©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Agonist-stimulated Ligand Binding by the Platelet Integrin IIb3 in a Lymphocyte Expression System (*)

(Received for publication, May 4, 1995; and in revised form, June 5, 1995)

Elwyn Loh (1)(§) Katherine Beaverson (1) Gaston Vilaire (1) Weiwei Qi (1) Mortimer Poncz (2) Joel S. Bennett (1)(¶)

From the  (1)Hematology-Oncology Division, Hospital of the University of Pennsylvania, the (2)Division of Hematology, Children's Hospital of Philadelphia, and the Departments of Medicine and Pediatrics, the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The ligand binding activity of the platelet integrin alphaIIbbeta3 is initiated by agonist-generated intraplatelet signals. We studied this process in vitro by expressing recombinant alphaIIbbeta3 in Epstein-Barr virus-immortalized B lymphocytes. We found that phorbol ester stimulation induced the adhesion of lymphocytes expressing alphaIIbbeta3 to immobilized fibrinogen. Moreover, replacement of the transmembrane and cytoplasmic domains of the alpha and beta subunits of alphaIIbbeta3 with those of alphaLbeta2 significantly increased adherence, whereas replacement of only the cytoplasmic domains significantly decreased adherence. This suggests that transmembrane segments are involved in the agonist-induced modulation of alphaIIbbeta3 activity. Similar results were seen when the alphaIIbbeta3 activation-dependent monoclonal antibody PAC-1 was substituted for immobilized fibrinogen. We also found that the adherence of lymphocytes expressing beta3 with either of the two alphaIIb/alphaL chimeras was similar to that of cells expressing alphaIIbbeta3, whereas the adherence of cells expressing alphaIIb with either of the two beta3/beta2 chimeras was substantially decreased, suggesting that the identity of the cytoplasmic domain of beta3, but not of alphaIIb, is critical for alphaIIbbeta3 function. This report indicates that B lymphocytes contain signal transduction pathways involving protein kinase C that can increase the ligand binding activity of alphaIIbbeta3 and demonstrates the utility of these cells as an expression system for the study of agonist-stimulated alphaIIbbeta3 function.


INTRODUCTION

The ligand binding activity of many integrins is enhanced by cell stimulation(1) . A paradigm for this phenomenon is the interaction of the platelet integrin alphaIIbbeta3 (GPIIb-IIIa) with fibrinogen and von Willebrand factor(2) . Although alphaIIbbeta3 on resting platelets does not bind soluble fibrinogen or von Willebrand factor, binding readily occurs when platelets are stimulated by agonists such as thrombin and ADP. The agonist-generated ``inside-out'' signals that convert alphaIIbbeta3 from an inactive to a ligand binding conformation do so by interacting with one or both cytoplasmic domains of the alphaIIbbeta3 heterodimer(3) . The signals are then propagated across the length of alphaIIbbeta3 to expose a ligand binding site located in its extracellular domain(4) . The nature of the signaling pathways, their targets on the cytoplasmic domains of alphaIIbbeta3, and the mechanism by which the signals are transmitted across the molecule are largely unknown. Moreover, it has not been possible to reproduce agonist-induced alphaIIbbeta3 activation in vitro using recombinant heterodimers because conventional expression systems do not support agonist-induced alphaIIbbeta3 activation(5) .

Like alphaIIbbeta3 in platelets, the ligand binding activity of integrins in leukocytes is enhanced by agonists(1) . For example, the activity of alphaLbeta2 (LFA-1) in lymphocytes can be up-regulated by cross-linking the T-cell receptor (6) or by stimulating the cell with a phorbol ester such as phorbol 12-myristate 13-acetate (PMA)(^1)(7) . It has also been reported that agonists up-regulate the ligand binding activity of lymphocyte alphavbeta3 (8) . Accordingly, we hypothesized that it might also be possible to generate signals capable of activating alphaIIbbeta3 in lymphocytes and tested this hypothesis by expressing recombinant alphaIIbbeta3 in Epstein-Barr virus (EBV)-immortalized B lymphocytes. In addition, because of the possibility that recognition of alphaIIbbeta3 by the signal transduction pathways in lymphocytes might require the presence of the cytoplasmic and transmembrane domains of alphaLbeta2, we also expressed chimeras of alphaIIbbeta3 and alphaLbeta2 in these cells.


EXPERIMENTAL PROCEDURES

Construction of alphaIIb/alphaL and beta3/beta2 Chimeras

Chimeric integrin subunit cDNAs were constructed in the EBV-based episomal plasmids pREP4 and pREP9 (9) (gifts of Dr. M. Tykocinski, Case Western Reserve University) by a modified overlap polymerase chain reaction (PCR) procedure(10) .

To construct a cDNA encoding the extracellular domain of alphaIIb and the transmembrane and cytoplasmic domains of alphaL (Fig. 1, 2LL), a full-length alphaIIb cDNA was first cloned into pREP9 using KpnI and HindIII. An overlap PCR fragment containing the transmembrane and cytoplasmic domains of alphaL was then synthesized using the primers GCTCCGGGCCTTGGAGGAGCGCCAGATGCTCTACCTCTACGTGCTG and TGTGAAGCTTCAGACATTCTCTTCCAAGG with cDNA from the EBV-transformed B cell line GM1500 (11) (a gift of Dr. Y. Tsujimoto, Osaka Medical College, Japan) as a template. The resulting DNA fragment was used as a primer along with the alphaIIb 5`-primer GCGGAATTCACAAGCGGGATCGCAGACAG to generate a chimeric fragment using alphaIIb as the template. The PCR product was sequenced and subcloned into alphaIIb using BglII and HindIII.


