From the
Department of Biochemistry and Molecular Biology, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, the
Service of Inmunology, Hospital de la Princesa, Universidad Autónoma de Madrid, 28006 Madrid, and the ¶Department of Biochemistry and Molecular Biology, Universitat de Barcelona, 08108 Barcelona, Spain
Received for publication, December 19, 2002 , and in revised form, February 12, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An advance in the understanding of CD45 function was the discovery of specialized membrane domains, called rafts, ganglioside-enriched membranes (GEMs), or detergent-resistant membranes (DRMs). These membranes contain a high density of sphingolipids and cholesterol (24) and serve as attachment sites for a variety of lipid-modified proteins (including GPI) and also integral membrane and cytoplasmic proteins (e.g. Src family kinases Lck and Fyn, CD4, CD8, and LAT (linker for activation of T cells)). A compartmentation model has been proposed in which the immunological synapse initiates the clustering and stabilization of the TCR by the formation of a large lipid microdomain that accumulates (e.g. CD4 and CD8) and segregates (e.g. CD45 and LFA-1) several membrane proteins (6). As the phosphorylation decreases within minutes after the initial response, other phosphatases (not CD45) should be recruited to these rafts later (5).
However, the role of the extracellular domain of CD45 remains elusive despite its high Mr and structure, which strongly suggest ligand-receptor interactions (6). The diversity of the structures and sizes of different CD45 isoforms is cell type-dependent and developmentally regulated. Upon T cell activation, naive T cells switch from isoforms containing A, B, or C epitopes, with post-translational (O- and N-linked glycosylation) information, to the lowest Mr isoform, CD45R0, which lacks sequences coded by 4/A, 5/B, or 6/C exons (68). Several experiments reported distinct CD45 interactions on naive and memory/effector (CD45R0+) cells. CD45 T cell lines transfected with cDNAs of different CD45 isoforms or cells from transgenic and knock-out mice had differential responses to Ag. CD45 has also been reported to associate with several surface molecules such as Thy-1, TCR, CD2, CD3, CD4, CD7, CD8, CD26, CD28, LFA-1, B cell receptor, lymphocyte phosphatase-associated protein, endoplasmic reticulum protein glucosidase II, and CD45 itself (69). As the different CD45 isoforms have similar PTP activities, these data suggest that they may interact differentially with other surface molecules that alter PTP accessibility to substrates, modifying in this way the signals received not only through Ag receptors but also through cytokine receptors and integrin-mediated adhesion, to either augment or inhibit T cell activation (912).
The surface CD26 glycoprotein is identical to dipeptidyl-peptidase IV (EC 3.4.14.5 [EC] ). T cells expressing high levels of CD26 constitute a subpopulation of CD45R0+ cells with helper activities in B cell Ig synthesis, proliferative responses to soluble Ags and allogeneic cells, secretion of TH1-type cytokines, and transendothelial migration capacity (13, 14). This study describes the distribution of CD26 and CD45R0 molecules in plasma membrane microdomains of fresh and activated human T cells; it also shows that IL-12 (an inducing TH1 response cytokine) dramatically changes CD45R0 membrane compartmentation through a CD26-CD45R0 association. The significance of this finding is discussed.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Isolation and CultureBuffy coats were kindly provided by the Centro de Transfusión de Galicia (Santiago, Spain). Blood was donated by healthy volunteers and human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque PLUS (Amersham Biosciences) density gradient centrifugation as described elsewhere (20). Cells were cultured (106 PBMCs/ml) in RPMI 1640 (Sigma) supplemented with 10% inactivated fetal calf serum (Invitrogen), 100 µg/ml streptomycin, and 100 IU/ml penicillin (Sigma) in a humidified atmosphere of 5% CO2 at 37 °C. PBMCs were activated with 11.5 µg/ml P. vulgaris lectin in the presence or absence of cytokines for the indicated time. The cell lines used were cultured under the same conditions except that Geneticin (100 µg/ml, Sigma) was added to the medium of CD26-transfected cell lines.
