Bidirectional Transmembrane Modulation of Integrin alpha IIbbeta 3 Conformations*

Tina M. LeisnerDagger , June D. Wencel-DrakeDagger §, Wei WangDagger , and Stephen C.-T. LamDagger

From the Dagger  Department of Pharmacology and § School of Biomedical and Health Information Sciences, University of Illinois, Chicago, Illinois 60612

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Activation of blood platelets by physiological stimuli (e.g. thrombin, ADP) at sites of vascular injury induces inside-out signaling, resulting in a conformational change of the prototype integrin alpha IIbbeta 3 from an inactive to an active state competent to bind soluble fibrinogen. Furthermore, ligand occupancy of alpha IIbbeta 3 initiates outside-in signaling and additional conformational changes of the receptor, leading to the exposure of extracellular neoepitopes termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies. To date, the mechanism of bidirectional transmembrane signaling of alpha IIbbeta 3 has not been established. In this study, using our newly developed anti-LIBScyt1 monoclonal antibody, we showed that extracellular ligand binding to alpha IIbbeta 3 on blood platelets induces a transmembrane conformational change in alpha IIbbeta 3, thereby exposing the LIBScyt1 epitope in the alpha IIb cytoplasmic sequence between Lys994 and Asp1003. In addition, a point mutation at this site (P998A/P999A) renders alpha IIbbeta 3 constitutively active to bind extracellular ligands, resulting in fibrinogen-dependent cell-cell aggregation. Taken collectively, these results demonstrated that the extracellular ligand-binding site and a cytoplasmic LIBS epitope in integrin alpha IIbbeta 3 are conformationally and functionally coupled. Such bidirectional modulation of alpha IIbbeta 3 conformation across the cell membrane may play a key role in inside-out and outside-in signaling via this integrin.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Interaction of adhesive proteins with transmembrane integrin adhesion receptors is essential for diverse biological processes including embryogenesis, angiogenesis, immune response, and hemostasis (1, 2). It is generally agreed that inside-out signaling processes regulate the affinity state of integrins for binding extracellular ligands (2, 3). Thus, upon activation of blood platelets by physiological stimuli (e.g. thrombin or ADP) at sites of vascular injury, the prototype integrin alpha IIbbeta 3 undergoes a conformational change from an inactive to an active state competent to bind soluble fibrinogen (1-5). The cytoplasmic domain of alpha IIbbeta 3 appears to play a regulatory role in alpha IIbbeta 3 activation, since truncations of the entire cytoplasmic sequence of either the alpha IIb or beta 3 subunit, including their membrane-proximal regions, were found to increase the ligand binding affinity of the mutant receptor expressed on Chinese hamster ovary (CHO)1 cells (6, 7). More recently, a potential salt bridge hinge formed between the alpha IIb and beta 3 cytoplasmic sequences has been suggested to maintain alpha IIbbeta 3 in a default low affinity state; disruption of this structure may result in receptor activation (8).

It is well established that binding of adhesive ligands to integrins initiates outside-in signaling processes that mediate post-ligand binding events including cytoskeleton reorganization, receptor clustering, and gene transcription (2, 3, 9). Although the mechanisms regulating outside-in signaling of integrins remain elusive, the binding of cytoskeletal proteins and signaling molecules to the receptor's cytoplasmic domain as well as the receptor's conformational state have been implicated to play an important role in this process. In this regard, it has been suggested that ligand occupancy of the alpha 5beta 1 integrin may induce a transmembrane conformational change of the receptor, thereby unmasking specific regions in the receptor cytoplasmic domain mediating cytoskeletal attachment, which ultimately leads to receptor localization to focal contacts (10). However, to date, ligand-induced transmembrane conformational changes of an integrin receptor have not been demonstrated.

