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Related Collections Hematopoiesis and Stem Cells Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 93 No. 8 (April 15), 1999: pp. 2559-2568 A Mutation in the Extracellular Cysteine-Rich Repeat Region of the 3 Subunit Activates Integrins IIb3 and V3 By Hirokazu Kashiwagi, Yoshiaki Tomiyama, Seiji Tadokoro, Shigenori Honda, Masamichi Shiraga, Hajime Mizutani, Makoto Handa, Yoshiyuki Kurata, Yuji Matsuzawa, and Sanford J. Shattil From The Second Department of Internal Medicine, Osaka University Medical School, and Department of Transfusion, Osaka University Hospital, Osaka, Japan; the Blood Center, Keio University Hospital, Tokyo, Japan; and the Department of Vascular Biology and Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA.     ABSTRACT TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIb3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIb3. One clone (AM-1) expressed constitutively active IIb3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIb3 antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of 3(T562N) was responsible for receptor activation. In fact, T562N also activated V3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIb3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of 3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIb3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of 3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling. © 1999 by The American Society of Hematology.     INTRODUCTION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES THE 3 SUBFAMILY OF heterodimeric integrin receptors includes IIb3, which is specific for platelets and critical for adhesive events in hemostasis, and V3, 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 3 integrins in turn generate outside-in signals, such as activation of the protein tyrosine kinases Src and pp125FAK, which act in concert with signals from growth factor receptors to regulate many anchorage-dependent cell functions.6-8 The 3 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 IIb3, 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 3 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 3,21,22 raising 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 3 that results in constitutive activation of both IIb3 and V3.     MATERIALS AND METHODS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Antibodies and plasmids.   PAC1, a ligand-mimetic mouse monoclonal IgM antibody, is specific for the activated IIb3 complex.23 Two antibodies to LIBS within 3, LIBS1 and LIBS6,20,24 were obtained from Dr Mark H. Ginsberg (The Scripps Research Institute, La Jolla, CA). AP5 (anti-3 LIBS),22 AP3 (anti-3; non-function blocking),25 and AP2 (anti-IIb3; function blocking)26 were obtained from Dr Thomas J. Kunicki (The Scripps Research Institute). PT25-2, an IIb3 activating antibody, and its Fab fraction have been characterized.27 LM142 (anti-V; non-function blocking)28 were obtained from Dr David A. Cheresh (The Scripps Research Institute). IIb cDNA and 3 cDNA inserted in pCDM8 (resulting in the expression plasmids, CD2b and CD3a)17 were obtained from Dr M.H. Ginsberg. IIb cDNA and 3 cDNA inserted in pcDNA3 (IIb/pcDNA3, 3/pcDNA3) were obtained from Dr Peter J. Newman (The Blood Center of Southeastern Wisconsin, Milwaukee, WI). V cDNA inserted in pcDNA1 was obtained from Dr D.A. Cheresh. Cell lines.   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 IIb3 were obtained by single cell sorting by a FACStar flow cytometry (Becton Dickinson, San Jose, CA). As shown previously, IIb3 transfectants 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. Nucleotide sequence analysis of IIb and 3.   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 IIb and 3 were amplified using 4 and 3 paired sets of primers, respectively.31,32 Amplified 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. Site-directed mutagenesis of 3 cDNA.   To introduce the T562N mutation in 3 cDNA, two-step ligation was performed. First, 3/pcDNA3 was cut with BamHI and Afl II, yielding three fragments, Frag1 (BamHI and Afl II ends: 6 kb), Frag2 (BamHI and BamHI ends: 1.5 kb), and Frag3 (BamHI and Afl II ends: 0.5 kb). After agarose gel electrophoresis, Frag1 and Frag2 were cut out with Gel extraction kit (Qiagen). The 3 cDNA fragment from AM-1 cells including the mutated site (T562N) was amplified using primers, IIIa3 and IIIa4-AflII, and digested with BamHI 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 with BamHI 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 3 cDNA, PCR-based mutagenesis was performed as described.33 In brief, first-round PCR was performed using 3/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 Table 1. 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 3(T562N, T564A) double mutant, 3(T562N)/pcDNA3 was used as a template instead of 3/pcDNA3. All plasmids were sequenced to confirm authenticity.                                View this table: [in this window] [in a new window]   Table 1. Oligonucleotides Used in the Construction of the 3 Mutants Transient transfection.   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 and fibrinogen binding assay.   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 IIb3 antagonist from Dr Jiro Seki, Fujisawa Pharmaceutical Co, Osaka, Japan)34 or 10 µg/mL PT25-2, an IIb3 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 IIb3-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 V3, 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 V3, or 1 mmol/L MnCl2, which induces a high-affinity state of V3.2 LM142 (10 µg/mL), a non-function blocking anti-V antibody, was added simultaneously to the tubes to monitor expression of V3. 