A Membrane-distal Segment of the Integrin alpha IIb Cytoplasmic Domain Regulates Integrin Activation*

Mark H. GinsbergDagger §, Brian YaspanDagger , Jane ForsythDagger , Tobias S. Ulmer||, Iain D. Campbell**, and Marina SlepakDagger

From the Dagger  Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 and the  Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom

Received for publication, March 2, 2001, and in revised form, April 11, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. We have constructed and validated recombinant structural mimics of the heterodimeric alpha IIbbeta 3 cytoplasmic domain. The mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) formed in mixtures of the alpha IIb and beta 3 cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact alpha IIbbeta 3, indicating that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is also formed by introducing the activation-blocking beta 3(Y747A) mutation into the beta 3 tail. A membrane-distal heptapeptide sequence in the alpha IIb tail (997RPPLEED) is responsible for this effect on beta 3. Membrane-permeant palmitoylated peptides, containing this alpha IIb sequence, specifically blocked alpha IIbbeta 3 activation in platelets. Thus, this region of the alpha IIb tail causes the beta 3 tail to resemble that of beta 3(Y747A) and suppresses activation of the integrin.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The integrin family of adhesion receptors is essential for the development and functioning of multicellular animals (1). Integrin-mediated adhesion is rapidly and precisely regulated, a process that is often central to integrin functions (2). One important regulatory mechanism is cellular modulation of integrin affinity for ligand (activation). These affinity changes arise from both changes in the conformation of the extracellular domain and lateral clustering of integrins in the plane of the membrane (3). Integrin activation has been widely documented among members of this receptor family and controls cell adhesion, migration, and the assembly of the extracellular matrix (4, 5). Thus, integrin activation plays an important role in mediating integrin functions.

Integrins are noncovalent heterodimers of type I transmembrane protein subunits termed alpha  and beta . Each subunit has a large (>700 residues) N-terminal extracellular domain, a single membrane-spanning domain, and a generally short (13-70 residues) cytoplasmic domain (5). In general, integrin alpha  and beta  subunits contain a remarkably conserved 7-10-residue motif near the junction of the transmembrane and cytoplasmic domains (membrane-proximal segment) (6). The remainders of the cytoplasmic domain sequences are generally less well conserved. Integrin beta  cytoplasmic domains are required for activation because mutations or truncations of specific membrane-distal sequences in beta  cytoplasmic tails can disrupt activation (7-10). A particularly sensitive site is a highly conserved NPX(Y/F) motif in the beta  cytoplasmic domain where substitution of the Tyr with Ala (e.g. beta 3(Y747A)) blocks activation (11). This region of the beta  tail is important for binding to cytoskeletal proteins, such as talin because a Tyr right-arrow Ala substitution in the NPX(Y/F) motif also disrupts talin binding to beta  tails in vitro (12, 13). Moreover, overexpression of an integrin-binding fragment of talin in cells activates integrin alpha IIbbeta 3 (14). Thus, interactions of membrane distal portions of the beta  cytoplasmic domain with proteins such as talin appear to be important in integrin activation.

In addition to a role for the membrane-distal portion of the beta  cytoplasmic domain, interactions between integrin alpha  and beta  subunit cytoplasmic tails may regulate activation. Deletion of the membrane-proximal region of either alpha  or beta  tail activates integrins (7, 10, 15-17). In addition, specific point mutations in this segment of both the alpha  and beta  subunits promote constitutive bidirectional signaling in integrin alpha IIbbeta 3 (18) and other integrins (19). Complementary mutations in the alpha  and beta  subunits suggest that these activating mutations disrupt an interaction between the highly conserved membrane-proximal portions of the alpha  and beta  cytoplasmic tails (18). In vitro integrin alpha  and beta  cytoplasmic domain interactions have been reported by surface plasmon resonance analysis (20, 21) and by alterations in circular dichroism and intrinsic fluorescence (22). Furthermore, replacement of the alpha  and beta  cytoplasmic domains with acidic and basic peptides that form an alpha -helical coiled-coil caused inactivation of integrin alpha Lbeta 2 (23). In contrast, replacement of these cytoplasmic domains with two basic peptides that do not form an alpha -helical coiled-coil activated alpha Lbeta 2. Thus, there is evidence to suggest that an interaction between integrin alpha  and beta  tails regulates activation.

We previously used a synthetic strategy to produce a model of the cytoplasmic domain of integrin alpha IIbbeta 3 (24). The integrin cytoplasmic domains are tethered at their N termini to membrane spanning presumptive alpha -helices. Moreover, they are laterally constrained because the subunits interact with each other. More importantly, they have vertical constraints, because they are initially parallel to each other and are in a specific vertical stagger as they exit the membrane. The model protein was made by the covalent ligation of two synthetic polypeptides (called "minisubunits") consisting of an integrin cytoplasmic tail at the C terminus, connected to a 28-residue stretch comprised of four heptad repeats derived from tropomyosin. The parallel orientation and stagger of the two tails is defined by the formation of the covalent linkage and by a noncovalent helical coiled-coil structure between heptad repeats on each minisubunit. We now describe a completely recombinant model of the alpha IIbbeta 3 cytoplasmic domain. By using recombinant proteins, we avoided limitations of polypeptide length and modest yield encountered in the initial synthetic approaches. Moreover, we modified the design of the model protein to contain four heptad repeats derived from the GCN4 transcription factor in place of those derived from tropomyosin. We used two variant sequences that preferentially heterodimerize (25) to obviate an inherent 50% loss in yield of heterodimer.