Inhibition of CD26 Expression with Antisense Morpholino Oligos Antisense morpholino oligos bound to partially complementary DNA and ethoxylated polyethileneimine (EPEI) were provided by Gene Tools as a Special Delivery Protocol kit. Morpholino subunits are assembled by phosphorodiamidate linkages to obtain an oligo with a modified non-ionic nuclease-resistant backbone. The CD26 antisense sequence, 5'-GAACCTTCCACGGTGTCTTCATCGT-3', was designed to bind to the target sequence in the region containing AUG of the post-spliced mRNA for the highest effectivity of translation blocking. The fluoresceinated 5'-CCTCTTACCTCAGTTACAATTTATA-3' morpholino oligo was used as a standard control for nonspecific effects. EPEI electrostatically binds the anionic morpholino-DNA duplex, generating a cationic complex that approaches the anionic cell surface, leading to endocytosis. Subsequent acidification within the endosome increases EPEI ionization driving to membrane permeabilization and oligo release into the cytosol.
The ratio of EPEI/morpholino oligos/PBMCs was tested for optimal internalization. Briefly, Morpholino-DNA duplex and EPEI (1.6 nM and 0.64 nM, final concentrations) were mixed and preincubated for 20 min in MilliQ (Millipore) water at room temperature, the mixture was added to the cells (4 x 106) in serum-free RPMI medium, and the samples were incubated for 3 h at 37 °C in a humidified atmosphere of 5% CO2 in air. Cells were then washed with RPMI and cultured as described above.
Immunostaining and ImmunofluorescenceCell surface Ag expression was measured by direct or indirect immunofluorescence as described (21). CD45RA, CD45RB, CD45RC, and sometimes TP1/16 mAbs were used as hybridoma supernatant and revealed with FITC- or PE-labeled GAM Abs. The other Abs were applied as primary antibodies. Viable lymphocytes were identified according to their forward and right angle scattering. The percentage of cells positive for the Ag was evaluated by setting negative controls as the omission of primary antibody or the inclusion of isotype controls. Direct immunofluorescent protocol was used for two-color experiments (21). For studies of detergent resistance of proteins associated with rafts, the work of Janes et al. (22) was adapted to flow cytometry. Briefly, cells were treated with 1% Triton X-100 for 5 min on ice, with 10 mM methyl--cyclodextrin (M
CD; Sigma), which depletes cellular cholesterol, for 15 min at 37 °C, or with M
CD followed by Triton X-100 extraction, before fixation (3% PFA in PBS for 30 min at room temperature) and staining as described above. Samples were processed on a BD Biosciences FACScalibur cytometer, except where indicated. WinMDI software (a kind gift of J. Trotter, Scripps Institute, La Jolla, CA) was used to analyze data.
Protein Concentration DeterminationThe protein concentration of samples was determined using the Bradford procedure (Sigma). Bovine serum albumin was used as a standard.
Membrane Phosphatase and Peptidase in Vitro AssaysCells were washed twice in RPMI 1640 and resuspended at 3 x 106 cells/ml in hypotonic lysis buffer (HLB) (25 mM Tris-HCl, pH 7.5, 25 mM sucrose, 0.1 mM EDTA, 5 mM MgCl2, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin) before sonication. Individual samples were made up in duplicate in the presence and absence of 1 mM Na3VO4. The membranes were sedimented from post-nuclear supernatants at 100,000 x g for 60 min at 4 °C, the resulting pellet was resuspended in 200 µl HLB by sonication, and the protein concentration was determined. Plasma membrane (PM) protein (20 µg) was incubated as described by Cayota et al. (23) in a reaction mixture of 5 mM p-nitrophenyl phosphate (Sigma 104® substrate), 80 mM MES, pH 5.5, 10 mM EDTA, and 10 mM dithiothreitol at 37 °C. After a 20-min incubation, reaction was stopped by the addition of 1 ml of 0.2 N NaOH. PTP activity was expressed as absorbance units at 405 nm after removal of values of the samples duplicated in the presence of vanadate. Dipeptidyl-peptidase IV activity was measured as described previously (24) and expressed as absorbance units at 405 nm. Briefly, PM protein (2040 µg) was incubated for 1 h at 37 °C in the presence of 0.5 mg/ml Gly-Pro p-nitroanilide tosylate (Sigma).