It has previously been shown that ligand binding to alpha IIbbeta 3 induces further conformational changes of the receptor extracellular domain, resulting in the exposure of neoantigenic sites termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies (mAbs) (11-16). Furthermore, certain anti-LIBS mAbs were found to activate alpha IIbbeta 3 to bind soluble fibrinogen (14, 15). In this study, we postulated that bidirectional conformational changes of alpha IIbbeta 3 transducing through the receptor's transmembrane segment occur as a result of cellular activation and ligand binding. To test this possibility, we examined whether extracellular ligand binding induces the exposure of LIBS epitope(s) in the cytoplasmic domain of alpha IIbbeta 3. In addition, we evaluated the functional role of a putative cytoplasmic LIBS epitope in regulating alpha IIbbeta 3 ligand binding affinity.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Peptides and Antibodies-- Peptides, represented as sequences of single letter amino acid codes (17), were synthesized by solid-phase synthesis using an ABI model 431 peptide synthesizer or were purchased from Research Genetics, Inc. (Huntsville, AL). The amino acid composition of each peptide was consistent with its desired sequence. The anti-alpha IIbbeta 3 antibodies PMI-1 (18), anti-V41 (19), anti-LIBS1 (13), and mAb 15 (13) were from Dr. M. H. Ginsberg, and AP-2 (20) was from Dr. T. J. Kunicki of the Scripps Research Institute (La Jolla, CA). Anti-alpha IIbC, an antipeptide polyclonal antibody raised in rabbits against the alpha IIb cytoplasmic sequence Phe992-Glu1008, was a generous gift of Dr. X. Du of the University of Illinois (Chicago, IL).

Production of Anti-LIBScyt1-- For the production of antipeptide mAbs against the alpha IIb cytoplasmic sequence, the full-length P2b peptide (CKVGFFKRNRPPLEEDDEEGE) was coupled to keyhole limpet hemocyanin using m-maleimidobenzoic acid N-hydroxysuccinimide ester and used as immunogen for BALB/c mice. Isolated splenocytes were fused with P3-X63Ag8.653 myeloma cells. Hybridomas were grown in selective media (hypoxanthine/aminopterin/thymidine), and their supernatants were tested in an ELISA for the presence of antibodies reactive with RGD affinity-purified alpha IIbbeta 3 (13, 21). A positive hybridoma 3F5, which secreted anti-alpha IIb antibodies belonging to the IgG1kappa subclass, was subcloned twice at limiting dilutions of 0.5 cell/well. The antibody was produced as ascites and purified by chromatography on protein A-Sepharose CL-4B (Amersham Pharmacia Biotech).

Immunoprecipitation-- Gel-filtered platelets were surface-labeled with Na125I and solubilized in lysis buffer containing 50 mM octyl glucoside (21). Cell lysates were incubated with GRGDSP, GRGESP, or vehicle buffer for 30 min at 37 °C. Antibodies were then added and incubated overnight at 4 °C. The immunoprecipitated proteins were collected on protein G-Sepharose, electrophoresed on SDS-7% polyacrylamide gels under nonreducing conditions, and analyzed by autoradiography.

Indirect Immunofluorescent Microscopy-- Washed human platelets resuspended in Tyrode's solution (2.5 × 108 cells/ml) were incubated with the indicated reagents (see legend of Fig. 2) at 37 °C for 30 min and subsequently fixed with 1% paraformaldehyde on ice for 1 h. After blocking unreacted aldehyde with Tris-buffered saline (30 mM Tris, 120 mM NaCl, pH 7.4) containing 0.5 M NH4Cl, cells were allowed to settle onto polylysine-coated glass coverslips and incubated with 0.2 mg/ml lysophosphatidylcholine (LPC) for 5 min to render them permeable. Permeabilized cells were rinsed with Tris-buffered saline containing 0.1% bovine serum albumin and incubated with anti-LIBScyt1 followed by rhodamine-conjugated goat anti-mouse IgG. Samples were mounted with a droplet of FITC guard, and platelets were viewed with a Jenaval phase/fluorescence microscope (Jenoptik Jena GmbH) and photographed with Eastman Kodak Tri-X panchromatic film (22).