Figure 1: Schematic diagram of the subunits of alphaIIbbeta3 and of chimeric subunits consisting of the extracellular domains of alphaIIbbeta3 (EX) and the transmembrane (TM) and cytoplasmic (CY) domains of alphaLbeta2.



A cDNA encoding the extracellular domain of beta3 and the transmembrane and cytoplasmic domains of beta2 (Fig. 1, 3LL) was constructed after a full-length beta3 cDNA was cloned into pREP4 using KpnI and XhoI. An overlap PCR fragment containing the transmembrane and cytoplasmic domains of beta2 was then made using the primers GTGGTAGAAGAGCCAGAGTGTGTGGCCGGCCCCAACATCGC and GGTCCTCGAGGGATGTCATTTTATACCCTGAC with Jurkat cell cDNA as a template. The resulting fragment was used as a primer along with the beta3 5`-primer CACGAATTCGAGATTGAGTCAGTGAAAGAGC to generate a chimeric fragment using beta3 as a template. The PCR product was sequenced and subcloned into beta3 in pREP4 using AflII and XhoI.

A related strategy was used to construct cDNAs encoding the extracellular and transmembrane domains of alphaIIb and beta3 and the cytoplasmic domains of alphaL and beta2, respectively. To construct a cDNA encoding the extracellular and transmembrane domains of alphaIIb and the cytoplasmic domain of alphaL (Fig. 1, 22L), PCR mutagenesis was used to introduce a DraI site into the sequence encoding the amino acids Phe-Lys at the junction between the transmembrane and cytoplasmic domains of both alphaIIb (12) and alphaL(13) . The modified cDNAs were then digested with DraI, and the proximal fragment of alphaIIb and distal fragment of alphaL were ligated and subcloned into pREP4. To construct a cDNA encoding the extracellular and transmembrane domains of beta3 and the cytoplasmic domain of beta2 (Fig. 1, 33L), a BclI site was introduced into the sequence encoding for amino acids Leu-Ile-Thr at the junction between the transmembrane and cytoplasmic domains of beta3 (14) and a BglII site into the sequence encoding for amino acids Arg-Ile-His of beta2(15) . The two portions were digested with their respective enzymes, ligated at the compatible BclI/BglII ends, and subcloned into pREP9.

Expression of alphaIIbbeta3 and Chimeras of alphaIIbbeta3 and alphaLbeta2 in Human B Lymphocytes

pREP4 and pREP9 containing cDNAs encoding wild-type alphaIIb and beta3 or the alpha and beta subunit chimeras described above were cotransfected into 7.5 10^6 GM1500 B lymphocytes by electroporation (Gene Pulser, Bio-Rad, 250 V and 960 millifarads). Stable cotransfectants were selected by growth in RPMI media containing 20% fetal calf serum and both G418 (750 µg/ml) and hygromycin (250 µg/ml). The presence of alphaIIbbeta3 and the chimeras of alphaIIbbeta3 and alphaLbeta2 on the lymphocyte surface was detected by flow cytometry and by immunoprecipitation. For flow cytometry, cells were stained with either fluoroscein-conjugated anti-murine IgG alone or with the alphaIIbbeta3-specific murine monoclonal antibody (mAb) A2A9 (16) followed by fluorosceinconjugated anti-murine IgG. Flow cytometry was performed using a FACScan flow cytometer (Becton Dickinson) as previously described(17) . For immunoprecipitation, the lymphocyte surface was labeled with I using lactoperoxidase(17) . alphaIIbbeta3 and the chimeric integrins were then immunoprecipitated using the beta3-specific mAb SSA6 (17) and visualized following SDS-polyacrylamide gel electrophoresis and autoradiography as previously described(18) .

Measurement of Lymphocyte Adherence to Immobilized Fibrinogen

The function of alphaIIbbeta3 and the chimeras of alphaIIbbeta3 and alphaLbeta2 in B lymphocytes was tested by measuring the adherence of PMA (Sigma)-stimulated transfected cells to immobilized fibrinogen. In some experiments, bovine serum albumin or the mAb PAC-1 (19) was substituted for fibrinogen. For these assays, the wells of Immulon 2 flat bottom microtiter plates (Dynatech Laboratories) were coated with either 50 µg/ml purified human fibrinogen in 50 mM NaHCO(3) buffer, pH 8.0, containing 150 mM NaCl, or 40 µg/ml PAC-1 in the same buffer. Unoccupied protein binding sites on the wells were then blocked with 5 mg/ml bovine serum albumin dissolved in the same buffer. 1.5 10^5 lymphocytes, metabolically labeled overnight with [S]methionine, were suspended in 100 µl of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl(2), 0.1% glucose, and 1% bovine serum albumin and added to the protein-coated wells. Following an incubation at 37 °C without agitation, the plates were vigorously washed four times with the lymphocyte suspension buffer, and adherent cells were dissolved using 2% SDS. The SDS solutions were then counted for S in a liquid scintillation counter.


