeled with BM, and the first BM labeled residue was at Phe⁷²⁷ marking the intracellular border of β₃ TM. The intensity of BM labeling on the cysteine substitutions from F727C to F730C was similar suggesting the peptide after Phe⁷²⁷ enters the aqueous medium abruptly.

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).

Functional Analysis of Cysteine-substituted Integrin α_IIbβ₃

Functional effects of cysteine substitutions in the TM and the MP regions of integrin α_IIbβ₃ have recently been reported (16), in that cysteine substitution of any of the residues in the ⁹⁹¹GFFKRN⁹⁹⁶ region of α_IIb activated the receptor to various degrees, whereas substitution in the remaining portion of the α_IIb intracellular tail had no functional effect. Surprisingly, in the MP region of β₃, only one cysteine substitution at amino acid Lys⁷¹⁶ substantially activated integrin α_IIbβ₃. Because most of the functional studies of integrin α_IIbβ₃ were performed in the CHO cells, we expressed the cysteine-substituted constructs in the CHO cells and subjected them to FACS analysis with PAC1 antibody that recognizes the active state of integrin α_IIbβ₃. Fig. 4 shows that, indeed, our findings were in agreement with the previous report.

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.”

Intracellular Border of Integrin α_IIb TM in Fully Active State

Functional results indicated that the intracellular border of integrin α_IIb TM represented mixed states of active/inactive receptors. Substitution of β₃ Lys⁷¹⁶ with Cys or Pro was shown to activate integrin α_IIbβ₃ close to the full activation level (16) in the HEK 293 cells. To obtain the intracellular border of α_IIb TM in the active state, we co-expressed α_IIb cysteine-substituted constructs with β₃ K716P and subjected them to whole cell labeling with BM. Fig. 5 shows that activation of integrin α_IIbβ₃ had no effect on the labeling in the region of Pro⁹⁹⁸ to Glu¹⁰⁰⁸; however, the region of Lys⁹⁹⁴-Arg⁹⁹⁷ was no longer labeled with BM, and the first labeled residues started with Pro⁹⁹⁸, which differs significantly from the previous results obtained in the mixed active/inactive states of the receptor. We further tested BM labeling on the α_IIb cysteine-substituted constructs co-expressed with a truncated form of β₃ at Lys⁷¹⁶, which also produces a highly active state of α_IIbβ₃ (14). The same results were observed (data not shown). To test whether breaking of the predicted outer membrane clasp (highly activates integrin α_IIbβ₃) affects conformation of the region, we whole cell labeled the α_IIb cysteine-substituted constructs co-expressed with β₃ G708I (20). Again, we observed a similar pattern of labeling (data not shown), indicating that this conformational change in α_IIb is irrespective to the breaking of either the inner or the outer membrane clasp.

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.

It was previously reported that the GFFKR region of α_IIb may associate with intracellular peripheral proteins such as calcium- and integrin-binding protein (30) which potentially shield the substituted cysteines from being labeled. To test this, we isolated the plasma membranes from cells expressing the α_IIb cysteine-substituted constructs (Phe⁹⁹³-Phe⁹⁹⁸) with β₃ K716P and subjected to chemical stripping with 0.1 m Na₂CO₃ prior to BM labeling. Compared with the unstripped plasma membranes, Na₂CO₃ treatment effectively removed multiple protein bands from the membrane samples as shown on the Coomassie Blue-stained SDS-PAGE (Fig. 6A). Fig. 6, B and C, shows no differences in BM labeling were detected compared with the fully active state of α_IIbβ₃. We then tested BM labeling on the membranes stripped with 3 m Na₂CO₃, a stringent condition inducing both counter-ion and ionic strength that could effectively expose a deeply buried residue in the sodium bicarbonate co-transporter 1 (25). Again, we did not observe any labeling on the substituted cysteines in Phe⁹⁹³-Arg⁹⁹⁷ (data not shown). Interestingly, the Na₂CO₃-treated α_IIbβ₃ protein was no longer immunoprecipitated by the antibody recognizing the protein complex, but instead, by an antibody that recognizes α_IIb, suggesting that a conformational change in the extracellular domain of the receptor must have been induced by the Na₂CO₃ treatment. Our data indicated that activation of α_IIbβ₃ triggers repartition of the α_IIb MP region from aqueous into the lipid bilayer.