Competitive ELISA-- Microtiter wells were coated with the full-length P2b peptide (5 µg/well) and blocked with 3% bovine serum albumin. Anti-LIBScyt1 was incubated with 10 µM inhibitory peptides at 37 °C for 30 min and added to the P2b-coated wells. Antibody binding to the adsorbed P2b proceeded at 37 °C for 1 h. The wells were washed, and bound antibody was detected with horseradish peroxidase-conjugated goat anti-mouse IgG using o-phenylenediamine as substrate (12). Absorbance at 490 nm (A490) was measured, and percentage inhibition was calculated relative to control without inhibitor.

Site-directed Mutagenesis-- The expression constructs encoding wild type alpha IIb (CD2b) and beta 3 (pc3A) have been previously described (23, 24). To generate the pc2b construct encoding wild type alpha IIb, a 3.3-kilobase fragment of alpha IIb containing the entire coding sequence and the 3'-untranslated region was excised from CD2b by digestion with XbaI and ligated into the expression vector pcDNA3. The resultant construct was designated as pc2b. Both pc2b and pc3A were kindly provided by Dr. J. C. Loftus at the Mayo Clinic (Scottsdale, AZ). The P998A/P999A mutation in alpha IIb was generated by splice overlap extension mutagenesis (25). Overlapping fragments containing this mutation were first made by polymerase chain reaction amplifications on pc2b using the following oligonucleotide pairs: (a) 5'-CACAAGCGGGATCGCAGACAGATCTTCCTGCCAGA-3' and (b) 5'-CTTCTTCCAGGGCTGCCCGGTTCCGCTTG-3'; (c) 5'-CAAGCGGAACCGGGCAGCCCTGGAAGAAG-3' and (d) 5'-GGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAA-3' as primers. The overlapping fragments were combined, denatured by heating at 94 °C for 5 min, and reannealed by cooling to 55 °C. The ends were filled in with Pfu, and the double-stranded fragments were then amplified by polymerase chain reaction using the oligonucleotide pair a and d. The amplified product was digested with BamHI and XbaI and reinserted into a BamHI-XbaI-digested pc2b vector fragment. The mutant construct was identified by automated DNA sequencing, purified by chromatography on QIAGEN Tip-100, and co-transfected with the wild type beta 3 construct (pc3A) into CHO-K1 cells (ATCC, Rockville, MD) by liposome-mediated transfection as described (7). Surface expression of mutated alpha IIbbeta 3 was analyzed by flow cytometry using FITC-conjugated AP-2. Stable cell lines were selected in medium containing 0.75 mg/ml G418 (Sigma), and single cell sorting was performed to obtain stable clonal lines, which were high expressors of the mutant alpha IIbbeta 3.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Ligand Binding Induces A Transmembrane Conformational Change of alpha IIbbeta 3-- To examine the possibility that ligand binding induces the exposure of LIBS epitopes in the cytoplasmic domain of alpha IIbbeta 3, we developed anti-peptide mAbs against the receptor's cytoplasmic sequences and screened for antibodies that preferentially bind to the ligand-occupied conformer of alpha IIbbeta 3. In the present study, we focused on the alpha IIb cytoplasmic tail. Initially, mAbs reactive with RGD affinity-purified alpha IIbbeta 3 in an ELISA system were further characterized by immunoblotting and immunoprecipitation studies. The mAb obtained from clone 3F5 recognizes a ligand-induced binding site in the cytoplasmic domain of alpha IIbbeta 3 (LIBScyt), and therefore this mAb is designated as anti-LIBScyt1. Fig. 1A shows that anti-LIBScyt1 specifically immunoblotted the 140-kDa nonreduced alpha IIb subunit in RGD affinity-purified alpha IIbbeta 3 (lane 1) and in a detergent extract of platelet proteins (lane 2). Upon reduction of purified alpha IIbbeta 3 and proteins in the platelet lysate, anti-LIBScyt1 immunoblotted the 27-kDa light chain of alpha IIb, which contains its cytoplasmic sequence (lanes 3 and 4). To determine whether the interaction of anti-LIBScyt1 with nondenatured alpha IIbbeta 3 is dependent on ligand occupancy, we performed immunoprecipitation experiments using lyates of surface-radioiodinated platelets in the presence and absence of an RGD peptide. As judged by densitometric scanning of the immunoprecipitated 125I-labeled protein bands, incubation of platelet lysates with GRGDSP caused a 7.2-fold increase in the amount of alpha IIbbeta 3 immunoprecipitated by anti-LIBScyt1 (Fig. 1B). In contrast, the variant GRGESP peptide was much less effective (1.5-fold). As controls, we used the well characterized anti-LIBS1 mAb (13), which demonstrated a similar effect in RGD-dependent immunoprecipitation of alpha IIbbeta 3. However, using the control mAb 15, whose binding to alpha IIbbeta 3 is not markedly influenced by ligand occupancy (13), we observed that GRGDSP incubation induced only a slight (1.5-fold) increase in the immunoprecipitation of alpha IIbbeta 3. Thus, these results suggest that interaction of GRGDSP with the extracellular ligand binding site of alpha IIbbeta 3 induces a conformational change in the receptor's cytoplasmic domain. In support of our finding that the cytoplasmic domain of alpha IIbbeta 3 can exist in different conformational states, the cytoplasmic sequences of alpha IIb and beta 3 have been shown to interact with each other, and at least two docking models with different tertiary structures of the alpha IIbbeta 3 cytoplasmic domain have been proposed (26-28).