In the present work, we utilized these model proteins as a tool to probe the potential role of integrin alpha beta tail interaction in regulating activation. Antibodies raised against these model proteins revealed the presence of combinatorial epitopes formed by a mixture of the alpha IIb and beta 3 tails but not present in either isolated tail. These combinatorial epitopes were present in intact integrin alpha IIbbeta 3 isolated from platelets, suggesting that the alpha IIb and beta 3 tail can interact in the intact receptor. Moreover, the same set of epitopes was present in the beta 3 tail bearing a Tyr747 right-arrow Ala mutation, even in the absence of the alpha IIb tail. This result indicates that alpha IIb cytoplasmic domain causes the beta 3 tail to resemble the activation-defective beta 3(Y747A) mutant. Furthermore, we mapped the alpha IIb residues required for this interaction into a minimal heptapeptide sequence and found that palmitoylated peptides containing this sequence specifically blocked the agonist-induced activation of integrin alpha IIbbeta 3 in platelets. Thus, our data provide evidence that the alpha IIb and beta 3 tails can interact in the native integrin and indicate that the interaction opposes integrin activation by altering the structure of the beta 3 cytoplasmic domain.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Model Protein Synthesis and Peptide Production-- Polymerase chain reaction as described (12) was used to create a cDNA encoding the modified GCN4 heptad repeat protein sequences reported by John et al. (25). The cDNAs were ligated into a NdeI-HindIII-restricted modified pET15b vector (12) (Novagen). alpha IIb and beta 3 integrin cytoplasmic domain cDNAs were generated by polymerase chain reaction from appropriate cDNAs using forward oligonucleotides introducing a 5'-HindIII site and reverse oligonucleotide creating a 3'-BamHI site directly after the stop codon. Integrin cytoplasmic domains were joined to the helix as HindIII-BamHI fragments and verified by sequencing. Recombinant expression in BL21(DE3)pLysS cells (Novagen) and purification of the recombinant products was performed as described with an additional final purification step on a reverse phase HPLC1 column (Vydac) (12). Typical yields were ~10 mg/liter of bacterial culture. To form heterodimers, a mixture of 45 µM beta 3 and 90 µM alpha IIb cytoplasmic tail model proteins were denatured for 20 min at 100 °C in the presence of 10 mM dithiothreitol. The dithiothreitol was removed by gel filtration through a PD 10 column (Bio-Rad), and the mixture was then air-oxidized by stirring overnight. The disulfide-bonded products were purified by reverse phase C18 HPLC. The products were analyzed by electrospray ionization mass spectrometry using an API-III quadrupole spectrometer (Sciex). Typically we recovered ~30 mg of heterodimer/preparation. The identity of each model protein is specified by the nature of the GCN4 helix (Jun-like = J, Fos-like = F), the number of Gly spacers (G0-G4), and the identity of the integrin tail (alpha IIb and beta 3), e.g. JG3alpha IIb.

Synthetic peptides were prepared by the Scripps Microchemistry Core using a Gilson AMS 422 or ABI 430A Peptide Synthesizers. One beta 3 cytoplasmic domain peptide (beta 3(Ile719-Thr762)) was a generous gift of Drs. Ed Plow and Tom Haas (Cleveland Clinic). The peptides were routinely >90% homogeneous as judged by reverse phase HPLC using an analytical Vydac C-18 column. Their mass varied by less than 0.1% from that predicted from their sequence as judged by electrospray ionization mass spectroscopy. Peptides were desalted prior to use, and peptide concentrations were verified by quantitative amino acid analysis.

Antibodies-- Antibodies to the alpha IIbbeta 3 cytoplasmic domain heterodimer model protein (see Fig. 1) (anti-alpha IIbbeta 3-Cyt) were raised in 2.5-kg New Zealand White rabbits. Antibodies were also produced against the beta 3(Y747A) model protein (anti-beta 3(Y747A)). 100 µl of a water solution containing 1 mg/ml of the model protein was emulsified in 1 ml of incomplete Freund's adjuvant and administered subcutaneously to the rabbits. Two additional injections at 2-week intervals were followed by bleeding (~50mls) at monthly intervals until the rabbits were sacrificed. The blood was permitted to clot at room temperature, and the serum was recovered by centrifugation. Serum was heat-inactivated at 56 °C for 30 min and stored at -20 °C. A preimmune serum, obtained prior to immunization, was used as a control. Antibodies against a synthetic peptide containing the last 20 residues of the beta 3 cytoplasmic domain (anti-beta 3-Cyt, rabbit 8275; Ref. 15) and the extracellular domain of alpha IIbbeta 3 (D57; Ref. 26) have been described.

Enzyme-linked Immunosorbent Assays-- alpha IIbbeta 3 was purified by gel filtration as described previously (26) with omission of the heparin and Con A affinity chromatography steps. The final product was greater than 95% homogenous as judged by SDS-PAGE. In the enzyme-linked immunosorbent assay (ELISA) the alpha IIbbeta 3 was used at a concentration of 5 µg/ml in a coating buffer containing 0.1 M NaHCO3 and 0.05% NaN3. 50 µl/well was used to coat Immulon II microtiter wells at 4 °C overnight. After removal of the coating solution, 150 µl of blocking buffer (coating buffer containing 5% bovine serum albumin) was added. After an additional 1-h incubation at 4 °C, the blocking buffer was removed, and the plates were washed three times with wash buffer (0.01 M Tris, 0.15 M NaCl, 0.01% thimerisol, 0.05% Tween 20, pH 8.0). Preliminary titration experiments established a dilution of anti-model protein antibody resulting in 75% maximal binding. 25 µl of the competitor was added to each well followed by 25 µl of this dilution of the anti-model protein antibody. Following mixing, the plate was covered for 1 h at 37 °C and washed four times with wash buffer. To quantify bound antibody, 50 µl of horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) was diluted to a concentration of 1:1000 in wash buffer containing 1 mg/ml bovine serum albumin. These plates were then incubated at 37 °C for 1.5 h. After four washes, bound antibody was assayed by measuring peroxidase activity with O-phenylenediamine as a substrate and quantifying reaction product by its optical density at 490 nm. The data were expressed as B/B0 where B = A490 in the presence of competitor and B0 = A490 in its absence. In some experiments, varying concentrations of alpha IIb peptides were added to a fixed, saturating quantity of the beta 3 model protein (10-50 nM). Competition was again expressed as B/B0; however, B0 was A490 in the presence of the beta 3 protein and no added alpha IIb peptide. EC50 was defined as the dose of alpha IIb resulting in B/B0 = 0.5.

Immunoprecipitations-- The generation of Chinese hamster ovary cells expressing recombinant integrins alpha IIbbeta 3 and alpha IIbDelta 996beta 3Delta 717 has been described (10). Chinese hamster ovary cell lines were cell surface-labeled with sulfo-NHS-Biotin (Pierce) following the manufacturer's instructions. Cells were lysed on ice for 30 min in an immunoprecipitation buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM benzamidine HCl, 0.02% sodium azide, 1% Triton X-100, 0.05% Tween 20, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). After clarification by centrifuging at 12,000 rpm for 20 min at 4 °C, cell lysate was then incubated with protein G-Sepharose (Amersham Pharmacia Biotech) coated with antibody overnight at 4 °C. The beads were washed with the immunoprecipitation buffer four times, and the precipitated polypeptides were extracted with SDS sample buffer. Precipitated cell surface biotin-labeled polypeptides were separated by SDS-PAGE under nonreducing conditions and detected with streptavidin-peroxidase followed by ECL (Amersham Pharmacia Biotech).