RESULTS

Expression of alphaIIbbeta3 and Chimeras of alphaIIbbeta3 and alphaLbeta2 in B Lymphocytes

Heterodimers composed of alphaIIbbeta3 or chimeras of alphaIIbbeta3 and alphaLbeta2 (Fig. 1) were stably expressed in EBV-immortalized human GM1500 B lymphocytes using the EBV-based plasmids pREP9 and pREP4. As shown in Fig. 2A, the alpha and beta subunits of these heterodimers were immunoprecipitated from detergent extracts of surface-labeled cells using the anti-beta3 mAb SSA6, demonstrating that the expressed heterodimers had been transported to the lymphocyte surface. Moreover, the presence of the alphaIIb heavy chain (alphaIIbalpha) in the immunoprecipitates indicates that the heterodimers had undergone correct intracellular processing in these cells(12) . Faint bands corresponding to beta3 and the heavy chain of the vitronectin receptor, alphaV, were present in immunoprecipitates from mock-transfected cells, consistent with the previous reports that B lymphocytes constitutively express low levels of alphaVbeta3(8) . As shown in Fig. 2B, flow cytometry using the alphaIIbbeta3 heterodimer-specific mAb A2A9 revealed that comparable levels of alphaIIbbeta3 and the chimeras were expressed on the lymphocyte surface.


Figure 2: Demonstration of alphaIIbbeta3 and chimeras of alphaIIbbeta3 and alphaLbeta2 on the surface of transfected lymphocytes by immunoprecipitation and flow cytometry. A, alphaIIbbeta3 and the 2LL/3LL and 22L/33L chimeras were immunoprecipitated using the beta3-specific mAb SSA6 from Triton X-100 extracts of transfected lymphocytes whose surface had been labeled with I as described under ``Experimental Procedures.'' alphaIIbalpha corresponds to the heavy chain of alphaIIb; beta corresponds to the beta subunit of the heterodimer. B, lymphocytes transfected with the plasmids pREP 4 and pREP 9 (mock-transfected cells) and lymphocytes expressing alphaIIbbeta3 or the 2LL/3LL and 22L/33L chimeras were stained with either fluoroscein-conjugated anti-murine IgG alone (No A2A9) or with the alphaIIbbeta3 heterodimer-specific mAb A2A9 followed by fluoroscein-conjugated anti-murine IgG (A2A9) and analyzed by flow cytometry as described under ``Experimental Procedures.'' The histograms of mock-transfected cells stained under both sets of conditions overlap, indicating no detectable A2A9 binding to these cells.



Adherence of B Lymphocytes Expressing alphaIIbbeta3 to Immobilized Fibrinogen

The ability of alphaIIbbeta3 in B lymphocytes to interact with ligands was tested by measuring the adherence of phorbol ester-stimulated lymphocytes to the wells of microtiter plates coated with purified fibrinogen. Because the ligand binding activity of the endogenous lymphocyte integrin alphaLbeta2 requires the presence of Mg(20) , whereas the interaction of alphaIIbbeta3 with fibrinogen occurs in the presence of either Mg or Ca(21) , alphaLbeta2-mediated lymphocyte function was prevented by performing the assays in a Mg-free buffer containing 0.5 mM Ca. As shown by the photomicrographs in Fig. 3, few unstimulated lymphocytes expressing alphaIIbbeta3 attached to immobilized fibrinogen over a 30-min period. However, stimulation of these cells with 50 nM PMA resulted in a marked increase in the number of adherent cells over the same time period. Adherence was inhibited by the presence of 1 mM EDTA, indicating that it was calcium-dependent. Because flow cytometry before and after PMA stimulation revealed no differences in the extent of alphaIIbbeta3 expression (data not shown), it is likely that the increased adherence induced by PMA resulted from an increase in the avidity of alphaIIbbeta3 for fibrinogen.


Figure 3: Photomicrographs of B lymphocytes expressing alphaIIbbeta3 adherent to immobilized fibrinogen. The adherence of unstimulated lymphocytes (No PMA), lymphocytes stimulated with 50 nM PMA (PMA), and lymphocytes stimulated with 50 nM PMA in the presence of 1 mM EDTA (PMA + EDTA) to fibrinogen immobilized on microtiter plates was examined as described under ``Experimental Procedures.'' The plates were then washed four times and examined by light microscopy.



To further characterize the adherence of lymphocytes expressing alphaIIbbeta3 to fibrinogen, cells were metabolically labeled with [S]methionine and the number of adherent cells quantitated under a variety of conditions. Fig. 4A shows the time course of lymphocyte adherence. In the absence of PMA stimulation, adherence was minimal throughout a 60-min period of incubation. In contrast, PMA stimulation resulted in a significant increase in adherence within 5 min (p = 0.014), and adherence was maximal at 30 min. To confirm that adherence was mediated by the extracellular domain of alphaIIbbeta3, lymphocytes were preincubated with 50 µg/ml A2A9, a concentration of antibody sufficient to prevent fibrinogen binding to platelet alphaIIbbeta3(16) . In the presence of A2A9, PMA-stimulated adherence was reduced to the level observed when albumin was substituted for fibrinogen or when the assay was performed in the presence of 1 mM EDTA (Fig. 4B). Moreover, an identical concentration of the beta3-specific mAb SSA6, an antibody that has no effect on fibrinogen binding to platelet alphaIIbbeta3(17) , had no effect on lymphocyte adherence. Fibrinogen binding to alphaIIbbeta3 is also inhibited by peptides containing the amino acid sequence RGDS (22) . RGDS, at a concentration of 5 mM, completely inhibited PMA-stimulated adherence, whereas the same concentration of the control peptide RGES had only a minimal effect (Fig. 4B). The IC of RGDS in this assay was approx400 µM (Fig. 4C), approx20-fold greater than its IC for inhibiting thrombin-stimulated platelet adhesion to immobilized fibrinogen(23) , suggesting that the avidity of lymphocytes expressing alphaIIbbeta3 for immobilized fibrinogen is substantially greater than that of platelets.