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Fig. 1.   Immunoblot and immunoprecipitation characterization of anti-LIBScyt1. A, RGD affinity-purified alpha IIbbeta 3 (lanes 1 and 3) and octyl glucoside extracts of platelet proteins (lanes 2 and 4) were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and probed with anti-LIBScyt1 followed by detection with 125I-labeled goat anti-mouse IgG. Positions of molecular mass markers in kDa are indicated on the right. B, lysates of surface-radioiodinated platelets were incubated with vehicle buffer (lane 1), 1 mM GRGESP (lane 2), or 1 mM GRGDSP (lane 3) for 30 min at 37 °C. Immunoprecipitation with the indicated antibodies proceeded overnight at 4 °C. The immunoprecipitated proteins were electrophoresed on SDS-7% polyacrylamide gels under nonreducing conditions and analyzed by autoradiography.

To examine whether RGD occupancy also induces transmembrane conformational changes of alpha IIbbeta 3 in situ, we performed indirect immunofluorescence microscopy using whole platelets preincubated with or without GRGDSP followed by paraformaldehyde fixation and LPC permeabilization to allow antibody access. As shown in Fig. 2A, incubation of platelets with GRGDSP (panel a) resulted in significant intracellular staining of anti-LIBScyt1 as opposed to control platelets incubated with vehicle buffer (panel b) or GRGESP (panel c). Furthermore, the observed rim staining pattern with GRGDSP-treated permeabilized platelets is suggestive of anti-LIBScyt1 localization to the inner face of the plasma membrane, since minimal staining was observed with nonpermeabilized cells (not shown). To investigate whether binding of the physiological ligand fibrinogen to alpha IIbbeta 3 on activated platelets also induces the exposure of LIBScyt1, we performed indirect immunofluorescence studies with ADP-stimulated platelets in the presence and absence of exogenous fibrinogen. Again, anti-LIBScyt1 staining was performed following fixation and cell permeabilization. The addition of fibrinogen to ADP-stimulated platelets dramatically increased anti-LIBScyt1 staining as compared with activation of platelets with ADP alone (Fig. 2B, panels a and b). In control samples, resting platelets failed to stain for anti-LIBScyt1 in the presence and absence of fibrinogen (Fig. 2B, panels c and d). Therefore, these results demonstrate that anti-LIBScyt1 recognizes the ligand-occupied but not the activated unoccupied conformer of alpha IIbbeta 3. By immunogold staining with AP6, an anti-LIBS mAb directed against the beta 3 extracellular domain, Nurden et al. (16) previously reported that a pool of alpha IIbbeta 3 in the alpha -granules of unactivated platelets exists in the ligand-occupied state. However, using anti-LIBScyt1, we failed to detect immunofluorescent staining of alpha IIbbeta 3 in the alpha -granules of resting platelets. This may be due to the association of alpha IIbbeta 3 with cytoskeletal components that mediate internalization and transport of the fibrinogen-alpha IIbbeta 3 complex to the platelet alpha -granules (22, 29, 30), thus blocking interaction of anti-LIBScyt1 with the cytoplasmic domain of ligand-occupied alpha IIbbeta 3 in the alpha -granule membranes. Nonetheless, the observation that fibrinogen binding to alpha IIbbeta 3 on the platelet surface induces a transmembrane conformational change of the receptor provides a possible mechanism by which ligand occupancy of alpha IIbbeta 3 mediates a variety of post-ligand binding function of blood platelets including clot retraction, receptor internalization, and cytoskeletal attachment.