NMR Spectroscopy-- The beta 3 minisubunit of the alpha IIbbeta 3 heterodimer model protein was biosynthetically labeled with 15N as described.2 NMR signal intensities were taken from a two-dimensional 15N-1H heteronuclear single-quantum coherence (28, 29) spectrum of 1.2 mM heterodimer model protein in 10 mM acetic acid-d3, pH 4.5, 37 °C on a home-built spectrometer operating at a 1H frequency of 750 MHz. Backbone assignment has been described previously, and 3JHNalpha coupling constants for the tails have been reported previously2 and were determined now for the coiled-coil region from the same data set as described previously.

Fibrinogen Binding Assays-- Fibrinogen binding to platelets was performed as described previously (30). Briefly, 100 nM 125I-labeled fibrinogen and the indicated concentration of peptide inhibitor were preincubated for 30 min at 22 °C with a suspension of washed platelets (2 × 108/ml) in Tyrode's solution containing added 10 mM Hepes, 2 mg/ml bovine serum albumin, 2 mg/ml glucose, and 2 mM CaCl2. 20 µM ADP and 20 µM epinephrine were added to this suspension, and the resulting mixture was incubated for an additional 30 min. Bound 125I-labeled fibrinogen was separated from free by centrifugation through a 20% sucrose cushion and was quantified by gamma  scintillation spectrometry.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Construction and Characterization of Recombinant Heterodimer Integrin Cytoplasmic Domain Model Proteins-- To test potential interactions between the alpha IIb and beta 3 cytoplasmic tails, we first produced model proteins designed to mimic their normal arrangement in integrin alpha IIbbeta 3. Recombinant alpha -helical coiled-coils were previously used to model dimerized integrin beta  cytoplasmic domains (12); this strategy was employed to produce heterodimeric integrin cytoplasmic domains. Heptad repeats of identical length derived from GCN4 were used to form the nearly symmetrical, parallel coiled-coil. To favor heterodimer formation, Lys residues were placed in the in the g1 and e2 positions of the first and second heptad repeats to make a "Jun-like" helix (25). A complementary "Fos-like" helix was prepared by introducing Glu residues in these positions. These complementary helices produce preferentially heterodimerizing parallel coiled-coils (25). We also introduced a unique Cys residue N-terminal of each helix so that the formation of a cystine bridge would ensure parallel orientation and correct stagger of the coiled-coil. (Fig. 1A).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Structure and properties of alpha IIbbeta 3 cytoplasmic domain model protein. A, strategy for cytoplasmic domain mimics. Depicted is the amino acid sequence of the recombinant minisubunit. An N-terminal polyhisitidine (His-Tag) is used to immobilize and purify the construct. A thrombin cleavage site permits release of the His tag, and a unique cysteine is used to form covalent parallel dimers in a defined vertical stagger. Heptad repeats, from GCN4, nucleate the formation of a parallel dimeric coiled-coil. The presence of Lys residues at the g1 and e2 positions of the heptad repeats (bold type) render the helix Jun-like. Glu substitutions at this position result in a Fos-like helix that preferentially forms heterodimers with the Jun-like helix (25). Cytoplasmic domains, such as that of integrin alpha IIb depicted here, were joined to the coiled-coil. Three Gly residues were inserted between the coiled-coil and the integrin tail to disrupt induced helical structure in the cytoplasmic domain (12). At the bottom of the panel is a list of the integrin-specific sequences of all recombinant constructs used in this study. All integrin peptides correspond to published human integrin sequences in Swissprot. To preserve a HindIII site, a Val residue in the cytoplasmic domain of the human integrin alpha IIb chain was replaced by Leu. The membrane-promixal conserved segment of the alpha IIb and beta 3 cytoplasmic domains are underlined and in bold type. B, schematic of the JG3alpha IIb-FG3beta 3 model protein. The heterodimeric model protein was formed from a JG3alpha IIb and FG3beta 3 minisubunit. The hexahistidine tag was removed from the alpha IIb subunit to facilitate its separation from the heterodimer during purification. C, ion spray mass spectrum of an alpha IIbbeta 3 model protein. The predicted mass based on the expected covalent structure was 19008.2 Da (assuming no 13C) and the measured mass was 19013 + 3.1 Da. D, SDS-PAGE analysis of JG3alpha IIb-FG3beta 3 model protein. Purified JG3alpha IIb-FG3beta 3 was separated on a 4-20% SDS-PAGE in the presence (R) or absence (NR) of 10 mM dithiothreitol. The positions of the JG3alpha IIb (J-alpha IIb) and FG3beta 3 (F-beta 3) minisubunits are indicated. E, verification of the coiled-coil. NMR signal intensities from a two-dimensional 15N-1H heteronuclear single-quantum coherence spectrum at 37 °C and 750 MHz of 1.2 mM JG3alpha IIb-FG3beta 3 in which the FG3beta 3 minisubunit is 15N-labeled. The heterodimeric JG3alpha IIb-FG3beta 3 was dissolved in 10 mM acetic acid-d3, pH 4.5. The GCN4 coiled-coil element exhibits uniform intensity, whereas the beta 3 tail exhibits more intense signals with intensities increasing toward the C terminus. This behavior and the low 3JHNalpha coupling constants of the coiled-coil (<5.5 Hz) are indicative of the coiled-coil being well folded in a helical conformation.

A dimer was formed in which the Jun-like helix was fused to the alpha IIb cytoplasmic domain and a Fos-like helix was fused to the beta 3 cytoplasmic domain (Fig. 1B) (JG3alpha IIb-FG3beta 3) model protein. A three-Gly spacer was inserted between the helices and the integrin alpha  or beta  cytoplasmic domains to prevent the propagation of helical structure into the integrin tails (12).2 The mass of the heterodimer differed by less than 0.1% from that predicted for the desired covalent structure (Fig. 1C), and SDS-PAGE confirmed that disulfide bonds covalently linked the heterodimers (Fig. 1D). Furthermore, NMR analysis confirmed formation of the symmetrical, parallel coiled-coil domain by the heptad repeats and that the helical structure of this domain did not propagate into the beta 3 tail as will be discussed in full detail elsewhere.2 In particular, the coiled-coil behaved as a rigid, folded unit, whereas the motions of the beta 3 tail residues appear largely uncoupled from each other as judged from NMR signal intensities of the FG3beta 3 subunit of JG3alpha IIb-FG3beta 3 (Fig. 1E). Because of the symmetry of the coiled-coil all properties of the Fos-like heptad-repeats can be inferred to be present the Jun-like heptad repeats. The value of the 3JHNalpha coupling constant reflects the backbone conformation (ranging from helical to extended conformations) at the residue in question (31). For the Fos-like heptad repeats, values below 5.5 Hz show distinctly helical (and folded) conformations. Thus, the recombinant JG3alpha IIb-FG3beta 3 model protein had the expected properties.