Figure 4: Characterization of the adherence of lymphocytes expressing alphaIIbbeta3 to immobilized fibrinogen. The adherence of lymphocytes metabolically labeled with [S]methionine and expressing alphaIIbbeta3 was quantitated by dissolving adherent cells in 2% SDS and measuring the amount of S in the SDS solution as described under ``Experimental Procedures.'' Each measurement of cell adherence was made in quadruplicate. A, time course of unstimulated and PMA-stimulated lymphocyte adherence. B, effect of various inhibitors on PMA-stimulated lymphocyte adherence. PMA-stimulated lymphocyte adherence to immobilized fibrinogen or bovine serum albumin was quantitated as described above. The consequences of preincubating the cells with 50 µg/ml mAb A2A9, 1 mM EDTA, 50 µg/ml mAb SSA6, or 5 mM of the tetrapeptides Arg-Gly-Asp-Ser (RGDS) or Arg-Gly-Glu-Ser (RGES) on adherence to fibrinogen were then determined. The data were normalized to 100% adherence in the absence of inhibitors to facilitate comparison. C, effect of RGDS concentration on PMA-stimulated adherence. 100% represents adherence in the absence of RGDS.



Next, we compared the adherence of lymphocytes expressing alphaIIbbeta3 to that of cells expressing the 2LL/3LL and 22L/33L chimeras (Fig. 5). In the absence of PMA stimulation, 0.3% of mock-transfected cells, 3.8% of cells expressing alphaIIbbeta3, 7.3% of cells expressing 2LL/3LL, and 4.5% of cells expressing 22L/33L were adherent to fibrinogen. Following PMA stimulation, the adherence of the mock-transfected cells did not change, while the adherence of cells expressing alphaIIbbeta3, 2LL/3LL, and 22L/33L increased to 26.2, 37.4, and 17.9%, respectively. Adherence of both the unstimulated and PMA-stimulated cells expressing alphaIIbbeta3, 2LL/3LL, and 22L/33L was reduced to that of the mock-transfected cells by the addition of 1 mM EDTA or the mAb A2A9 (data not shown). Moreover, the differences in adherence of the PMA-stimulated cells expressing alphaIIbbeta3 versus 2LL/3LL and 2LL/3LL versus 22L/33L were statistically significant at p < 0.02 and p < 0.0005, respectively (t test for unpaired samples). Thus, these data indicate that in B lymphocytes, the ligand binding activity of alphaIIbbeta3 is unaffected by replacing its cytoplasmic domains with those of alphaLbeta2. Furthermore, the difference in adherence of cells expressing 2LL/3LL versus 22L/33L suggests that transmembrane segments are involved in agonist-induced modulation of alphaIIbbeta3 activity.


Figure 5: Comparison of the unstimulated and PMA-stimulated adherence of lymphocytes expressing either alphaIIbbeta3 or the alphaIIbbeta3-alphaLbeta2 chimeras to immobilized fibrinogen. Mock-transfected lymphocytes and lymphocytes expressing alphaIIbbeta3 or the 2LL/3LL and 22L/33L chimeras were metabolically labeled with [S]methionine and incubated with immobilized fibrinogen as described in Fig. 3and Fig. 4and under ``Experimental Procedures.'' Each measurement of cell adherence was performed in quadruplicate. The data are the mean and standard error of 4-8 separate experiments.



Adherence of B Lymphocytes Expressing alphaIIbbeta3 and alphaIIbbeta3-alphaLbeta2 Chimeras to Immobilized PAC-1

Platelet agonists, including PMA, activate alphaIIbbeta3 by inducing a conformational change in the molecule that exposes its ligand binding site(24) . Similarly, PMA enhances lymphocyte adherence to ICAM-1 by converting alphaLbeta2 from a low affinity to a high affinity state(7, 25) . Nevertheless, it has been suggested that PMA-stimulated adhesion is a post-receptor event(26) , perhaps the result of a cytoskeleton-mediated rearrangement of integrins on the cell surface (27) . To address the mechanism of PMA-stimulated lymphocyte adherence to fibrinogen, we repeated the lymphocyte adherence assay, substituting the mAb PAC-1 for immobilized fibrinogen (Fig. 6). PAC-1 is an IgM mAb that only interacts with alphaIIbbeta3 in its activated conformation(19) . In the absence of PMA stimulation, 4.4% of cells expressing alphaIIbbeta3, 4.7% of cells expressing 2LL/3LL, and 4.4% of cells expressing 22L/33L were adherent to immobilized PAC-1. Following PMA stimulation, the adherence of cells expressing alphaIIbbeta3, 2LL/3LL, and 22L/33L increased to 29.8, 35.1, and 24.7%, respectively. Adherence of each cell type declined to 0.5, 0.7, and 0.2, respectively, in the presence of 1 mM EDTA (data not shown), and there was essentially no adherence of the cells to immobilized non-immune IgM, regardless of whether they were unstimulated or stimulated with PMA. Although the differences in the magnitude of adherence of the PMA-stimulated cells expressing alphaIIbbeta3 versus 2LL/3LL and 2LL/3LL versus 22L/33L were smaller than when fibrinogen was the ligand, the differences remain statistically significant at p < 0.001 and p < 0.0001, respectively. Thus, these data support a conclusion that the PMA-stimulated adherence we observed resulted from a conformational change in the extracellular domain of alphaIIbbeta3. Whether a cytoskeletal rearrangement was also involved in this process remains to be determined.