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Fig. 2.   Indirect immunofluorescent microscopy studies to detect the exposure of the anti-LIBScyt1 epitope in ligand-occupied alpha IIbbeta 3 of permeabilized platelets. A, platelets were treated with 1 mM GRGDSP, 1 mM GRGESP, or vehicle buffer prior to fixation and permeabilization with LPC. B, resting or activated platelets (10 µM ADP) were incubated with or without fibrinogen (3 µM), fixed, and permeabilized with LPC (original magnification, × 400).

To identify specific residues within the alpha IIb cytoplasmic sequence mediating interaction with anti-LIBScyt1, we performed competitive ELISA analyses using peptides corresponding to the full-length or partial sequences of the alpha IIb cytoplasmic tail. As shown in Table I, the full-length P2b peptide, as well as the truncated 15-mer (KVGFFKRNRPPLEED) effectively blocked anti-LIBScyt1 binding to immobilized P2b peptide. Moreover, using two overlapping peptides, we further localized the anti-LIBScyt1 epitope to the KRNRPPLEED sequence. Molecular modeling suggests that this region in both alpha IIb and alpha v subunits would form a tight beta -turn (28, 31). Since Pro998-Pro999 may facilitate this beta -turn formation, we tested the ability of KRNRAALEED to inhibit anti-LIBScyt1 binding. The inhibitory effect of the peptide was found to be significantly diminished by substitution of the two proline residues with alanine, indicating that anti-LIBScyt1 recognizes a structural motif dependent on these two proline residues.

                              
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Table I
Competitive ELISA to localize the anti-LIBScyt1 epitope within the alpha IIb cytoplasmic sequence
Results of A490 values are means ± S.D. of triplicate determinations.

A Site-directed Mutation of the Anti-LIBScyt1 Epitope Activates alpha IIbbeta 3 to Bind Extracellular Ligands-- The ability of certain anti-LIBS mAbs to activate alpha IIbbeta 3 (14, 15) suggests that these LIBS epitopes may regulate the ligand binding affinity state of the receptor. Therefore, we evaluated the functional significance of Pro998-Pro999 in the regulation of extracellular ligand binding to alpha IIbbeta 3. In these studies, a double P998A/P999A mutation in alpha IIb was generated by splice overlap extension mutagenesis (25), and the mutant alpha IIb construct was co-transfected with a wild type beta 3 construct into CHO cells. A stable clonal cell line (G4) expressing the mutant alpha IIbbeta 3 was established, and comparative analyses were performed with the control A5 cell line expressing wild type alpha IIbbeta 3 (32). As determined by flow cytometry using FITC-conjugated AP-2, a complex-specific anti-alpha IIbbeta 3 mAb (20), both G4 and A5 cells expressed similar amounts of alpha IIbbeta 3 (mean fluorescence intensity: G4, 73.9; A5, 74.9). It has previously been shown that truncation of the entire alpha IIb cytoplasmic sequence at residue 991 resulted in an increase of the ligand binding affinity of alpha IIbbeta 3 (6); therefore, we examined whether the P998A/P999A mutation might result in proteolytic cleavage of the alpha IIb cytoplasmic domain, which would lead to receptor activation. Initially, we compared the molecular mass of the P998A/P999A mutant alpha IIb light chain with those of wild type and truncated alpha IIb by immunoblotting with anti-V41, an antipeptide antibody directed against the amino terminus of the alpha IIb light chain (19). As shown in Fig. 3A, the light chain of the P998A/P999A mutant migrated with a similar molecular mass as the inactive wild type alpha IIb. In contrast, the constitutively active Delta 991 truncation mutant of alpha IIb (6) migrated with an increased mobility on SDS-polyacrylamide gel electrophoresis. Furthermore, Fig. 3B shows that both wild type and the P998A/P999A mutant alpha IIb reacted with PMI-1, a mAb directed against an extracellular epitope in the alpha IIb heavy chain (18, 33), and with anti-alpha IIbC, an antipeptide polyclonal antibody raised against the alpha IIb cytoplasmic sequence (Phe992-Glu1008). Collectively, these results indicated that the P998A/P999A mutation did not result in proteolytic cleavages of the alpha IIb cytoplasmic tail. As expected, anti-LIBScyt1 immunoblotted the wild type but not the mutant alpha IIb.


