A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β₃ Subunit Activates Integrins _IIbβ₃ and _Vβ₃

THE β₃ SUBFAMILY OF heterodimeric integrin receptors includes α_IIbβ₃, which is specific for platelets and critical for adhesive events in hemostasis, and α_Vβ₃, which is more widely distributed and involved in regulation of cell growth, migration, and programmed death.1-4 The ligand binding function of these integrins is tightly regulated by two processes often referred to collectively as inside-out signaling: (1) affinity modulation, which involves structural changes intrinsic to the heterodimer; and (2) avidity modulation due to lateral diffusion and clustering of heterodimers into oligomers.5,6 Ligand binding to and clustering of β₃ integrins in turn generate outside-in signals, such as activation of the protein tyrosine kinases Src and pp125^FAK, which act in concert with signals from growth factor receptors to regulate many anchorage-dependent cell functions.6-8 

The β₃ integrin subunit consists of 762 amino acids encompassing a large extracellular domain, a single membrane-spanning region, and a 47 amino acid cytoplasmic tail.9 The extracellular domain is notable for an I-domain like ligand-binding region (residues 110-294)10 and a cysteine-rich repeat region (residues 423-622) that contains 31 of the subunit’s 56 cysteine residues.9,11 Studies of individuals with variant Glanzmann thrombasthenia, who bleed due to functional defects of α_IIbβ₃, and of recombinant integrins expressed in mammalian cells have focused attention on the ligand-binding region and the cytoplasmic tail as particularly relevant to the process of inside-out signaling.12-19 In addition, antibodies known as ligand-induced binding site (LIBS), which bind better to β₃ after fibrinogen binds, can in some cases increase receptor affinity without the need for signals from inside the cells.20,21 Some of these antibodies recognize epitopes within the cysteine-rich repeats of β₃,21,22raising the possibility that this region may also be involved in propagating activating signals from inside the cell to the ligand binding region of the receptor. In this report, we provide the first direct evidence for this idea by characterizing a single amino acid substitution in the cysteine-rich repeat region of β₃that results in constitutive activation of both α_IIbβ₃ and α_Vβ₃.

PAC1, a ligand-mimetic mouse monoclonal IgM antibody, is specific for the activated α_IIbβ₃ complex.23Two antibodies to LIBS within β₃, LIBS1 and LIBS6,20,24 were obtained from Dr Mark H. Ginsberg (The Scripps Research Institute, La Jolla, CA). AP5 (anti-β₃LIBS),22 AP3 (anti-β₃; non-function blocking),25 and AP2 (anti-α_IIbβ₃; function blocking)26 were obtained from Dr Thomas J. Kunicki (The Scripps Research Institute). PT25-2, an α_IIbβ₃ activating antibody, and its Fab fraction have been characterized.27 LM142 (anti-α_V; non-function blocking)28 were obtained from Dr David A. Cheresh (The Scripps Research Institute). α_IIb cDNA and β₃ cDNA inserted in pCDM8 (resulting in the expression plasmids, CD2b and CD3a)17were obtained from Dr M.H. Ginsberg. α_IIb cDNA and β₃ cDNA inserted in pcDNA3 (α_IIb/pcDNA3, β₃/pcDNA3) were obtained from Dr Peter J. Newman (The Blood Center of Southeastern Wisconsin, Milwaukee, WI). α_V cDNA inserted in pcDNA1 was obtained from Dr D.A. Cheresh.

Chinese hamster ovary (CHO) and 293 cells were maintained as described.29,30 CD2b and CD3a were cotransfected into a CHO-AA8 cell line (Clontech, Palo Alto, CA), and stable transfectants expressing α_IIbβ₃ were obtained by single cell sorting by a FACStar flow cytometry (Becton Dickinson, San Jose, CA). As shown previously, α_IIbβ₃transfectants obtained in this manner do not bind PAC1 or fibrinogen and are in a low-affinity/avidity state.17,30 Then, one of these clones (24/12) was stained with PAC1 and subjected to another round of single-cell sorting to select for rare variants that now bound PAC1. 192 cells were sorted and PAC1 binding of 48 clones was reassessed. Only one clone, AM-1, showed constitutive binding of PAC1, as described below.