Figure 6: Comparison of the unstimulated and PMA-stimulated adherence of lymphocytes expressing either alphaIIbbeta3 or the 2LL/3LL and 22L/33L chimeras to immobilized PAC-1 or non-immune IgM. The wells of microtiter plates were coated with either purified PAC-1 or non-immune murine IgM (Sigma), each at a concentration of 40 µg/ml. The adherence of unstimulated lymphocytes and lymphocytes stimulated with 50 nM PMA was measured as described in Fig. 5. Each measurement of cell adherence was performed in quadruplicate. The data are the mean and standard error of 2-8 separate experiments.



Adherence of B Lymphocytes Expressing either alphaIIb or beta3 and a Complementary Chimeric Subunit to Immobilized Fibrinogen

The preceding experiments indicated that alphaIIbbeta3 activation in lymphocytes was most efficient when the transmembrane and cytoplasmic domains of the heterodimer were derived from the same integrin. To study the role of individual transmembrane and cytoplasmic domains in this process, we measured the adherence of lymphocytes expressing alphaIIb with either 3LL or 33L and beta3 with either 2LL or 22L to immobilized fibrinogen (Fig. 7). In the absence of PMA stimulation, 6.5% of lymphocytes expressing 2LL/beta3 and 7.6% of lymphocytes expressing 22L/beta3 were adherent to fibrinogen. Following PMA stimulation, adherence increased to 28.6 and 33.9%, respectively. In contrast, only 0.9 and 1.5% of cells expressing alphaIIb/3LL or alphaIIb/33L were adherent in the absence of PMA stimulation, and adherence increased to only 10.5 and 13.3% following PMA stimulation. Moreover, because GM1500 cells express a low level of endogenous beta3 (Fig. 2), it is possible that part of the adherence of cells expressing alphaIIb/3LL or alphaIIb/33L was actually mediated by alphaIIbbeta3. Thus, the measured adherence may overstate the function of the alphaIIb/3LL and alphaIIb/33L chimeras. Accordingly, these experiments indicate that the cytoplasmic domain of beta3 is specifically involved in alphaIIbbeta3 activation in lymphocytes. They also suggest that compatibility of the alphaIIbbeta3 cytoplasmic domains is important in this process.


Figure 7: Comparison of the unstimulated and PMA-stimulated adherence of lymphocytes expressing heterodimers composed of beta3 and the 2LL and 22L chimeras or alphaIIb and the 3LL and 33L chimeras to immobilized fibrinogen. The adherence of unstimulated lymphocytes and lymphocytes stimulated with 50 nM PMA was measured as described in Fig. 5. Each measurement of cell adherence was performed in quadruplicate. The data are the mean and standard error of the mean of 2-10 separate experiments.




DISCUSSION

Intracellular signals initiated by platelet agonists convert the platelet integrin alphaIIbbeta3 from a functionally inactive to a functionally active conformation, enabling it to interact with ligands such as fibrinogen(2) . The signaling pathways responsible for inducing this conformational change may be unique to platelets because it has not been possible to use cellular agonists to activate the endogenous alphaIIbbeta3 expressed by megakaryocyte-like cell lines or recombinant alphaIIbbeta3 expressed in heterologous cells(5) . Nevertheless, the ability of cells to modulate integrin function is a general phenomenon (1) . Consequently, we postulated that lymphocytes, as cells of hematopoietic origin, might contain signal transduction pathways capable of activating alphaIIbbeta3 and expressed this integrin in EBV-immortalized B lymphocytes. Using an adherence assay similar to that used by others to study the activity of alphaLbeta2 in lymphocytes (7) , we found that the adherence of lymphocytes expressing alphaIIbbeta3 to immobilized fibrinogen increased nearly 7-fold when they were incubated with the phorbol ester PMA. Moreover, like platelets, the interaction of the transfected lymphocytes with fibrinogen was inhibited by calcium chelation, by the mAb A2A9, and by the peptide RGDS(16, 21, 22) . Nevertheless, it has been observed that unstimulated platelets can adhere to fibrinogen but by a process that is independent of platelet metabolic activity(23, 28) . Similarly, we found that approx4% of transfected lymphocytes adhere to fibrinogen in the absence of PMA stimulation. Thus, it is possible that the PMA-stimulated adherence we observed simply represents an enhanced interaction of inactive alphaIIbbeta3 with fibrinogen. To address this possibility, we measured lymphocyte adherence to the activation-dependent anti-alphaIIbbeta3 mAb PAC-1 (19) and found that adherence to PAC-1 correlated well with adherence to fibrinogen. Accordingly, these data suggest that the alphaIIbbeta3 in a fraction of the unstimulated lymphocytes is present in an activated state and that the increase in adherence we observed following PMA stimulation resulted from an increase in the number of lymphocytes expressing active alphaIIbbeta3.