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 V3 (FL2). Cell aggregation.   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). Cell surface labeling and immunoprecipitation.   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 IIb3 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). Tyrosine phosphorylation of pp125FAK.   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 IIb3, 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. pp125FAK 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).     RESULTS TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES Establishment of the AM-1 cell line expressing a constitutively active form of IIb3.   IIb3 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 IIb3 activation, a stable CHO cell line that expresses IIb3 (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 (ctivated utant-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 IIb3 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 IIb3 in AM-1 cells was independent of metabolic energy (data not shown). View larger version (25K): [in this window] [in a new window]   Fig 1. Assessment of affinity state of IIb3 by PAC1 (A) and fibrinogen (B) binding. In (A), 24/12 cells (a through c) or AM-1 cells (d through f) were preincubated with 10 µmol/L FK633 (a and d), 10 µg/mL PT25-2 (c and f), or buffer (b and e) for 30 minutes on ice. Then, 250× diluted PAC1 ascites were added and incubated for another 30 minutes at room temperature. After washing, cells were incubated with FITC-conjugated antimouse IgM for 25 minutes on ice. To exclude dead cells, PI were added to the cells and incubated for 5 minutes. After washing, cells were resuspended in buffer and flow cytometric analysis was performed. In (B), cells were preincubated with 10 µmol/L FK633 (dotted lines), 10 µg/mL PT25-2 (solid lines), or buffer (bold lines) for 30 minutes on ice. Cells were then incubated with 150 µg/mL of FITC-labeled fibrinogen for 25 minutes at room temperature and then with PI for 5 minutes. After washing, flow cytometric analysis was performed. 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-IIb3 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 IIb3 in AM-1 cells is in a constitutively activated state, fully capable of supporting spontaneous cell aggregation in the presence of fibrinogen. View larger version (85K): [in this window] [in a new window]   Fig 2. Spontaneous, fibrinogen-dependent aggregation of AM-1 cells. Cells were resuspended in Tyrode's buffer in the presence of 300 µg/mL of fibrinogen and with or without 20 µmol/L FK633 or 20 µg/mL PT25-2. Cells were then rotated for 20 minutes on a horizontal rotator. Aggregate formation was stopped by adding equal amount of 0.5% formaldehyde and incubating for 30 minutes. Shown are photographs representative of three independent experiments. IIb3 in AM-1 cells is in a high-affinity state.   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 IIb3 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 3 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 IIb3 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 IIb3 in AM-1 cells, even in the absence of added ligand. View larger version (32K): [in this window] [in a new window]   Fig 3. LIBS expression of 24/12 (the left row) and AM-1 cells (the right row). Cells were preincubated with 5 µmol/L FK633 (solid lines) or buffer (bold lines) for 30 minutes on ice, and then 5 µg/mL of LIBS1 (upper row), LIBS6 (middle row), or AP5 (lower row) was added. After 30 minutes of incubation, cells were washed and then incubated with FITC-conjugated antimouse IgG for 30 minutes. After washing, flow cytometric analysis was performed. Dotted lines indicate MOPC21, a control mouse IgG antibody, binding. Molecular analysis of IIb3 in AM-1 cells.   To begin to analyze the conformational change of IIb3 in AM-1 cells, IIb3 was immunoprecipitated with AP2. In contrast to 3 in the parental cells, 3 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 3 of AM-1 cells (Fig 4). Next, the sequence of the entire coding region of IIb and 3 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 IIb and 3 DNA. A consecutive dinucleotide change in 3 cDNA integrated in AM-1 cells (1732AA) 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 3, and it would establish a new putative N-glycosylation site at the mutated residue (562RTRT: 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 3. By contrast, they are poorly conserved between human 3 and other human  integrins (Fig 5B). View larger version (56K): [in this window] [in a new window]   Fig 4. Immunoprecipitation of IIb3 from 24/12 and AM-1 cells. Cell surface was labeled with sulfo-NHS-LC-biotin and lysed with 1% Triton X-100 lysis buffer. Lysates from 24/12 cells (lanes 1 and 3) and AM-1 cells (lanes 2 and 4) were incubated with anti-IIb3 antibody, AP2, and immunoprecipitates were separated on 6% SDS-PAGE under reducing (lanes 1 and 2) or nonreducing conditions (lanes 3 and 4). After transfer, the membrane was incubated with peroxidase-conjugated avidin and developed with chemiluminescence. View larger version (23K): [in this window] [in a new window]   Fig 5. Sequence analysis of 3 cDNA from 24/12 or AM-1 cells. (A) The results of sequencing using an antisense primer are shown. The same results were obtained using a sense primer for sequencing (data not shown). The mutated nucleotides and the changed amino acid are in bold. (B) Amino acid alignment around the mutated site is shown. T562 in 3 and corresponding amino acids in other  integrins are in bold. NRT (underlined) is a consensus sequence for N-glycosylation. Amino acid sequences were obtained from Wippler et al,44 Mimura et al,53 and Ransom et al.54 Substitution of asparagine for threonine at position 562 of 3 causes a high-affinity state of IIb3.   