Genomic DNA from 24/12 and AM-1 cells was isolated (Qiagen DNA extraction kit; Qiagen Inc, Chatsworth, CA), and the entire coding regions of α_IIb and β₃ were amplified using 4 and 3 paired sets of primers, respectively.31,32Amplified DNA fragments were purified and subjected to direct cycle sequence analysis using an Applied Biosystems Automated sequencer (Perkin Elemer-Japan, Chiba, Japan) according to the manufacturer’s directions.

To introduce the T562N mutation in β₃ cDNA, two-step ligation was performed. First, β₃/pcDNA3 was cut withBamHI and Afl II, yielding three fragments, Frag1 (BamHI and Afl II ends: 6 kb), Frag2 (BamHI andBamHI ends: 1.5 kb), and Frag3 (BamHI and AflII ends: 0.5 kb). After agarose gel electrophoresis, Frag1 and Frag2 were cut out with Gel extraction kit (Qiagen). The β₃cDNA fragment from AM-1 cells including the mutated site (T562N) was amplified using primers, IIIa3 and IIIa4-AflII, and digested withBamHI and Afl II. The 0.5-kb fragment [Frag3(T562N)] was cut out and ligated to Frag1 with a ligation kit (Boehringer Mannheim, Mannheim, Germany). After transformation to JM109 competent cells (Takara, Shiga, Japan), miniprep DNA was cut withBamHI and Frag2 was inserted. After transformation, a clone containing Frag2 in the correct orientation was selected by polymerase chain reaction (PCR), amplified, and purified with a cesium chloride gradient. The entire coding region of the plasmid was sequenced to confirm aquisition of T562N mutation and absence of other nucleotide change.

To introduce T562A, T562Q, and T564A into β₃ cDNA, PCR-based mutagenesis was performed as described.33 In brief, first-round PCR was performed using β₃/pcDNA3 as a template with antisense primer containing mutated nucleotide(s) or sense primer containing mutated nucleotide(s) and IIIa4-AflII. The sequence of primers used is shown in Table1. After purification of PCR fragments with a purification kit (Qiagen), second-round PCR was performed using mixture of first-round PCR products as template with primers, IIIa3 and IIIa4-AflII. PCR products were cut with BamHI and Afl II, and the appropriate fragments were purified and ligated to Frag1 and Frag2. To make the β₃(T562N, T564A) double mutant, β₃(T562N)/pcDNA3 was used as a template instead of β₃/pcDNA3. All plasmids were sequenced to confirm authenticity.

A human embryonic kidney cell line, 293 (obtained from American Type Cell Culture, Rockville, MD), was used for transient transfection assays. Transfections were performed using the calcium phosphate method as described.29 Functional assays were performed 48 hours after transfection.

PAC1 binding to cells was assessed as described30 with minor modification. Cells (5 × 10⁵) were preincubated in 45-μL aliquots containing Tyrode’s buffer supplemented with 2 mmol/L CaCl₂ and 2 mmol/L MgCl₂ in the presence or absence of 10 μmol/L FK633 (a peptidomimetic α_IIbβ₃ antagonist from Dr Jiro Seki, Fujisawa Pharmaceutical Co, Osaka, Japan)34 or 10 μg/mL PT25-2, an α_IIbβ₃ activating antibody. Then, 5 μL of a 1:25 dilution of PAC1 ascites was added to each tube, and incubations were performed for another 30 minutes at room temperature. After washing, cells were resuspended in a 1:20 dilution of fluorescein isothiocyanate (FITC)-conjugated antimouse IgM (μ-chain specific; Caltag Lab, Burlingame, CA) for 25 minutes on ice. Then, 1 μL of 1 mg/mL of propidium iodide (PI; Sigma, St Louis, MO) was added, and 5 minutes later the cells were washed and resuspended in 500 μL of ice-cold Tyrode’s and analyzed on a FACScan flow cytometer (Becton Dickinson). PAC1 binding (FL1) was analyzed on the gated subset of single, live cells (PI-negative, FL3). To compare the PAC1 binding results from one experiment with those from another, PAC1 binding was expressed as an activation index, defined as (Fx − Fi)/(Fm − Fi), where Fx is the median fluorescence intensity of PAC1 binding in the absence of inhibitor, FK633; Fi is the median fluorescence intensity of PAC1 binding in the presence of inhibitor, FK633; and Fm is median fluorescence intensity of PAC1 binding in the presence of the activating antibody, PT25-2.30 