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).

Intracellular Border of Integrin β₃-TM in Fully Active State

The flow cytometry assay showed that none of the cysteine substitutions except K716C in the β₃ TM/MP region activated α_IIbβ₃. Therefore, the first labeled cysteine-substituted residue, F727C, marks the intracellular border of β₃ TM in α_IIbβ₃ inactive state in living cells. To determine the intracellular border of β₃ TM in activated α_IIbβ₃, we co-expressed β₃ cysteine-substituted constructs with α_IIb F992A/F993A, a construct that fully activates the receptor (16). Fig. 3, B and C, shows that activation of α_IIbβ₃ did not expose any β₃ endogenous cysteines to aqueous. Interestingly, F727C again remained the first residue labeled with BM, indicating its aqueous accessibility. We were somewhat surprised that no differences were observed in the β₃ TM BM labeling between the active and the inactive state of α_IIbβ₃, and therefore we performed additional BM labeling assays on the co-expressed β₃ cysteine-substituted constructs with a truncated form of α_IIb at Gly⁹⁹¹, which generates the constitutively active state of α_IIbβ₃ (6). Indeed, the same results were obtained (data not shown). These results suggested that β₃ TM/MP regions do not repartition between lipid bilayer and aqueous medium upon the receptor activation as seen in α_IIb subunit.

Aqueous Accessibility of the Predicted Membrane-anchored Integrin β₃ Intracellular Tail

A recent NMR analysis on the complex of fragmented α_IIb and β₃ intracellular tails has suggested that regions of Phe⁷²⁷-Trp⁷³⁹ and Tyr⁷⁴⁷-Tyr⁷⁵⁹ in the β₃ tail form membrane-anchored α-helices in loose contact with the lipid bilayer (31). To determine whether these predicted membrane-anchored helices are accessible to BM labeling, we individually substituted amino acids from Glu⁷³¹ to Thr⁷⁶² (in wild-type β₃) with cysteines that cover the whole intracellular tail of β₃. The cysteine-substituted constructs were co-expressed with wild-type α_IIb in HEK 293 cells and subjected to whole cell BM labeling. Fig. 7 shows that all of the substituted cysteines are increasingly strongly labeled. Therefore, these predicted membrane-anchored helices are accessible to aqueous that differs from Leu⁷¹²-Glu⁷²⁶ region that is embedded in the lipid bilayer.

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).

DISCUSSION

Intracellular Borders of Integrin α_IIb and β₃ TMs in Living Cells

Here, we present for the first time the application of SCSAM in analyzing the conformational changes of integrin α_IIbβ₃, a membrane adhesion receptor in living cells. The intracellular MP region of integrin α_IIbβ₃ has been the target of intensive study because of its critical role in controlling the receptor inside-out activation. In this study, we have unambiguously resolved the intracellular borders of integrin α_IIb and β₃ TM domain in the receptor active and inactive states on the surface of living cells. Our data revealed that, unlike the results from in vitro analysis, the intracellular borders of the active state integrin α_IIb and β₃ TMs reside at amino acids Pro⁹⁹⁸ and Phe⁷²⁷, respectively. This located the proposed MP regions of both subunits in the lipid bilayer in the active state. The conclusion is supported by two sets of BM labeling experiments by using highly active mutation (K716P and G708I) or truncation (at Lys⁷¹⁶) in the β₃ subunit, and highly active mutation (F992A/F993A) or truncation (at Gly⁹⁹¹) in the α_IIb subunit that generates constitutive active states of integrin α_IIbβ₃.