Immunochemical Characterization of the JG3alpha IIb-FG3beta 3 Protein-- As noted above, there is substantial mutational and in vitro data to suggest that integrin alpha IIb and beta 3 cytoplasmic domains interact with each other. An immunochemical approach was used to determine whether such interactions could occur within the intact receptor. Antibodies were raised against JG3alpha IIb-FG3beta 3 protein (anti-alpha IIbbeta 3-Cyt). As expected, those antibodies reacted with native alpha IIbbeta 3 as judged by immunoprecipitation of the intact receptor. Furthermore, the capacity of these antibodies to immunoprecipitate the receptor depended on the presence of the cytoplasmic domains, because deletion of both cytoplasmic domains abolished reactivity (Fig. 2A).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Immunochemical analysis of the anti-alpha IIbbeta 3 model protein antibody. A, the antibody reacts with the cytoplasmic domains of native integrin alpha IIbbeta 3. Surface biotinylated Chinese hamster ovary cells expressing recombinant integrin alpha IIbbeta 3 (alpha IIbbeta 3) or alpha IIbbeta 3 lacking its cytoplasmic domains (alpha IIb(Delta 996)beta 3(Delta 717)) were lysed in immunoprecipitation buffer. The lysates were immunoprecipitated with anti-model protein antibody (alpha IIbbeta 3-Cyt), an antibody (rabbit 8275) directed against the cytoplasmic domain of beta 3 (beta 3-Cyt), or D57, an anti-alpha IIbbeta 3 monoclonal antibody (alpha IIbbeta 3). Lysates were also precipitated with normal rabbit serum (NRS). The immunoprecipitates were fractionated by SDS-PAGE, transferred to nitrocellulose filters, and developed with avidin peroxidase. B, rationale for immunochemical analysis. On the left are depicted linear integrin cytoplasmic tails, and on the right are depicted the tails in their assumed relationship in the intact receptor. The classes of epitopes expected are those present in the linear peptide and the intact receptor (1). If the cytoplasmic tails of the receptor interact, then additional classes of potential epitopes are those manifest in the linear peptide but lost in the native receptor (2) and combinatorial epitopes formed in the native receptor but absent from the linear peptides (3). Note that epitope classes 1 and 2 could be present on either the alpha  or beta  subunit cytoplasmic domain peptides. C, immunochemical analysis of the anti-model protein antibody. A competitive ELISA was used to measure the binding of the polyclonal anti-model protein antibody (anti-alpha IIbbeta 3-Cyt) to purified platelet alpha IIbbeta 3. The results are expressed as B/B0, where B = A490 in the presence of competitor and B0 = A490 in the absence of competitor. The data depict competition with full-length alpha IIb cytoplasmic domain peptide (alpha IIb), beta 3(Ile719-Thr762) cytoplasmic domain peptide (beta 3), or an equimolar mixture of the alpha IIb and beta 3 peptides (alpha IIb+beta 3). Depicted are the means of triplicate determinations. The curves represent best fits of the data to B/B0 = 1 - (E*C/(K + C)), where E is the fraction of antibodies that can bind the competitor, C is the concentration of the competitor, and K is the average apparent dissociation constant for the binding of the antibodies to the competitor. For the alpha IIb peptide E = 0.15 ± 0.01, for beta 3 E = 0.63 ± 0.007, and for alpha IIb + beta 3 E = 1.00 ± 0.01. D, immunochemical analysis of the anti-beta 3 cytoplasmic domain antibody. ELISA was used to measure binding of an antibody directed against a linear beta 3 cytoplasmic domain peptide (anti-beta 3-Cyt, rabbit 8275) to purified platelet alpha IIbbeta 3. The data depict competition with full-length alpha IIb cytoplasmic domain peptide (alpha IIb) or beta 3(Thr720-Thr762) cytoplasmic domain peptide (beta 3).

Having established that antibodies raised against JG3alpha IIb-FG3beta 3 reacted with the cytoplasmic domain of the native receptor, we sought to analyze potential interactions between the tails. We reasoned (Fig. 2B) that if the alpha  and beta  cytoplasmic domains interact with each other, then this interaction could create novel, combinatorial epitopes. To detect potential combinatorial epitopes, we assayed the effect of linear peptides comprising the alpha IIb or beta 3 cytoplasmic domains on the binding of anti-alpha IIbbeta 3-Cyt to integrin alpha IIbbeta 3 purified from human platelets. A full-length alpha IIb cytoplasmic domain peptide produced negligible inhibition (Fig. 2C). The beta 3 cytoplasmic domain peptide competed to a maximum of 63 ± 0.7%. Thus, antibodies reactive with intact integrin alpha IIbbeta 3 were resistant to competition by either of the linear peptides (Fig. 2C). These residual antibodies, however, could be competed by a mixture of the alpha IIb and beta 3 peptides (Fig. 2C). Consequently, these antibodies recognize epitopes expressed in the native alpha IIbbeta 3 and formed by the interaction of the alpha IIb and beta 3 cytoplasmic domains.

To exclude potential solubility artifacts, we examined the binding of an antibody against a linear beta 3 cytoplasmic domain peptide to the native alpha IIbbeta 3 integrin (Fig. 2D). In contrast to the result with anti-alpha IIbbeta 3-Cyt, the beta 3 peptide could completely inhibit antibody binding. Furthermore, an alpha IIb peptide completely inhibited the binding of an anti-alpha IIb cytoplasmic domain to alpha IIbbeta 3 (data not shown). These combinatorial antibodies were produced by only two of the four New Zealand White rabbits immunized with JG3alpha IIb-FG3beta 3. They may represent an unusual specificity because immunization of several strains of inbred mice (e.g. Balb C, C57Bl 6) failed to elicit such antibodies (data not shown). Thus, these immunochemical results indicate that the alpha IIb and beta 3 cytoplasmic domains can form a combinatorial epitope(s) in the intact integrin.