Fibrinogen was labeled with FITC as described.35 In the case of α_IIbβ₃-stable cell lines, the binding of FITC-fibrinogen (150 μg/mL) was assessed in the same manner as described for PAC1. In the case of 293 cells transiently transfected with α_Vβ₃, cells were suspended in calcium-free Tyrode’s buffer supplemented with 1 mmol/L MgCl₂ and pretreated for 30 minutes on ice with or without 1 mmol/L RGDW, an inhibitor of fibrinogen binding to α_Vβ₃, or 1 mmol/L MnCl₂, which induces a high-affinity state of α_Vβ₃.2 LM142 (10 μg/mL), a non-function blocking anti-α_V antibody, was added simultaneously to the tubes to monitor expression of α_Vβ₃. After washing, cells were incubated with 150 μg/mL of FITC-fibrinogen and phycoerythrin (PE)-conjugated antimouse IgG (Serotec, Oxford, UK) for 25 minutes at room temperature. Then, after 5 minutes of incubation with PI, cells were washed and three-color analysis was performed on FACScan. In this case, fibrinogen binding (FL1) was analyzed on the gated subset of single, live cells (PI-negative, FL3) that stained positively for α_Vβ₃ (FL2).

Fibrinogen-dependent cell aggregation was monitored as described.36 In brief, 5 × 10⁶/mL cells were added to wells of a 24-well culture dish precoated with 1 mg/mL of bovine serum albumin (BSA). Fibrinogen (300 μg/mL) was added with or without 20 μmol/L FK633 or 20 μg/mL PT25-2 and the dish was rotated at 100 rpm for 20 minutes on a horizontal rotator (Multi-Mixer; Lab-Line Instruments Inc, Melrose Park, IL). Aggregate formation was stopped by adding an equal volume of 0.5% formaldehyde, and aggregates were visualized and photographed under a phase-contrast microscope (Olympus, Tokyo, Japan).

Cells were labeled with sulfo-NHS-biotin (Pierce, Rockford, IL) and lysed in Triton X-100 buffer (1% Triton X-100, 25 mmol/L Tris-Cl, 100 mmol/L NaCl, pH 7.4, 0.1 mg/mL leupeptin, 4 μg/mL pepstatin A, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L benzamide). Two hundred micrograms of protein from each sample was immunoprecipitated with an α_IIbβ₃ complex-specific antibody, AP2, as described.32 Immunoprecipitates were resolved in 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon; Millipore, Bedford, MA). After incubation with peroxidase-conjugated avidin (Vectastain ABC kit; Vector Lab, Burlingame, CA), immunoreactive bands were visualized by enhanced chemiluminescence (Renaissance; NEN Life Science, Boston, MA).

Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.5% fetal bovine serum for 18 hours and then resuspended to 3 × 10⁶ cells/mL in DMEM. To test the effects of soluble fibrinogen to α_IIbβ₃, suspended cells were incubated at 37°C for 30 minutes in the presence or absence of 10 μg/mL of Fab fragments of PT25-2, and then 250 μg/mL of fibrinogen was added to the cells. After 15 minutes of incubation at 37°C, the cells were washed with phosphate-buffered saline (PBS) and lysed with Triton lysis buffer supplemented with 1 mmol/L sodium vanadate. For studies of adherent cells, 1 mL of suspended cells were seeded onto plastic dishes that had been precoated with 100 μg/mL fibrinogen or poly-L lysine (Iwaki Glass, Tokyo, Japan). After 30 minutes at 37°C, plates were washed twice with ice-cold PBS. Then adherent cells were lysed on the plates with Triton lysis buffer containing sodium vanadate and scraped into microcentrifuge tubes. Lysates were incubated on ice for 30 minutes and clarified supernatants were processed for immunoprecipitation.

pp125^FAK was immunoprecipitated with 1 μg of rabbit polyclonal antibody, FAK(C903) (Santa Cruz Biotech, Santa Cruz, CA), and protein-G sepharose (Pharmacia, Uppsala, Sweden). Precipitates were separated on 7.5% SDS-PAGE and transferred to a PVDF membrane. Phosphotyrosine was detected with monoclonal antibody, 4G10. To monitor loading of gel lanes, the blots were stripped (2% SDS, 62.5 mmol/L Tris, pH 6.7, 100 mmol/L 2-mercaptoethanol for 30 minutes at 70°C) and reprobed with FAK(C903).