In addition to stimulating the adherence of lymphocytes expressing alphaIIbbeta3 to fibrinogen, we found that PMA stimulated the adherence of lymphocytes expressing chimeras of alphaIIbbeta3 and alphaLbeta2 to the same substrate. This result allowed us to examine the contribution of individual cytoplasmic domains to alphaIIbbeta3 function. Despite substantial differences in the sequences of the cytoplasmic domains of alphaIIb and alphaL (Fig. 8), we found that PMA-stimulated adherence mediated by heterodimers composed of beta3 and either the 22L or 2LL alpha subunit chimeras was equivalent to adherence mediated by alphaIIbbeta3. This result is consistent with previous studies of the function of the integrin alpha2beta1 in which it was found that replacing the cytoplasmic domain of alpha2 with the cytoplasmic domains of either alpha4 or alpha5 did not affect constitutive or PMA-stimulated alpha2beta1 activity in RD or K562 cells(29) . However, truncation of the alpha2 cytoplasmic domain distal to its GFFKR sequence (Fig. 8) eliminated both constitutive and PMA-stimulated alpha2beta1 activity, demonstrating that the presence of an alpha subunit cytoplasmic domain is necessary for alpha2beta1 function. On the other hand, it has been reported that truncation of the cytoplasmic domain of alphaL distal to GFFKR has no effect on alphaLbeta2 function in COS cells(7) , and truncation of the cytoplasmic domain of alphaIIb proximal to GFFKR or replacing the cytoplasmic domain with the cytoplasmic domain of alpha5 results in constitutive alphaIIbbeta3 activity in Chinese hamster ovary cells(5) . Nevertheless, our results indicate that like alpha2, the exact identity of the cytoplasmic domain of alphaIIb is not critical for alphaIIbbeta3 function, perhaps because the GFFKR sequence was preserved.


Figure 8: Alignment of the amino acid sequences of the cytoplasmic domains of alphaIIbbeta3 and alphaLbeta2. The : indicates the position of identical amino acids in the alignment. The numbers represent the carboxyl-terminal amino acid of each sequence(12, 13, 14, 15) . The positions of the membrane-proximal sequences postulated to be involved in integrin activation are underlined.



In contrast to the lack of effect of exchanging the alphaIIb and alphaL cytoplasmic domains on lymphocyte adherence to fibrinogen, we found that both unstimulated and PMA-stimulated adherence was significantly diminished when the cytoplasmic domain of beta3 was replaced by that of beta2. This result is consistent with the virtual elimination of constitutive and PMA-stimulated lymphocyte adherence to ICAM-1 following deletion of the beta2 cytoplasmic domain (7) and suggests that specific amino acids in the cytoplasmic domain of beta3 are involved in modulating alphaIIbbeta3 function. Mutagenesis of the beta2 cytoplasmic domain indicated that the carboxyl-terminal amino acids 757-761 (ATTTV) and Phe are required for alphaLbeta2 function (Fig. 8)(30) . The corresponding amino acids in the beta3 cytoplasmic domain are ATSTF (residues 750-754) and Tyr(14) . Moreover, a naturally occurring mutation in beta3, Ser Pro, prevents platelet aggregation(31) , confirming the importance of this residue for alphaIIbbeta3 activity. However, conversion of beta2 Thr Ser or beta2 Phe Tyr, such that this portion of the beta2 cytoplasmic domain resembles that of beta3, has no effect on alphaLbeta2 function(30) . Consequently, it is unlikely that differences in the carboxyl-terminal regions of the beta2 and beta3 cytoplasmic domains can account for the results of our experiments. It has also been reported that amino acids located in the membrane-proximal region of the beta1 cytoplasmic domain, corresponding to the amino acid sequence DRKEFAKFEEE in human beta3 (Fig. 8), are involved in localizing beta1-containing integrins in focal adhesions (32) . The secondary structure of this beta1 sequence was predicted to be an alpha-helix(32) . Thus, it is conceivable that a similar helix in beta3 interacts with as yet unidentified signaling molecules, with the cytoskeleton, or with a complementary sequence in the alpha subunit cytoplasmic domain to up-regulate alphaIIbbeta3 function. The stretch of amino acids in beta2 analogous to beta3, DLREYRRFEKE, contains notable differences, perhaps accounting for the diminished ability of the beta2 cytoplasmic domain to support alphaIIbbeta3 function.

In our initial experiments, we expressed double chimeras in which the cytoplasmic and transmembrane domains of alphaIIb and beta3 were replaced by the corresponding domains of alphaL and beta2. We found that the adherence of lymphocytes expressing 2LL/3LL to fibrinogen was increased compared to lymphocytes expressing alphaIIbbeta3, perhaps reflecting a preference of the lymphocyte signaling mechanism for beta2 integrins. Conversely, we found that replacing the transmembrane domains of 2LL/3LL with those of alphaIIbbeta3 significantly reduced adherence, implying that transmembrane domains play a role in alphaIIbbeta3 activity. This inference is underscored by the complete conservation of the transmembrane domains of human, rat, and mouse alphaIIb and beta3(33, 34) and the nearly complete conservation of the transmembrane domains of human and rat alphaL and beta2(35, 36) . Furthermore, the ability of heterodimers composed of 2LL and beta3 to respond to PMA as well as those composed of 22L and beta3 suggests that alphaIIb can tolerate other acceptable sequences in its transmembrane region. Similar results have been reported for the insulin (37) and epidermal growth factor (EGF) receptors (38) but not for the platelet-derived growth factor (39) and nerve growth factor receptors(40) , implying that there are differences in the way various transmembrane segments contribute to receptor activity. We also found little difference in lymphocyte adherence mediated by heterodimers composed of alphaIIb and 33L or 3LL. However, the absence of a beta3 cytoplasmic domain in this circumstance likely obscures the contribution of the beta3 transmembrane domain to alphaIIbbeta3 function.