The Interaction of alpha IIb and beta 3 Cytoplasmic Domains Alters the Antigenicity of the beta 3 Cytoplasmic Domain-- In the presence of the alpha IIb tail there is reduced targeting of beta 3-containing integrins to cytoskeletal structures and a suppression of bidirectional signaling (15, 32, 33). Similar effects result from changing a Tyr in the first NPXY motif of beta 3 to an Ala (beta 3(Y747A)); Refs. 11, 34, and 35). These functional similarities prompted us to test the capacity of the beta 3(Y747A) mutant to compete for the set of antibodies recognizing combinatorial epitopes in the cytoplasmic domain of alpha IIbbeta 3. The beta 3(Y747A) mutant competed nearly completely for the binding of anti-alpha IIbbeta 3-Cyt to integrin alpha IIbbeta 3 (Fig. 3A). Furthermore, addition of the alpha IIb cytoplasmic domain peptide to the beta 3(Y747A) produced little increase in competition. In contrast, as noted previously, a population of antibodies was resistant to inhibition by the wild type beta 3 cytoplasmic domain. Thus, a point mutation in the beta 3 tail that disrupts its signaling function and its capacity to interact with the cytoskeleton causes the beta 3 tail to exhibit combinatorial epitopes formed in the presence of the alpha IIb tail.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   The alpha IIb cytoplasmic domain alters the antigenicity of the beta 3 cytoplasmic domain. A, beta 3(Y747A) binds combinatorial antibodies in anti-alpha IIbbeta 3-Cyt. A competitive ELISA was used to measure the binding of the polyclonal anti-model protein antibody (anti-alpha IIbbeta 3-Cyt) to purified platelet alpha IIbbeta 3, and the data were analyzed as described in the legend to Fig. 2. The graphs depict competition with the FG3beta 3 minisubunit (beta 3) or FG3beta 3(Y747A). Also depicted is competition with an equimolar mixture of the FG3beta 3(Y747A) and full-length alpha IIb cytoplasmic domain peptide (beta 3(Y747A)+alpha IIb). Depicted are the means of triplicate determinations. B, anti-beta 3(Y747A) recognizes combinatorial determinants formed by the interaction of alpha IIb and beta 3 tails. A competitive ELISA was used to measure the binding of the polyclonal antibody against beta 3(Y747A) to purified platelet alpha IIbbeta 3. The graphs depict competition with FG3beta 3 cytoplasmic domain (beta 3) or FG3beta 3(Y747A). Also depicted is competition with an equimolar mixture of the FG3beta 3 protein and full-length alpha IIb cytoplasmic domain synthetic peptide (alpha IIb+beta 3). Depicted are the means of triplicate determinations.

The foregoing result suggested that the alpha IIb cytoplasmic domain causes the beta 3 tail to resemble the beta 3(Y747A) mutant. To test this idea, we raised polyclonal antibodies against the FG3beta 3(Y747A) model protein and tested the reactivity of those antibodies with native alpha IIbbeta 3. The capacity of those antibodies to bind alpha IIbbeta 3 was completely inhibited by the immunogen, FG3beta 3(Y747A) (Fig. 3B). No significant increase in competition was observed in the presence of the alpha IIb cytoplasmic domain peptide (data not shown). In sharp contrast, the wild type FG3beta 3 failed to compete completely (Fib. 3B). The addition of an alpha IIb cytoplasmic domain peptide to FG3beta 3 resulted in complete competition. Thus, the presence of the alpha IIb cytoplasmic domain caused the antigenicity of the beta 3 cytoplasmic domain to resemble that of beta 3(Y747A).

A Heptapeptide Sequence in the alpha IIb Cytoplasmic Domain Is Responsible for Its Effect on the beta 3 Cytoplasmic Domain-- The foregoing studies suggested that the alpha IIb cytoplasmic domain could change the antigenicity of the beta 3 tail. We next sought to evaluate the specificity of the alpha IIb cytoplasmic domain effect. The addition of a synthetic peptide containing the alpha IIb cytoplasmic domain sequence to 10 nM FG3beta 3 protein resulted in dose-dependent formation of combinatorial epitopes (EC50 = 15 nM) (Fig. 4A). In sharp contrast, the cytoplasmic domains of integrin alpha 4 or alpha 5 had no such effect, even though they share a highly conserved N-terminal seven residues with that of the alpha IIb cytoplasmic domain (6). We next analyzed a series of nested deletion mutants from the N and C termini of alpha IIb in this assay. To increase sensitivity, these experiments were conducted in the presence of 50 nM FG3beta 3. Deletion of the C-terminal 4 amino acids had no effect on this interaction; however, deletion of 2 additional residues of alpha IIb completely abolished activity (Fig. 4B). A series of N-terminal deletions were performed. The first 7 amino acids were dispensable for this activity as expected from the results with the alpha 4 and alpha 5 cytoplasmic domains. However, deletion of 3 additional amino acids abolished activity (Fig. 4B). Consequently, progressively smaller peptides were produced and a minimal sequence spanning Arg997-Asp1003 had residual activity. Furthermore, double Ala substitutions in this sequence at Pro998 and Pro999 blocked activity. To further confirm our identification of this as the critical site, we synthesized a loop out peptide deleting both Arg997 and Pro998 (alpha IIb(Delta Lys994-Pro998)) and that loop out peptide was devoid of activity (Fig. 4B). Thus, the capacity of the alpha IIb cytoplasmic domain to alter the antigenicity of the beta 3 tail exhibits structural specificity and can be mapped to a minimal heptapeptide sequence.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Mapping the sites in the alpha IIb tail that interact with the beta 3 cytoplasmic domain. A, alpha IIb peptide dose response. A competitive ELISA was used to measure the binding of the polyclonal anti-model protein antibody (anti-alpha IIbbeta 3-Cyt) to purified platelet alpha IIbbeta 3 in the presence of 10 nM FG3beta 3 model protein. Varying quantities of full-length alpha IIb cytoplasmic domain peptide (alpha IIb(Lys989-Gln1008)) were added prior to the addition of the antibody. The results are expressed as B/B0, where B = A490 in the presence of alpha IIb peptide and B0 = A490 in the absence of alpha IIb peptide. The results are the means of triplicate determinations. Note that B/B0 for alpha 4 and alpha 5 peptides are superimposed. B, mapping the interactive site. A competitive ELISA was used to measure the binding of the polyclonal anti-JG3alpha IIb-FG3beta 3 (anti-alpha IIbbeta 3-Cyt) to purified platelet alpha IIbbeta 3 in the presence of 50 nM FG3beta 3 model protein and varying doses of the indicated synthetic alpha IIb peptides. Depicted in the column to the right are the concentrations of alpha IIb peptides that produced 50% maximal response (EC50). The sequence of full length (alpha IIb(Lys989-Gln1008)) is shown, and the boundaries of N- and C-terminally truncated alpha IIb peptides are depicted as bars to the left of the EC50. Note that the minimal active peptide was 997RPPLEED. Furthermore, Ala substitution of Pro998 and Pro999 and the loop out of Lys994-Pro998 in abolished activity are shown.