α_IIbβ₃ expressed in resting platelets or in a CHO cell model system exists in a low-affinity/avidity state and does not bind soluble fibrinogen or the ligand-mimetic antibody, PAC1.6,17 To better understand the structural basis of α_IIbβ₃ activation, a stable CHO cell line that expresses α_IIbβ₃ (24/12) was stained with PAC1 and analyzed by flow cytometry to screen for rare variants that might bind PAC1 constitutively. In so doing, a stable cell line, AM-1 (Activated Mutant-1), that expressed this activated phenotype was established. Parental 24/12 cells did not bind to PAC1 spontaneously, but they did do so as expected in response to activating antibody, PT25-2 (activation index [AI], 0.04 ± 0.02 [mean ± SD]; n = 5). In contrast, AM-1 cells bound PAC1 even in the absence of PT25-2 (AI, 0.67 ± 0.09; n = 5) and the binding was completely inhibited by incubation with an α_IIbβ₃ specific antagonist, FK633 (Fig 1A). The same results were obtained when another activating antibody (LIBS6) was used to induce ligand binding21 or when another specific antagonist (Integrilin) was used to block ligand binding37 (data not shown). Preincubation of AM-1 cells with 0.2% sodium azide and 4 mg/mL 2-deoxy-d-glucose had no effect on the increase in PAC1 binding, indicating that the high-affinity/avidity state of the α_IIbβ₃ in AM-1 cells was independent of metabolic energy (data not shown).

To establish whether AM-1 cells also bind a soluble physiological ligand in a constitutive fashion, binding of FITC-labeled fibrinogen was examined by flow cytometry. Consistent with the PAC1 results, fibrinogen bound to AM-1 in the absence of PT25-2 and the binding was completely blocked by FK633 (Fig 1B). To determine the functional relevance of this spontaneous fibrinogen binding, the ability of AM-1 cells to aggregate was assessed. The parental 24/12 cells exhibited fibrinogen-dependent aggregation only after addition of the activating antibody. In contrast, AM-1 cells exhibited fibrinogen-dependent and FK633-inhibitable aggregation even in the absence of the activating antibody (Fig 2). AP2, a function blocking anti-α_IIbβ₃ antibody,26 also inhibited the fibrinogen-dependent aggregation of AM-1 cells, and this aggregation was divalent cations dependent, because the aggregation was not observed when cells were resuspended in divalent cation free Tyrodes buffer with 2 mmol/L EDTA (data not shown). These results indicate that α_IIbβ₃ in AM-1 cells is in a constitutively activated state, fully capable of supporting spontaneous cell aggregation in the presence of fibrinogen.

Integrins can become activated to bind ligands through conformational changes in the heterodimer (affinity modulation) and/or receptor clustering (avidity modulation).5-7 To determine whether the activation of α_IIbβ₃ in AM-1 cells was associated with conformational changes in the receptor, the binding of various anti-LIBS antibodies (LIBS1, LIBS6, and AP5) was examined. It has been shown that LIBS1, LIBS6, and AP5 preferentially recognize epitopes on β₃ after ligand-binding, and the epitope of LIBS6 and AP5 has been defined within the cysteine-rich repeat region and the N-terminal region, respectively.20,22 FK633 (5 μmol/L) was used to induce LIBS expression, because preliminary studies showed that it could induce full expression of LIBS epitopes in platelets and CHO cell lines (Kashiwagi et al, unpublished observation). In parental 24/12 cells the LIBS antibodies bound to α_IIbβ₃ only after cell incubation with FK633. In contrast, LIBS6 and AP5 bound fully to AM-1 cells in the absence of FK633 and LIBS1 showed a slight increase in binding (Fig 3). Thus, some but not all LIBS epitopes are constitutively exposed on α_IIbβ₃ in AM-1 cells, even in the absence of added ligand.