To prove that this single amino acid change was responsible for the constitutive activation of IIb3, we introduced this mutation into wild-type 3 and transiently transfected it into 293 cells. Indeed, in the absence of activating antibody PT25-2, PAC1 bound spontaneously to cells transfected with IIb3(T562N) (AI, 0.80 ± 0.10; n = 5), but not to cells transfected with wild-type IIb3 (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 3 that abrogates fibrinogen binding to IIb312 was introduced into 3(T562N), PAC1 failed to bind to IIb3(D119Y, T562N) either in the absence or presence of PT25-2 (data not shown). View larger version (81K): [in this window] [in a new window]   Fig 6. Assessment of the activation state of wild-type and mutant IIb3 in transiently transfected 293 cells. (A) Wild-type IIb cDNA was transfected with wild-type 3 cDNA (WT) (a through c) or mutant 3 cDNAs (T562N [d through f], T562A [g through i], T562Q [j through l]) to 293 cells, and PAC1 binding was determined. Plots in the upper row (a, d, g, and j) represent nonspecific PAC1 binding determined in the presence of FK633. Plots in the lower row (c, f, i, and l) represent maximal PAC1 binding in the presence of PT25-2. Plots in the middle row (b, e, h, and k) represent PAC1 binding in the absence of the antagonist and the activating antibody. (B) Wild-type IIb3, IIb3(T564A), or IIb3(T562N,T564A) transfected cells were incubated with PAC1 and biotinylated-AP3, a non-function blocking anti-3 antibody, followed by incubation with FITC-conjugated antimouse IgM and PE-conjugated streptoavidin, and analyzed by flow cytometry. The overlay histogram represents PAC1 binding to cells expressing high levels of IIb3 determined by AP3 (denoted by the rectangle in the dot blots) [wild-type IIb3, dotted line; IIb3(T564A), solid line; IIb3(T562N,T564A), bold line]. (C) Wild-type and mutant IIb3 were surface-labeled with biotin, and immunoprecipitation was performed with AP2. Immnoprecipitates were electrophoresed on 6% polyacrylamide gel under reducing conditions. After transfer, membrane was incubated with peroxidase-conjugated avidin and developed with chemiluminescence. To determine the mechanism of activation by the T562N mutation, we introduced T562A and T562Q mutations into 3. 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 IIb3(T562A) or IIb3(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 3 to disrupt the aberrant consensus sequence of N-glycosylation at residue 562. Immunoprecipitation of this form of 3 showed that 3(T564A) and 3(T562N,T564A) migrated on SDS gels similarly to the migration of 3(WT) (Fig 6C). Because the T564A mutation led to a mild reduction in the expression of IIb3, we monitored IIb3 expression by a non-function blocking anti-3 antibody, AP3, and analyzed only cells expressing high levels of IIb3 for PAC1 binding. Although IIb3(T564A) showed a slight increase in the activation index (0.28 ± 0.03; n = 3), the activation of IIb3(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 IIb3 in AM-1 cells. A lack of relationship between the aberrant N-glycosylation of 3(T562N) and IIb3 activation 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 IIb3 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 IIb3 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). The T562N mutation also leads to activation of V3.   To determine whether the activating mutation in 3 would affect the affinity state of the related integrin V3, this integrin was transiently expressed in 293 cells. In this case, FITC-labeled fibrinogen was used to determine the activation state of V3 and a non-function blocking anti-V antibody, LM142, was used to monitor V3 expression. When V was transfected with wild-type 3, fibrinogen bound only if integrin affinity was upregulated by the addition of manganese. In contrast, fibrinogen binding to cells expressing V3(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 IIb3 and V3. View larger version (29K): [in this window] [in a new window]   Fig 7. Soluble fibrinogen binding to V3 and V3(T562N) transfected cells. Cells were preincubated with 1 mmol/L RGDW, 1 mmol/L manganese, or buffer with 10 µg/mL of anti-V antibody, LM142. After 30 minutes of incubation, cells were washed and then incubated with 150 µg/mL FITC-conjugated fibrinogen and PE-conjugated antimouse IgG for 30 minutes and analyzed by flow cytometry. (A) Dot blots represent FITC-fibrinogen (horizontal) and LM142 (vertical) binding in the absence of RGDW and manganese. (B) Fibrinogen binding to cells expressing high levels of V3 (denoted by the rectangle in the dot blots) was analyzed on the histograms. (a through c) Wild-type V3 transfected cells; (d through f) V3(T562N) transfected cells. (a and d) With RGDW; (b and e) with buffer; (c and f) with manganese. Tyrosine phosphorylation of pp125FAK in AM-1 cells.   