Cell Permeable Peptides Containing the beta 3-interactive Site of the alpha IIb Tail Block Activation of alpha IIbbeta 3-- The foregoing experiments identified a short peptide sequence of the alpha IIb tail that alters that of beta 3, mimicking the immunochemical effects of a Tyr to Ala substitution in the first NPXY motif. The beta 3(Y747A) mutation blocks the activation of integrin alpha IIbbeta 3 in vivo (11). Consequently, we tested the effects of addition of a peptide containing the alpha IIb sequence on activation of integrin alpha IIbbeta 3 in platelets. The peptide was palmitoylated, a modification that promotes entry of peptides into the platelet cytoplasm (36). Activation of alpha IIbbeta 3 was suppressed by a palmitoylated decapeptide (KRNRPPLEED) that contained the minimal heptapeptide sequence (Fig. 5A). Nearly 100% inhibition was observed at a peptide concentration of 100 µM. Inhibition was specific, because a scrambled peptide of the same composition was nearly devoid of activity. Furthermore, a nonpalmitoylated peptide containing the same sequence caused little inhibition (Fig. 5B). Thus, a palmitoylated alpha IIb peptide that contains the beta 3-interactive site inhibits activation of integrin alpha IIbbeta 3 in platelets.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5.   Palmitoylated alpha IIb peptides block activation of platelet integrin alpha IIbbeta 3. A, specific inhibition by beta 3 interacting peptide. A suspension of washed platelets (3 × 108/ml) was incubated at 22 °C in the presence of the indicated concentration of palmitoylated alpha IIb(Lys994-Asp1003) (P-KRNRPPLEED) or a palmitoylated scrambled peptide of the same composition (P-NPDKRLEREP) and 300 nM 125I-labeled fibrinogen. Fibrinogen binding was measured 15 min after addition of 10 µM ADP + 20 µM epinephrine, and the data are expressed as percentages of inhibition = 100*(B0 - B)/B0 where B is fibrinogen binding in the presence of inhibitor and B0 is fibrinogen binding in its absence. The average B0 value was 7320 molecules/platelet. Depicted are the means ± S.E. of three independent experiments. B, palmitoylation is required for inhibition. The capacity of palmitoylated alpha IIb(Lys994-Asp1003) (Palmitoylated) or alpha IIb(Lys994-Asp1003) (Unmodified) synthetic peptides to inhibit fibrinogen binding were assayed as described for A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. In the present work, we have constructed and validated recombinant structural mimics of the heterodimeric alpha IIbbeta 3 cytoplasmic domains. These mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) in the presence of a mixture of the alpha IIb and beta 3 cytoplasmic domains but not present in either isolated tail. The presence of this epitope(s) within intact alpha IIbbeta 3 indicates that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is present in the activation-defective beta 3(Y747A) mutant. Thus, the alpha IIb tail causes the beta 3 tail to resemble that of beta 3(Y747A), suggesting that the interaction of the alpha IIbbeta 3 tails opposes activation of the integrin. Furthermore, mapping of the site in the alpha IIb tail responsible for this activity identified a membrane-distal region previously implicated in regulation of integrin activation. Finally, palmitoylated peptides containing the beta 3-interactive alpha IIb sequence specifically blocked alpha IIbbeta 3 activation in platelets. Thus, these studies provide direct evidence for an interaction of the alpha IIb and beta 3 cytoplasmic domains that regulates activation of the integrin in platelets.

Antibodies raised against the alpha IIbbeta 3 model protein contained a population of antibodies that recognize a combinatorial epitope formed in the presence of the alpha IIb and beta 3 tails. This polyclonal antibody reacted with the cytoplasmic domain of native alpha IIbbeta 3 (Fig. 2). However, its binding to alpha IIbbeta 3 was only partially competed by either the isolated alpha IIb tail or the beta 3 tail. The remaining population of antibodies was inhibited only in the presence of both cytoplasmic tails. These experiments were conducted at 25-75% saturating antibody concentrations, and similar results were observed at each concentration. They were not due to insolubility of the tails, because the isolated beta 3 cytoplasmic domain or alpha IIb cytoplasmic domains could efficiently compete for the binding of antibodies specifically directed only against the isolated cytoplasmic domains. Furthermore, antibodies raised against the FG3beta 3(Y747A) protein, which had no reactivity with the alpha IIb tail, also contained antibodies against these combinatorial determinants. Thus, immunization with the JG3alpha IIb-FG3beta 3 model protein can elicit the formation of antibodies against combinatorial epitopes in the alpha IIbbeta 3 cytoplasmic domain.

The presence of combinatorial epitopes in intact alpha IIbbeta 3 suggests that the cytoplasmic domains can interact in the native receptor. The combinatorial antibodies react with native alpha IIbbeta 3 in a manner dependent on its cytoplasmic domains. However, the immunochemical assay requires the presence of the antibody to detect interactions between the alpha IIb and beta 3 tails. Indeed, in detailed NMR analysis, we found that the unpaired beta 3 tail exhibits essentially the same structural and dynamic properties as the alpha IIb-paired beta 3 tail within the context of the coiled-coil (JG3alpha IIb-FG3beta 3).2 Furthermore, constructs with different lengths of the glycine linker resulting in relative vertical tail-tail shifts of -2 to +2 residues did not uncover any such structural differences. Thus, it is possible that the antibody is a necessary co-factor that enhanced the interaction. Within cells, such co-factors, like the antibody, could modify the strength of the interaction. One such co-factor might be a plasma membrane. Indeed, Vinogradova et al. (37) reported that when the alpha IIb tail was myristoylated and inserted into a phospholipid bilayer, it formed a stable structure. This is in sharp contrast to the unstructured nature of the alpha IIb peptide in aqueous solution (22, 24).2 Furthermore, Leisner et al. (38) produced an anti-alpha IIb tail antibody (anti-LIBScyt1) that manifested reduced binding to intact integrin alpha IIbbeta 3. One interpretation of this result is that the availability of the epitope for this monoclonal antibody was influenced by interaction with the beta 3 tail. The alpha IIb sequence implicated here in forming the combinatorial epitope (RPPLEED) is contained in the region recognized by the anti-LIBScyt1 antibody (38). Taken together, these results suggest that interactions between the integrin alpha  and beta  cytoplasmic domains occur within cells.