How might the alphaIIb and beta3 transmembrane domains contribute to alphaIIbbeta3 function? One possibility is that the transmembrane and cytoplasmic domains exist as a conformational unit such that the composition of the transmembrane domain influences the conformation of the cytoplasmic domain. As a precedent for this possibility, it was found that the ability of an EGF receptor-nerve growth factor receptor chimera to transduce EGF signals was lost when the transmembrane domain of the nerve growth factor receptor was replaced by that of the EGF receptor(40) . A second possibility is based on analogy to many cytokine and growth factor receptors where transmembrane signaling requires homo- or heterodimerization of receptor subunits(41, 42) . Examination of solubilized alphaIIbbeta3 by electron microscopy reveals that the tails of alphaIIb and beta3 containing their transmembrane segments are flexible and interact with each other in approx30% of images (43) . In view of the fluid nature of biological membranes(44) , it is likely that the transmembrane segments of alphaIIb and beta3 would interact in unstimulated platelets unless constrained by factors such as cytoskeletal associations or unfavorable interactions between its cytoplasmic domains(41, 45) . By relieving these constraints, agonist-generated signals could allow the transmembrane domains of alphaIIb and beta3 to associate, thereby transmitting the signals to the extracellular domain.

The identity of the signal transduction pathways that activate integrins in lymphocytes and platelets are unknown, but our data indicate that similar pathways involving phorbol ester-sensitive protein kinase C are present in both cells. On the other hand, it has not been possible to activate recombinant beta2 or beta3 integrins using agonists like PMA in fibroblast-like cells(5, 7, 46) , suggesting that there are differences between the repertoire of signal transduction pathways in cells of fibroblast origin and in cells of hematopoietic origin where activation of beta2 or beta3 integrins normally occurs. Previous studies of alphaIIbbeta3 activation have depended on the ability of mutations to induce constitutive alphaIIbbeta3 activity(5, 46) . Besides restricting such studies to activating mutations, the relationship of the changes in alphaIIbbeta3 induced by the mutations to the changes induced by cellular agonists remains problematic. Our results indicate that the ligand binding activity of alphaIIbbeta3 and of alphaIIbbeta3-alphaLbeta2 chimeras can be modulated by phorbol esters in human B lymphocytes. Accordingly, this lymphocyte system establishes a model that permits study of both agonist-initiated signal transduction pathways and the agonist-stimulated function of intact or mutant integrins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HL40387 (to J. S. B. and M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
A Council for Tobacco Research Scholar and the recipient of a Junior Faculty Research Award from the American Cancer Society.

To whom correspondence should be addressed: Hematology-Oncology Division, BRB1, Rm. 1005a, 422 Curie Blvd., Philadelphia, PA 19014. Tel.: 215-662-4028; Fax: 215-662-7617.

^1
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; EBV, Epstein-Barr virus; PCR, polymerase chain reaction; mAb, monoclonal antibody; EGF, epidermal growth factor.