A puzzling pattern of BM labeling was observed on the determination of α_IIb TM border in the receptor inactive state. When co-expressed with wild-type β₃ subunit, weak labeling was detected on α_IIb K994C; strong labeling on N996C, R997C, and P998C; however, no labeling on F992C, F993C, and R995C. This result is readily explained by the functional data from the current and the previous studies (16), which showed cysteine substitution of Arg⁹⁹⁵ activated the receptor close to the maximum level in the HEK 293 cells, whereas Phe⁹⁹², Phe⁹⁹³, Lys⁹⁹⁴, Asn⁹⁹⁶, Arg⁹⁹⁷, and Pro⁹⁹⁸ were ∼0–40% activation, indicating that >60% of the population of the mutant receptors on the cell surface were not active. Additionally, the recent Rosetta model on the α_IIb MP region showed that the side chain of Lys⁹⁹⁴ points away from the dimer interface in the receptor inactive state, suggesting its accessibility to the aqueous medium (16). Therefore, labeled K994C, N996C, R997C, and P998C represent the major population of inactive receptors. Consistent with this, no BM labeling was detected on α_IIb R995C co-expressed either with wild-type or activating β₃ subunit because both conditions activated the receptor at a high level. Based on these observations, we concluded that the labeled K994C defines the intracellular border of α_IIb TM in the inactive state. One possibility is that in the active state, intracellular proteins may bind to the unclasped α_IIb tail that shields the substituted cysteines to BM labeling. We ruled out this possibility by stripping the isolated membranes with 0.1 m or 3 m Na₂CO₃ that removes potential bound peripheral proteins prior to BM labeling and showed no detectable differences compared with the samples without treatment.

In contrast to α_IIb, determination of the β₃TM intracellular border was clear and compelling because none of the cysteine substitutions in the proposed MP region except Lys⁷¹⁶ activated the receptor. Our data showed that none of the cysteine substitutions prior to Phe⁷²⁷, including the highly charged ⁷²²HDRKE⁷²⁶ stretch, was accessible to BM labeling, suggesting their lipid embedment. Residues starting at Phe⁷²⁷ became strongly labeled, indicating their aqueous exposure. Therefore, we assigned the intracellular border of β₃ TM in the inactive state to Phe⁷²⁷. Indeed, the strong labeling of the cysteine-substituted β₃ tail membrane-anchored α-helices further supports that Phe⁷²⁷ marks the intracellular lipid/aqueous interface of β₃ TM in the living cells.

Comparison with the in Vitro Determined TM Borders

Our findings differ significantly from the results obtained in the in vitro studies, including glycosylation mapping in the cell-free system (32, 33) and the recent NMR structures of α_IIb (34) and β₃ TM (35) peptides in the lipid bicelles, which both placed the intracellular borders of α_IIb TM at Lys⁹⁹⁴ and β₃ TM at Asp⁷²³. Because integrin TMs are fully separated in the active state (36), these in vitro findings may represent the TM borders of the integrin active state. Because the isolated TM fragments do not contain the intracellular clasp in the MP region and more importantly do not carry the extracellular domains, the physiologic significance is questionable. It is known that the extracellular domain of integrin α_IIb and β₃ forms a dimer independent of the TM domains (13), and further, dimerization of the extracellular domains proceed the dimerization of the TM domains during biosynthesis. It is conceivable that the α_IIbβ₃ TM domain dimerization and lipid embedding are influenced or guided by the dimerized extracellular domain. In support of this, mutations that separated the dimerized α_IIbβ₃ TM fragment in the lipid bicelles failed to activate the intact receptor in the cells (20), confirming that the extracellular domain is involved in stabilizing the α_IIbβ₃ TM dimer and that the previous in vitro observations may not be physiological.

Our study has considerable advantages over the in vitro studies because the analyses were performed on intact integrin α_IIbβ₃ protein in living cells. Our data showed, in contrast to the in vitro findings, that Lys⁹⁹⁴ marks the intracellular border of inactive state α_IIb TM. When the receptor is active, the border shifts to Pro⁹⁹⁸, indicating a significant peptide repartitioning from aqueous medium into the lipid bilayer. The extracellular domain of integrin was shown to switch from a bent to an extended conformation in the active state (36). We suggest that the peptide repartitioning is caused by the pulling of the extended extracellular domain rather by a spontaneous repartition of the peptide itself. Indeed, upon close examination of the lipid-embedded extracellular end of α_IIb TM, two amphiphilic residues (Trp⁹⁹⁷, Trp⁹⁹⁸) immediately follow the first lipid embedded residue Ile⁹⁹⁶, making it possible for them to repartition into the aqueous medium as a result of the stretching forces induced by the extracellular domain extension. In support of this, when the extracellular domain was missing, the ⁹⁹⁴KRNRP⁹⁹⁸ stretch of α_IIb TM was not embedded in the lipid bilayer, indicating that spontaneous lipid repartitioning of the peptide is not favorable (32–34). This phenomenon can only be observed in intact integrin in a cellular environment.