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 pp125FAK in AM-1 cells was studied. pp125FAK 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 pp125FAK phosphorylation when adherent to plates coated with poly-L-lysine. On the other hand, both showed pp125FAK 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 pp125FAK; nonetheless, this mutant receptor is fully capable of mediating this outside-in signaling response upon cell adhesion. View larger version (33K): [in this window] [in a new window]   Fig 8. pp125FAK phosphorylation. (A) 24/12 cells (lanes 1 through 3) or AM-1 cells (lanes 4 through 6) were maintained in suspension for 15 minutes with no addition (S; lanes 1 and 4), with the addition of 250 µg/mL of fibrinogen (S, Fg; lanes 2 and 5), and with addition of fibrinogen and 10 µg/mL of Fab fragments of PT25-2 (S, Fg+PT; lanes 3 and 6). (B) 24/12 cells (lanes 1 through 3) or AM-1 cells (lanes 4 through 6) were allowed to become adherent to immobilized fibrinogen (Fg; lanes 3 and 6) or poly-L lysine (PL; lanes 2 and 5) or were maintained in suspension (S; lanes 1 and 4) for 30 minutes. The cells were lysed and pp125FAK was immunoprecipitated with an anti-FAK polyclonal antibody. Phosphotyrosine was detected with 4G10 (upper panel), and blots were reprobed with anti-FAK to assess gel loading (lower panel).     DISCUSSION TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES In this report, we analyzed a cell line in which the ligand-binding function of integrin IIb3 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 3, 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 V3 as well as IIb3. (5) Activation of IIb3 by T562N or binding of soluble fibrinogen to the mutant is not sufficient to trigger tyrosine phosphorylation of pp125FAK. 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 IIb3-specific antagonists, (B) it was abrogated by a mutation in 3(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 IIb3 have been described previously. (1) Bajt et al41 showed that swapping the ligand-binding site (residues 129-133) of 3 with the corresponding sequence in 1 led to a gain of function in IIb3. (2) The proximal regions of cytoplasmic tails of IIb and 3 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 3 resulted in an increase in affinity of IIb3. In this context, it has been shown that mild reducing agents, such as dithiothreitol, can increase the ligand-binding function of IIb3.43 In addition, it has been shown that some LIBS antibodies that bind to epitopes within the cysteine-rich repeats of 3, such as LIBS2, LIBS3, and LIBS6, lead to activation of IIb3 without ligand-binding.20-22 Wippler et al44 also demonstrated that recombinant IIb3 lacking the cysteine-rich repeats of 3 showed high-affinity binding to fibrinogen. Furthermore, sequence alignment of 3 integrins indicate that about 90% of noncysteine residues in the cysteine-rich repeats are conserved between rodents and human 3, whereas noncysteine residues in the region are poorly conserved among 1, 2, and 3 integrins (~15%).45 These results suggest that the cysteine-rich repeats may have an important role for regulation of 3 integrin functions. Our results provide the direct evidence that this region is involved in the activation of 3 integrins. Enhanced ligand binding to IIb3(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 IIb3, 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 V3 indicates that activation of IIb3 by the mutation did not require a unique interaction of 3 with the 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 IIb3, because (1) IIb3(T562N, T564A), which lacked the aberrant glycosylation observed with the single N562 mutation, showed an even more activated state than IIb3(T564A); and (2) tunicamycin, an inhibitor of N-glycosylation,38 had no effect on the activated state of IIb3(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 3(T562N) mutation will require a level of understanding of integrin atomic structure that is not yet available. However, the predominant effect of the 3(T562N) mutation may be on 3 integrin conformation. First, some LIBS epitopes are constitutively exposed on IIb3 in AM-1 cells. Second, although affinity and avidity modulation both influence the functions of IIb3, affinity modulation is the predominant regulator of ligand binding.47 Third, soluble fibrinogen binding to platelet or CHO cell IIb3 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 3(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,2 3-LIBS antibody,50 and purification of V3 by affinity chromatography51 can induce a high-affinity state of V3. However, the T562N mutation is the first one reported to induce spontaneous activation of V3. A number of reports indicate that V3 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 V3. In any case, the current study serves to emphasize the possible involvement of residues in the cysteine-rich region of 3 in affinity modulation of both IIb3 and V3. This will have to be taken into account in future refinements of models for integrin activation.52     ACKNOWLEDGMENT 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.     FOOTNOTES Submitted September 14, 1998; accepted December 9, 1998. Supported in part by grants from the Ministry of Education, Science and Culture of Japan; the Japan Society for the Promotion of Science; Research Foundation for Cancer and Cardiovascular Diseases, Osaka, Japan; and the National Institutes of Health (Bethesda, MD). 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