REFERENCES

  1. Hynes, R. O. (1992)Cell69,11-25 [Medline] [Order article via Infotrieve]
  2. Phillips, D. R., Charo, I. F., and Scarborough, R. M.(1991)Cell 65,359-362 [Medline] [Order article via Infotrieve]
  3. Sastry, S. K., and Horwitz, A. F.(1993)Curr. Opin. Cell Biol. 5,819-831 [Medline] [Order article via Infotrieve]
  4. Du, X., Gu, M., Weisel, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993)J. Biol. Chem.268,23087-23092 [Abstract/Free Full Text]
  5. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H.(1994) J. Cell Biol.124,1047-1059 [Abstract]
  6. Dustin, M. L., and Springer, T. A.(1989)Nature341,619-624 [CrossRef][Medline] [Order article via Infotrieve]
  7. Hibbs, M. L., Xu, H., Stacker, S. A., and Springer, T. A.(1991)Science 251,1611-1613 [Medline] [Order article via Infotrieve]
  8. Stupack, D. G., Shen, C., and Wilkins, J. A.(1992)Exp. Cell Res. 203,443-448 [Medline] [Order article via Infotrieve]
  9. Groger, R. K., Morrow, D. M., and Tykocinski, M. L.(1989)Gene (Amst.) 81,285-294 [Medline] [Order article via Infotrieve]
  10. Landt, O., Grunert, H. P., and Hahn, U.(1990)Gene (Amst.) 96,125-128 [CrossRef][Medline] [Order article via Infotrieve]
  11. Endo, K., Borer, C. H., and Tsujimoto, Y.(1991)Oncogene6,1391-1396 [Medline] [Order article via Infotrieve]
  12. Poncz, M., Eisman, R., Heidenreich, R., Silver, S. M., Vilaire, G., Surrey, S., Schwartz, E., and Bennett, J. S.(1987)J. Biol. Chem. 262,8476-8482 [Abstract/Free Full Text]
  13. Larson, R. S., Corbi, A. L., Berman, L., and Springer, T.(1989)J. Cell Biol. 108,703-712 [Abstract]
  14. Zimrin, A. B., Eisman, R., Vilaire, G., Schwartz, E., Bennett, J. S., and Poncz, M. (1988)J. Clin. Invest.81,1470-1475 [Medline] [Order article via Infotrieve]
  15. Kishimoto, T. K., O'Connor, K., Lee, A., Roberts, T. M., and Springer, T. A. (1987)Cell48,681-690 [Medline] [Order article via Infotrieve]
  16. Bennett, J. S., Hoxie, J. A., Leitman, S. F., Vilaire, G., and Cines, D. B.(1983) Proc. Natl. Acad. Sci. U. S. A.80,2417-2421 [Abstract]
  17. Silver, S. M., McDonough, M. M., Vilaire, G., and Bennett, J. S.(1987) Blood69,1031-1037 [Abstract]
  18. Poncz, M., Salahandrin, R., Coller, B. S., Newman, P. J., Shattil, S. J., Parrella, T., Fortina, P., and Bennett, J. S.(1994)J. Clin. Invest.93,172-179 [Medline] [Order article via Infotrieve]
  19. Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F.(1985)J. Biol. Chem.260,11107-11114 [Abstract/Free Full Text]
  20. Rothlein, R., and Springer, T. A.(1986)J. Exp. Med.163,1132-1149 [Abstract]
  21. Bennett, J. S., and Vilaire, G.(1979)J. Clin. Invest.64,1393-1401 [Medline] [Order article via Infotrieve]
  22. Gartner, T. K., and Bennett, J. S.(1985)J. Biol. Chem.260,11891-11894 [Abstract/Free Full Text]
  23. Haverstick, D. M., Cowan, J. F., Yamada, K. M., and Santoro, S. A.(1985) Blood66,946-952 [Abstract]
  24. Sims, P. J., Ginsberg, M. H., Plow, E. F., and Shattil, S. J.(1991)J. Biol. Chem.266,7345-7352 [Abstract/Free Full Text]
  25. Lollo, B. A., Chan, K. W. H., Hanson, E. M., Moy, V. T., and Brian, A. A.(1993) J. Biol. Chem.268,21693-21700 [Abstract/Free Full Text]
  26. Danilov, Y. N., and Juliano, R. L.(1989)J. Cell Biol.108,1925-1933 [Abstract]
  27. Haverstick, D. M., Sakai, H., and Gray, L. S.(1992)Am. J. Physiol. 262,C916-C926
  28. Savage, B., Shattil, S. J., and Ruggeri, Z. M.(1992)J. Biol. Chem. 267,11300-11306 [Abstract/Free Full Text]
  29. Kawaguchi, S., and Hemler, M. E.(1993)J. Biol. Chem.268,16279-16285 [Abstract/Free Full Text]
  30. Hibbs, M. L., Jakes, S., Stacker, S. A., Wallace, R. W., and Springer, T. A.(1991) J. Exp. Med.174,1227-1238 [Abstract]
  31. Chen, Y., Djaffar, I., Pidard, D., Steiner, B., Cieutat, A.-M., Caen, J. P., and Rosa, J.-P. (1992)Proc. Natl. Acad. Sci. U. S. A.89,10169-10173 [Abstract]
  32. Reszka, A. A., Hayashi, Y., and Horwitz, A. F.(1992)J. Cell Biol. 117,1321-1330 [Abstract]
  33. Poncz, M., and Newman, P. J.(1990)Blood75,1282-1289 [Abstract]
  34. Cieutat, A. M., Rosa, J.-P., Letourneur, F., Poncz, M., and Rifat, S.(1993) Biochem. Biophys. Res. Commun.193,771-778 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kaufmann, Y., Tseng, E., and Springer, T. A.(1991)J. Immunol.147,369-374 [Abstract/Free Full Text]
  36. Wilson, R. W., O'Brien, W. E., and Beaudet, A. L.(1989) Nucleic Acids Res.17,5397 [Medline] [Order article via Infotrieve]
  37. Yamada, K., Goncalves, E., Kahn, C. R., and Shoelson, S. E.(1992)J. Biol. Chem.267,12452-12461 [Abstract/Free Full Text]
  38. Carpenter, C. D., Ingraham, H. A., Cochet, C., Walton, G. M., Lazar, C. S., Sowadski, J. M., Rosenfeld, M. G., and Gill, G. N.(1991)J. Biol. Chem.266,5750-5755 [Abstract/Free Full Text]
  39. Escobedo, J. A., Barr, P. J., and Williams, L. T.(1988)Mol. Cell. Biol. 8,5126-5131 [Medline] [Order article via Infotrieve]
  40. Yan, H., Schlessinger, J., and Chao, M. V.(1991)Science252,561-563 [Medline] [Order article via Infotrieve]
  41. Bormann, B., and Engelman, D.(1992)Annu. Rev. Biophys. Biomol. Struct.21,223-242 [CrossRef][Medline] [Order article via Infotrieve]
  42. Heldin, C.-H. (1995)Cell80,213-223 [Medline] [Order article via Infotrieve]
  43. Weisel, J. W., Nagaswami, C., Vilaire, G., and Bennett, J. S.(1992) J. Biol. Chem.267,16637-16643 [Abstract/Free Full Text]
  44. Singer, S. J., and Nicolson, G. L.(1972)Science175,720-731 [Medline] [Order article via Infotrieve]
  45. Grasberger, B., Minton, A. P., DeLisi, C., and Metzger, H.(1986)Proc. Natl. Acad. Sci. U. S. A.83,6258-6262 [Abstract]
  46. O'Toole, T. E., Mandelman, D., Forsyth, J., Shattil, S. J., Plow, E. F., and Ginsberg, M. H.(1991)Science254,845-847 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.