Compared with the reported in vitro determined β₃ TM intracellular border, our data are more complex: exposure to the aqueous starts at amino acid Phe⁷²⁷, leaving the stretch of charged residues ⁷²²HDRKE⁷²⁶ in the membrane. In the in vitro system, to place such a highly charged peptide in the lipid environment is energetically unfavorable. The β₃ TM NMR structure showed that the TM helix terminates at Asp⁷²³ (20, 35), whereas cysteine cross-linking analyses in the intact protein suggested that the helical structure extended to Phe⁷³⁰ (16). Following the latter observation, our findings suggest that there is an additional helix turn at the C terminus of β₃ TM in the lipid bilayer, ∼5.4 Å distant from the aqueous medium. Residue Asp⁷²³ has been shown to face the dimer interface (16, 20); therefore Arg⁷²⁴, Lys⁷²⁵, and Glu⁷²⁶ would be 100 degrees apart, which potentially interacts with the negatively charged phosphate groups and the positively charged choline head of membrane phospholipids, which forms ionic interactions to stabilize the end of TM in the lipid bilayer. In agreement with this, we observed that activation of the receptor had no effect on membrane embedding of this region, suggesting that it is unlikely that the C terminus of β₃ TM could move out of the lipid bilayer upon activation. Lipid embedding of heavily charged peptides has been shown in the crystallized voltage sensor of the voltage-gated potassium channel (37), and potentially, these charged residues are hydrated (38).

A recent analysis of the integrin β₃ TM intracellular border using a β₃ TM fragment suggested that β₃ Lys⁷¹⁶ helps determine the integrin β₃ TM topography (39). Removal of the positive charge at this position (active state) induced aqueous exposure of the residues in the proposed β₃ MP region. Because our experiments analyzed the intact receptor in a cellular environment, the difference between our findings and this observation is potentially due to differences in the experimental approaches used.

Implications for Integrin Inside-out Activation

The TMs of integrin have been proposed to undergo “separation” (9), “piston” (40), or “scissors” (40, 41) movement upon inside-out activation. We found that, upon activation of integrin α_IIbβ₃ in the living cells, the intracellular border of α_IIb TM undergoes an upward shift that repartitions into the lipid bilayer, whereas the position of β₃ TM border remains unchanged. It has been shown that the α_IIb TM is perpendicular in the lipid bilayer whereas β₃ TM tilts at a significant angle when dimerized (16, 20). Our finding that the membrane embedded region of β₃ TM extends to Glu⁷²⁶ suggests that the TM tilts at more extended angles. Taking our in vivo findings together with the reported in vitro TM structures (16, 20, 23), we propose a new mechanism for integrin inside-out activation (Fig. 8). In the resting state, the MP region of α_IIb ⁹⁹¹GFFKR⁹⁹⁵ interacts with β₃ Lys⁷¹⁶ forming a clasp that keeps the receptor inactive. Upon inside-out activation, the breakage of the intracellular clasp leads to extensive conformational changes in the extracellular domain from a bent to an extended conformation. Because the two TMs are dissociated, the extended α_IIb extracellular domain pulls its TM upward causing four consecutive residues in the MP region repartition into the lipid bilayer. The NMR structure shows the region after α_IIb Val⁹⁹⁰ has a left reverse turn rather than a helical structure; this would minimize the energy required for the peptide repartition into the lipid bilayer. Additionally, the up shift of α_IIb TM would draw the negatively charged ¹⁰⁰¹EEDDEEGE¹⁰⁰⁸ stretch close to the surface of lipid bilayer, which may form ionic interactions with the positively charged choline head of membrane phospholipids to serve as a “break” for the prevention of “whiplash” of α_IIb extracellular domain, to keep integrin in the correct active state conformation on the surface of cells. Indeed, the unique reverse folding structure prior to the acidic stretch introduced by two prolines (998 and 999) in the α_IIb tail makes the break hypothesis plausible (42).

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. 16 and 34.