The interaction of alpha IIb and beta 3 tails alter the antigenicity of the beta 3 tail. In particular, we found that introducing a mutation that blocks integrin activation into the beta 3 tail resulted in expression of the combinatorial epitope(s). The beta 3 tail is largely unfolded in aqueous solution; however, the beta 3 Tyr747 is part of an NPXY motif that has a propensity to form beta  turns. This propensity is abolished by the Y747A mutation.2 The Y747A mutation also abolishes talin binding. The region of talin that binds to integrin beta  tails contains a FERM domain (14, 39), a domain that contains a lobe structurally similar to PTB domains (40). NPXY motifs, such as that present at beta 3 Asn744-Tyr747, are often favored binding sites of PTB domains (41). Thus, binding of talin (or other FERM or PTB domain-containing proteins) may stabilize a reverse turn at the NPXY motif in integrin beta  tails leading to integrin activation. The presence of the alpha IIb tail may oppose formation of this stable turn, thus antagonizing integrin activation. The beta  turn propensity at Asn744-Tyr747 is preserved in the JG3alpha IIb-FG3beta 3 model protein.2 However, the beta  turn is only present in a small population of beta 3 molecules at any time. Consequently, the alpha IIb tail might oppose stabilization of the turn in the presence of talin but not appreciably reduce the propensity to form a turn in the absence of talin. Thus, the alpha IIb cytoplasmic domain alters the antigenicity of the beta 3 tail, causing it to resemble the structurally and functionally altered beta 3(Y747A) mutant.

When the beta 3-interactive region of the alpha IIb tail was palmitoylated, it suppressed activation of alpha IIbbeta 3 in platelets. Platelets are not readily susceptible to genetic manipulation; however, previous work from Fitzgerald's laboratory (36) has established that palmitoylated peptides enter platelets and can regulate activation of alpha IIbbeta 3. Utilizing their approach, we found that the identified beta 3 interactive region of alpha IIb, when palmitoylated, suppressed alpha IIbbeta 3 activation in platelets. Specificity was established by the markedly reduced activity in either a nonpalmitoylated peptide, presumably because the latter fails to enter platelets. In addition, a palmitoylated-scrambled peptide also exhibited greatly reduced activity. Because both the authentic and scrambled peptides bore the same lipid modification and were of identical composition, similar distributions within the platelet seem likely. However, caution is warranted, because definitive proof of a completely identical subcellular localization for these two peptides is technically challenging. Nevertheless, a potential explanation of these results is that the palmitoylated alpha IIb peptide interacted with the beta 3 tail within the cell interior, rendering it less capable of supporting platelet activation. Such a mechanism would also account for the observation that a full-length myristoylated alpha IIb peptide inhibited alpha IIbbeta 3 activation in platelets (37). In the same work, an alpha IIb tail peptide inhibited alpha IIbbeta 3 activation in vitro; however, it failed to inhibit the activation of a receptor lacking its beta 3 cytoplasmic domain. Additional evidence for the view that the alpha IIb tail suppresses activation by interacting with the beta 3 tail comes from the present finding that the alpha IIb(P998A,P999A) mutation blocked formation of the combinatorial epitope. This same mutation abolishes inhibition of activation by the myristoylated alpha IIb peptide (37) and activates intact recombinant alpha IIbbeta 3(38). Thus, taken together, these data strongly suggest that the interaction of this region of alpha IIb cytoplasmic domain with the beta 3 tail opposes activation of alpha IIbbeta 3 in platelets.

The results presented here and in previous literature suggest a unifying hypothesis for the activation of integrins. The receptor is proposed to be restrained in a resting state through an interaction involving the membrane-proximal region of the alpha  and beta  cytoplasmic domains (6, 10, 23). The removal of either of these regions results in a constitutive activated receptor, and that activation is independent of other interactions of the beta  cytoplasmic domains or cellular energy (7, 15, 42). Furthermore, introduction of the lipidated membrane-proximal sequence of the alpha IIb tail (KVGFFKR) into platelets results in alpha IIbbeta 3 activation, presumably by disrupting this membrane-proximal interaction (36). Thus, the membrane-proximal interaction forms a structural on-off switch for the receptor. However, normal activation mechanisms require the distal region of the beta 3 cytoplasmic domain (7, 9, 17, 27). An interaction of talin with the beta 3 cytoplasmic domain can activate the receptor, and this depends on membrane distal sequences (14). The present results indicate interactions between the membrane distal alpha IIb sequence and the beta 3 tail within the native receptor that block integrin activation. Disruption of this membrane distal interaction could account for energy and beta  tail-dependent activation caused by alterations in membrane-distal alpha  cytoplasmic domain sequences (7, 42). Furthermore, the interaction with the alpha IIb tail may oppose stabilization of the reverse turn at beta 3(Asn744-Tyr747) by cytoplasmic proteins such as talin. Thus, detailed analyses of the structural effects of interactions of proteins, such as talin, with integrin beta  cytoplasmic domains are likely be informative in testing this model of integrin activation. In addition, the identification of physiologic mechanisms that regulate the interactions of integrin tails with each other and with proteins such as talin will be central to the understanding of this important biological process.

    ACKNOWLEDGEMENTS

We acknowledge Drs. Ed Plow and Tom Haas for the generous gift of the beta 3(Ile719-Thr762) peptide. We also gratefully acknowledge Drs. Martin Schwartz and Sandy Shattil for critical reading of the manuscript.

    FOOTNOTES

* This is Publication 13951-VB from the Scripps Research Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by grants from the NHLBI and NIAMS, National Institutes of Health. To whom correspondence should be addressed: Dept. of Vascular Biology, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7124; Fax: 858-784-7343; E-mail: Ginsberg@scripps.edu.

|| Supported by the German Academic Exchange Service (Deutscher Akademischer Austauschdienst) and Biotechnology and Biological Sciences Research Council.

** Supported by the Wellcome Trust and, through the Oxford Center for Molecular Sciences, from the Medical Research Council, Biotechnology and Biological Sciences Research Council, and Engineering and Physical Sciences Research Council.

Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M101915200

2 T. S. Ulmer, B. Yaspan, M. H. Ginsberg, and I. D. Campbell, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
2. Ginsberg, M. H., Du, X., and Plow, E. F. (1992) Curr. Opin. Cell Biol. 4, 766-771[Medline] [Order article via Infotrieve]
3. Hato, T., Pampori, N., and Shattil, S. J. (1998) J. Cell Biol. 141, 1685-1695[Abstract/Free Full Text]
4. Hughes, P. E., and Pfaff, M. (1998) Trends Cell Biol. 8, 359-364[CrossRef][Medline] [Order article via Infotrieve]
5. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Annu. Rev. Cell Dev. Biol. 11, 549-599[CrossRef][Medline] [Order article via Infotrieve]
6. Williams, M. J., Hughes, P. E., O'Toole, T. E., and Ginsberg, M. H. (1994) Trends Cell Biol. 4, 109-112[CrossRef]
7. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R. N., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract]
8. Reszka, A. A., Hayashi, Y., and Horwitz, A. F. (1992) J. Cell Biol. 117, 1321-1330[Abstract]
9. Hibbs, M. L., Xu, H., Stacker, S. A., and Springer, T. A. (1991) Science 251, 1611-1613[Medline] [Order article via Infotrieve]
10. Hughes, P. E., O'Toole, T. E., Ylanne, J., Shattil, S. J., and Ginsberg, M. H. (1995) J. Biol. Chem. 270, 12411-12417[Abstract/Free Full Text]
11. O'Toole, T. E., Ylanne, J., and Culley, B. M. (1995) J. Biol. Chem. 270, 8553-8558[Abstract/Free Full Text]
12. Pfaff, M., Liu, S., Erle, D. J., and Ginsberg, M. H. (1998) J. Biol. Chem. 273, 6104-6109[Abstract/Free Full Text]
13. Kaapa, A., Peter, K., and Ylanne, J. (1999) Exp. Cell Res. 250, 524-534[CrossRef][Medline] [Order article via Infotrieve]
14. Calderwood, D. A., Zent, R., Grant, R., Rees, D. J. G., Hynes, R. O., and Ginsberg, M. H. (1999) J. Biol. Chem. 274, 28071-28074[Abstract/Free Full Text]
15. O'Toole, T. E., Mandelman, D., Forsyth, J., Shattil, S. J., Plow, E. F., and Ginsberg, M. H. (1991) Science 254, 845-847[Medline] [Order article via Infotrieve]
16. Rabb, H., Michishita, M., Sharma, C. P., Brown, D., and Arnaout, M. A. (1993) J. Immunol. 151, 990-1002[Abstract/Free Full Text]
17. Crowe, D. T., Chiu, H., Fong, S., and Weissman, I. L. (1994) J. Biol. Chem. 269, 14411-14418[Abstract/Free Full Text]
18. Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu, C., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571-6574[Abstract/Free Full Text]
19. Lu, C. F., and Springer, T. A. (1997) J. Immunol. 159, 268-278[Abstract]
20. Vallar, L., Melchior, C., Plancon, S., Drobecq, H., Lippens, G., Regnault, V., and Kieffer, N. (1999) J. Biol. Chem. 274, 17257-17266[Abstract/Free Full Text]
21. Laplantine, E., Vallar, L., Mann, K., Kieffer, N., and Aumailley, M. (2000) J. Cell Sci. 113, 1167-1176[Abstract/Free Full Text]
22. Haas, T. A., and Plow, E. F. (1996) J. Biol. Chem. 271, 6017-6026[Abstract/Free Full Text]
23. Lu, C., Takagi, J., and Springer, T. A. (2001) J. Biol. Chem. 276, 14642-14648[Abstract/Free Full Text]
24. Muir, T. W., Williams, M. J., Ginsberg, M. H., and Kent, S. B. H. (1994) Biochemistry 33, 7701-7708[Medline] [Order article via Infotrieve]
25. John, M., Briand, J.-P., Granger-Schnarr, M., and Schnarr, M. (1994) J. Biol. Chem. 269, 16247-16253[Abstract/Free Full Text]
26. Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C., and Ginsberg, M. H. (1991) Cell 65, 409-416[Medline] [Order article via Infotrieve]
27. Wang, R., Shattil, S. J., Ambruso, D. R., and Newman, P. J. (1997) J. Clin. Invest. 100, 2393-2403[Abstract/Free Full Text]
28. Boyd, J., Soffe, N., John, B., Plant, D., and Hurd, R. (1992) J. Magn. Reson. 98, 660-664
29. Grzesiek, S., and Bax, A. (1993) J. Am. Chem. Soc. 115, 12593-12594
30. Plow, E. F., Srouji, A. H., Meyer, D., Marguerie, G. A., and Ginsberg, M. H. (1984) J. Biol. Chem. 259, 5388-5391[Abstract/Free Full Text]
31. Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids , John Wiley & Sons, New York
32. Ylanne, J., Chen, Y.-P., O'Toole, T. E., Loftus, J. C., Takada, Y., and Ginsberg, M. H. (1993) J. Cell Biol. 122, 223-233[Abstract]
33. Leong, L., Hughes, P. E., Schwartz, M. A., Ginsberg, M. H., and Shattil, S. J. (1995) J. Cell Sci. 108, 3817-3825[Abstract/Free Full Text]
34. Ylanne, J., Huuskonen, J., O'Toole, T. E., Ginsberg, M. H., Virtanen, I., and Gahmberg, C. G. (1995) J. Biol. Chem. 270, 9550-9557[Abstract/Free Full Text]
35. Tahiliani, P. D., Singh, L., Auer, K. L., and LaFlamme, S. E. (1997) J. Biol. Chem. 272, 7892-7898[Abstract/Free Full Text]
36. Stephens, G., O'Luanaigh, N., Reilly, D., Harriott, P., Walker, B., Fitzgerald, D., and Moran, N. (1998) J. Biol. Chem. 273, 20317-20322[Abstract/Free Full Text]
37. Vinogradova, O., Haas, T., Plow, E. F., and Qin, J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1450-1455[Abstract/Free Full Text]
38. Leisner, T. M., Wencel-Drake, J. D., Wang, W., and Lam, S. C. (1999) J. Biol. Chem. 274, 12945-12949[Abstract/Free Full Text]
39. Patil, S., Jedsadayanmata, A., Wencel-Drake, J. D., Wang, W., Knezevic, I., and Lam, S. C. (1999) J. Biol. Chem. 274, 28575-28583[Abstract/Free Full Text]
40. Pearson, M. A., Reczek, D., Bretscher, A., and Karplus, P. A. (2000) Cell 101, 259-270[Medline] [Order article via Infotrieve]
41. Margolis, B., Borg, J. P., Straight, S., and Meyer, D. (1999) Kidney Int. 56, 1230-1237[CrossRef][Medline] [Order article via Infotrieve]
42. Chen, Y.-P., O'Toole, T. E., Shipley, T., Forsyth, J., LaFlamme, S. E., Yamada, K. M., Shattil, S. J., and Ginsberg, M. H. (1994) J. Biol. Chem. 269, 18307-18310[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.