To begin to analyze the conformational change of α_IIbβ₃ in AM-1 cells, α_IIbβ₃ was immunoprecipitated with AP2. In contrast to β₃ in the parental cells, β₃ in AM-1 cells migrated more slowly and as a broad band under both reduced and nonreduced conditions, suggesting that mutation(s) and/or change of glycosylation state existed in β₃ of AM-1 cells (Fig 4). Next, the sequence of the entire coding region of α_IIb and β₃ cDNA integrated in genomic DNA of AM-1 cells was determined. Amplification of genomic DNA using primer pairs that located in far separated exons excluded the possibility to amplify intrinsic hamster α_IIb and β₃ DNA. A consecutive dinucleotide change in β₃ cDNA integrated in AM-1 cells (¹⁷³²ACG→AAC) was discovered, leading to a single putative amino acid substitution, T562N (Fig 5A). No other sequence abnormalities were detected. This mutation is in the third of four cysteine-rich repeats in β₃, and it would establish a new putative N-glycosylation site at the mutated residue (⁵⁶²TRT→NRT: NXT/S is a consensus sequence of N-glycosylation). Amino acid alignment around the mutated site shows that amino acid residues in C560-C567, that may make a small loop in the third repeat of the cysteine-rich repeat region,11 are completely identical in Xenopus, chicken, rodent, and human β₃. By contrast, they are poorly conserved between human β₃ and other human β integrins (Fig 5B).

To prove that this single amino acid change was responsible for the constitutive activation of α_IIbβ₃, we introduced this mutation into wild-type β₃ and transiently transfected it into 293 cells. Indeed, in the absence of activating antibody PT25-2, PAC1 bound spontaneously to cells transfected with α_IIbβ₃(T562N) (AI, 0.80 ± 0.10; n = 5), but not to cells transfected with wild-type α_IIbβ₃ (AI, 0.06 ± 0.02; n = 5), indicating that the T562N mutation is responsible for constitutive activation of the receptor (Fig 6A). When a D119Y mutation in β₃ that abrogates fibrinogen binding to α_IIbβ₃12 was introduced into β₃(T562N), PAC1 failed to bind to α_IIbβ₃(D119Y, T562N) either in the absence or presence of PT25-2 (data not shown).

To determine the mechanism of activation by the T562N mutation, we introduced T562A and T562Q mutations into β₃. The T562Q mutant was constructed because asparagine and glutamine have the same amide in their side chain. PAC1 did not bind to cells transfected with either α_IIbβ₃(T562A) or α_IIbβ₃(T562Q) in the absence of PT25-2 (AI, 0.08 ± 0.03 and 0.07 ± 0.06, respectively; n = 3; Fig 6A), indicating that it was the acquisition of asparagine at residue 562 that was important for spontaneous receptor activation. Next, we introduced the T564A mutation into β₃ to disrupt the aberrant consensus sequence of N-glycosylation at residue 562. Immunoprecipitation of this form of β₃ showed that β₃(T564A) and β₃(T562N,T564A) migrated on SDS gels similarly to the migration of β₃(WT) (Fig 6C). Because the T564A mutation led to a mild reduction in the expression of α_IIbβ₃, we monitored α_IIbβ₃ expression by a non-function blocking anti-β₃ antibody, AP3, and analyzed only cells expressing high levels of α_IIbβ₃ for PAC1 binding. Although α_IIbβ₃(T564A) showed a slight increase in the activation index (0.28 ± 0.03; n = 3), the activation of α_IIbβ₃(T562N, T564A) was even greater (AI, 0.46 ± 0.01; n = 3), and this difference was statistically significant (P < .001; Fig 6B). These results suggest that the glycosylation at N562 may not be essential for the constitutive activation of α_IIbβ₃ in AM-1 cells. A lack of relationship between the aberrant N-glycosylation of β₃(T562N) and α_IIbβ₃activation was also suggested by the observation that 24 hours of incubation of the cells with 1 to 5 μg/mL of tunicamycin, a specific inhibitor of N-glycosylation,38 had no effect on the activation state of α_IIbβ₃ in AM-1 cells (AI, 0.67 ± 0.09, 0.67 ± 0.04, and 0.66 ± 0.02; 0, 1, and 5 μg/mL of tunicamycin, respectively; n = 3). On the other hand, the activation state of wild-type α_IIbβ₃ in 24/12 cells was slightly increased by tunicamycin treatment in a concentration-dependent manner (AI, 0.04 ± 0.02, 0.13 ± 0.04, and 0.18 ± 0.01; 0, 1, and 5 μg/mL of tunicamycin, respectively; n = 3).