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Fig. 3.   Immunoblot characterization of the P998A/P999A mutant alpha IIb. Lysates of CHO cells expressing beta 3 integrins complexed with wild type, P998A/P999A mutated, or Delta 991 truncated alpha IIb were subjected to immunoblotting with the indicated antibodies. A, proteins were resolved on SDS-18% polyacrylamide gel under reducing conditions and immunoblotted with anti-V41, an antipeptide antibody directed against the amino terminus of the alpha IIb light chain. B, proteins were resolved on SDS-7% polyacrylamide gels under nonreducing conditions and immunoblotted with PMI-1 directed against an extracellular epitope of alpha IIb, anti-alpha IIbC directed against the cytoplasmic sequence of alpha IIb (Phe992-Glu1008), or anti-LIBScyt1.

The affinity state of the mutant alpha IIbbeta 3 was then examined by the binding of FITC-conjugated PAC-1, an activation-specific mAb that, like fibrinogen, preferentially binds to activated alpha IIbbeta 3 (34). As reported previously (32), A5 cells expressing wild type alpha IIbbeta 3 bound minimal amounts of PAC-1 in the absence of receptor activation (Fig. 4A). In contrast, we observed constitutive binding of PAC-1 to the mutant alpha IIbbeta 3 on G4 cells, and this process was specifically blocked with 1 mM GRGDSP (Fig. 4A). Since the binding of fibrinogen to activated alpha IIbbeta 3 on platelets and transfected cells resulted in cell aggregation, we examined the ability of G4 cells to aggregate in the presence of fibrinogen. Fig. 4B shows that G4 but not A5 cells aggregated upon fibrinogen addition. Again, aggregation of G4 cells was specifically blocked with 1 mM GRGDSP. As a specificity control, we mutated the putative N744PLY beta -turn motif in the beta 3 cytoplasmic sequence to QALY. Cells expressing wild type alpha IIb and mutated beta 3 heterodimers failed to bind soluble fibrinogen and undergo aggregation (not shown). These results indicate that a structural change in the anti-LIBScyt1 binding site in the alpha IIb cytoplasmic tail induces a transmembrane conformational change of alpha IIbbeta 3, mimicking receptor activation due to inside-out signaling.


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Fig. 4.   Constitutive ligand binding function of G4 cells bearing the alpha IIb(P998A/P999A)beta 3 mutant receptor. G4 and A5 cells were harvested with trypsin and EDTA, washed, and resuspended in Tyrode's buffer. A, the binding of FITC-PAC-1 (15 nM) to G4 and A5 cells in the presence and absence of GRGDSP (1 mM) was assayed by flow cytometry as described by Wencel-Drake et al. (22). B, spontaneous aggregation of G4 cells in the presence of fibrinogen. Washed G4 and A5 cells (107/ml) were incubated with or without 3 µM fibrinogen in 24-well tissue culture plates and subjected to gyrorotation. In some samples, GRGDSP (1 mM) was added 30 min prior to the addition of fibrinogen.

It has previously been shown that ligand binding to integrin alpha IIbbeta 3 induces long range conformational changes in the extracellular domains of both alpha IIb and beta 3 subunits (12, 35). Our results demonstrated that such conformational changes transduce through the cell membrane to the cytoplasmic domain of the receptor. Besides alpha IIbbeta 3, several other integrins such as alpha vbeta 3 and alpha 5beta 1 have been shown to undergo extracellular conformational changes upon ligand occupancy (13, 36); therefore, it is tempting to speculate that ligand-induced conformational changes also occur in the cytoplasmic domains of other integrins. Since ligand binding to integrins results in cytoskeletal rearrangement and the generation of intracellular signals (2, 3, 9), the conformational state of integrin cytoplasmic domains may play a regulatory role in the assembly of cytoskeletal proteins and/or signaling molecules. In this regard, it has been shown that antibody-induced clustering of the alpha 5beta 1 integrin in the absence of ligand occupancy is sufficient for the intracellular accumulations of tensin and at least 20 signal transduction molecules (e.g. RhoA, Rac1, Ras, Raf, MEK, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and focal adhesion kinase) (37, 38). In contrast, both ligand occupancy and clustering of alpha 5beta 1 are required for transmembrane accumulations of several cytoskeletal proteins (e.g. talin, vinculin, and alpha -actinin). In light of these findings, our present data suggest that ligand-induced conformational changes of integrin cytoplasmic domains may play an essential role in the intracellular assembly of cytoskeletal proteins found in focal adhesions.

Emerging evidence has implicated ligand-induced oligomerization and/or conformational changes of transmembrane receptor complexes as potential mechanisms for receptor-mediated signal transduction. Specifically, it has been demonstrated that following ligand binding and dimerization of the platelet-derived growth factor receptor, there is a phosphorylation-dependent conformational change in the receptor cytoplasmic domain (39-41). Although integrin alpha IIbbeta 3 on platelets becomes tyrosine-phosphorylated as a result of ligand binding and cell aggregation, the monovalent RGD peptide has been shown to block receptor phosphorylation (42). Inasmuch as GRGDSP binding to alpha IIbbeta 3 is capable of inducing the exposure of the anti-LIBScyt1 epitope, receptor phosphorylation is apparently not required for the observed effect. Thus, our results provide the first evidence of a direct effect of ligand occupancy on the conformation of the cytoplasmic domain of an intact integrin receptor. Additionally, site-directed mutation of the identified LIBScyt1 epitope resulted in an increase of ligand binding affinity of alpha IIbbeta 3, indicating that the extracellular ligand-binding site and the cytoplasmic LIBS epitope of the receptor are functionally coupled. In sum, these findings suggest a bidirectional modulation of alpha IIbbeta 3 conformations across the cell membrane. Such conformational regulation may provide a novel mechanism for transmembrane receptor-mediated signal transduction.

    ACKNOWLEDGEMENTS

We thank Drs. X. Du, A. L. Frelinger III, M. H. Ginsberg, L. F. Lau, and J. C. Loftus for helpful discussions and critical comments on the manuscript. We also thank Dr. T. E. O'Toole for providing Chinese hamster ovary cell lines expressing wild type and truncated alpha IIbbeta 3.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL-41793 (to S. C.-T. L.) and HL-52755 (to J. D. W.-D.).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 an Established Investigator Award from the American Heart Association and Genentech. To whom correspondence should be addressed: Dept. of Pharmacology (M/C 868), University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-413-5928; Fax: 312-996-1225; E-mail: sclam{at}uic.edu.

    ABBREVIATIONS

The abbreviations used are: CHO, Chinese hamster ovary; LIBS, ligand-induced binding site(s); mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; LPC, lysophosphatidylcholine; FITC, fluorescein isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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