To determine whether the activating mutation in β₃ would affect the affinity state of the related integrin α_Vβ₃, this integrin was transiently expressed in 293 cells. In this case, FITC-labeled fibrinogen was used to determine the activation state of α_Vβ₃and a non-function blocking anti-α_V antibody, LM142, was used to monitor α_Vβ₃ expression. When α_V was transfected with wild-type β₃, fibrinogen bound only if integrin affinity was upregulated by the addition of manganese. In contrast, fibrinogen binding to cells expressing α_Vβ₃(T562N) could be detected even in the absence of manganese (Fig 7). These results indicate that the T562N mutation is capable of increasing the activation state of both α_IIbβ₃ and α_Vβ₃.

Ligand binding and clustering of integrins stimulate outside-in signaling, manifested by responses that include protein tyrosine phosphorylation and cytoskeletal reorganization.6-8 Focal adhesion kinase (FAK), a 125-kD cytoplasmic tyrosine kinase, is a component of focal adhesions and is a well-established component of integrin signaling pathways.39,40 Consequently, the tyrosine phosphorylation state of pp125^FAK in AM-1 cells was studied. pp125^FAK was not tyrosine-phosphorylated in AM-1 cells or in control 24/12 cells maintained in suspension for 15 minutes, either in the presence or absence of fibrinogen. Furthermore, neither cell type exhibited pp125^FAK phosphorylation when adherent to plates coated with poly-L-lysine. On the other hand, both showed pp125^FAK phosphorylation in response to cell adhesion to fibrinogen (Fig 8). These results indicate that receptor activation by the T562N mutation or the mere binding of soluble fibrinogen to the activated receptor is not sufficient to cause activation of pp125^FAK; nonetheless, this mutant receptor is fully capable of mediating this outside-in signaling response upon cell adhesion.

In this report, we analyzed a cell line in which the ligand-binding function of integrin α_IIbβ₃ was constitutively activated, in contrast to the usual, default low-affinity/avidity state of this integrin in platelets and transfected tissue culture cells.6,17 We found the following: (1) A single amino acid change in the extracellular cysteine-rich repeat region of β₃, T562N, is responsible for this constitutive activation. (2) The presence of the asparagine as opposed to the loss of the threonine appears responsible for this phenotype. (3) Although the T562N mutation leads to aberrant glycosylation, it is unlikely that this posttranslational modification actually causes the activated integrin phenotype. (4) The T562N mutation is capable of activating α_Vβ₃ as well as α_IIbβ₃. (5) Activation of α_IIbβ₃ by T562N or binding of soluble fibrinogen to the mutant is not sufficient to trigger tyrosine phosphorylation of pp125^FAK. Ligand binding as the result of the T562N mutation was similar to that induced through the more physiological process of inside-out signaling, because (A) it was completely blocked by synthetic α_IIbβ₃-specific antagonists, (B) it was abrogated by a mutation in β₃(D119Y) associated with a variant form of thrombasthenia and clinical bleeding,12 and (C) it mediated fibrinogen-dependent cell aggregation.

Three types of activating mutations in α_IIbβ₃ have been described previously. (1) Bajt et al41 showed that swapping the ligand-binding site (residues 129-133) of β₃ with the corresponding sequence in β₁ led to a gain of function in α_IIbβ₃. (2) The proximal regions of cytoplasmic tails of α_IIb and β₃ are highly conserved, and deletions or amino acid changes that may break the interaction between them lead to activation of integrins.13,17,18 (3) Liu et al42 recently reported that disruption of the C5-C435 disulfide bond in β₃ resulted in an increase in affinity of α_IIbβ₃. In this context, it has been shown that mild reducing agents, such as dithiothreitol, can increase the ligand-binding function of α_IIbβ₃.43 In addition, it has been shown that some LIBS antibodies that bind to epitopes within the cysteine-rich repeats of β₃, such as LIBS2, LIBS3, and LIBS6, lead to activation of α_IIbβ₃ without ligand-binding.20-22 Wippler et al44 also demonstrated that recombinant α_IIbβ₃lacking the cysteine-rich repeats of β₃ showed high-affinity binding to fibrinogen. Furthermore, sequence alignment of β₃ integrins indicate that about 90% of noncysteine residues in the cysteine-rich repeats are conserved between rodents and human β₃, whereas noncysteine residues in the region are poorly conserved among β₁, β₂, and β₃ integrins (∼15%).45 These results suggest that the cysteine-rich repeats may have an important role for regulation of β₃ integrin functions.

Our results provide the direct evidence that this region is involved in the activation of β₃ integrins. Enhanced ligand binding to α_IIbβ₃(T562N) was observed both in CHO cells and 293 cells, suggesting that the functional effect by the mutation is not cell type-specific. Because neither the T562A nor the T562Q mutations caused activation of α_IIbβ₃, the side-chain of asparagine 562 is clearly an important variable contributing to induction of the activated receptor. The finding that the T562N mutation also led to activation of α_Vβ₃ indicates that activation of α_IIbβ₃ by the mutation did not require a unique interaction of β₃ with the α_IIb subunit. Although T562N represents a new putative N-glycosylation site, it is unlikely that alternative glycosylation at this position is responsible for activation of α_IIbβ₃, because (1) α_IIbβ₃(T562N, T564A), which lacked the aberrant glycosylation observed with the single N562 mutation, showed an even more activated state than α_IIbβ₃(T564A); and (2) tunicamycin, an inhibitor of N-glycosylation,38 had no effect on the activated state of α_IIbβ₃(T562N) in AM-1 cells.

The binding of soluble ligands to integrins can be enhanced by two complementary mechanisms, conformational change within heterodimers (affinity modulation) and clustering of receptors into heteroligomers (avidity modulation).46,47 An understanding of the precise mechanism of integrin activation by the β₃(T562N) mutation will require a level of understanding of integrin atomic structure that is not yet available. However, the predominant effect of the β₃(T562N) mutation may be on β₃integrin conformation. First, some LIBS epitopes are constitutively exposed on α_IIbβ₃ in AM-1 cells. Second, although affinity and avidity modulation both influence the functions of α_IIbβ₃, affinity modulation is the predominant regulator of ligand binding.47 Third, soluble fibrinogen binding to platelet or CHO cell α_IIbβ₃ activated through affinity modulation by means of an activating LIBS antibody is not sufficient to trigger tyrosine phosphorylation of FAK; rather, integrin clustering and other post-ligand binding events are also required.47-49 Similarly, fibrinogen binding induced by the β₃(T562N) mutation was not sufficient to stimulate FAK phosphorylation, suggesting that this mutation was not primarily triggering receptor clustering.

It has been demonstrated that manganese,2β₃-LIBS antibody,50 and purification of α_Vβ₃ by affinity chromatography51 can induce a high-affinity state of α_Vβ₃. However, the T562N mutation is the first one reported to induce spontaneous activation of α_Vβ₃. A number of reports indicate that α_Vβ₃ plays an important role in angiogenesis, tumor invasion, and bone absorption.2-4 One obvious question now being pursued is whether this activating mutation affects any of these functions of α_Vβ₃. In any case, the current study serves to emphasize the possible involvement of residues in the cysteine-rich region of β₃in affinity modulation of both α_IIbβ₃ and α_Vβ₃. This will have to be taken into account in future refinements of models for integrin activation.52 

The authors are grateful to Drs M.H. Ginsberg, T.J. Kunicki, J. Seki, D.A. Cheresh, and P.J. Newman for providing the materials.