Isolation of GPI-enriched Membranes by Equilibrium Density Gradient CentrifugationThe following steps were carried out at 4 °C unless indicated otherwise, basically following the works of Ilangumaran et al. (25, 26). Cells (50 x 106) were washed twice in PBS and once in TKM buffer (50 mM Tris-HCl, pH 7.5, 25 mM KCl, 5 mM MgCl2, and 1 mM EDTA). Detergent lysates were prepared in TKM containing 0.5% Triton X-100 and the protease inhibitors Pefabloc SC (2 mM), leupeptin (10 µg/ml), and aprotinin (5 µg/ml) for 20 min on ice. For equilibrium gradient centrifugation, cell extracts were adjusted to 40% sucrose, loaded into SW55Ti tubes (Beckman L8-M), overlaid with 2.7 ml of 36% sucrose, and finally completed with 1.575 ml of 5% sucrose in TKM buffer. After centrifugation at 200,000 x g for 18 h, 450-µl fractions were collected and stored at 20 °C. Fraction proteins were evaluated by Western blotting or immunoprecipitation or were detected by dot immunoassay. Serial dilutions in PBS (200 µl) of detergent-lysates or sucrose density fractions were applied to the wells and dotted onto nitrocellulose filter sheets using a Bio-Rad dot-blot apparatus (25).
Western Blotting Analysis and ImmunoprecipitationDifferentially activated cells were washed in cold PBS and harvested in lysis buffer at 4 x 106 cells/100 µl (20 mM Tris, pH 7.4, 0.15 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1% Triton X-100; or alternatively, 20 mM triethanolamine, pH 7.8, 0.15 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.12% Triton X-100, and 1% digitonin as described by Torimoto et al. (27)). After 30 min on ice, nuclei and debris were removed by centrifugation at 13,000 x g for 15 min, and cleared lysates were assayed for protein content. Samples, normalized for total protein or for number of cells, were run on 7.5% SDS-PAGE and electrotransferred to nitrocellulose (Schleicher & Schuell) or polyvinylidene difluoride membranes (Amersham Biosciences) for analysis with the appropriate primary Ab (TP1/16, D3/9, or UCHL-1) and HRP-labeled secondary Ab. For Western blotting with D3/9 or TP1/16 Abs, samples were treated under nonreducing conditions with SDS-PAGE buffer at 37 °C for 15 min. Detection was carried out by the chemiluminescent system (ECL, Amersham Biosciences).
For immunoprecipitation studies, cell surface proteins were sometimes biotinylated following the manufacturer's instructions (Pierce). Briefly, 25 x 106 lymphocytes/ml were resuspended in PBS, pH 8.0, containing 0.5 mg/ml sulfo-NHS-biotin for 30 min at room temperature. When required, the depletion of microdomain-linked proteins was carried out according to Cheng et al. (28). Briefly, washed cells (50 x 106) were resuspended in 1 ml of ice-cold Hanks' balanced salt solution with 1 µg/ml HRP-conjugated cholera toxin B subunit (which binds membrane GM1; Sigma) for 30 min at 37 °C. Then cells were washed twice and resuspended in 1 ml of 0.5 mg/ml 33'-diaminobenzidine in Hanks' balanced salt solution with or without H2O2 for 45 min at 4 °C. After lysis, polymerized raft proteins were removed as described above.
Immunoprecipitation was performed with anti-CD26 TP1/16 (in contrast to 1F7, anti-CD26 TP1/16 was more effective with Triton than digitonin lysates), anti-CD45 D3/9, or anti-CD45R0 mAbs, previously coupled for 1 h at 4 °C to anti-mouse-agarose beads (Sigma; 50 µl of 1:1 lysis buffer). Occasionally, precleared lysates were incubated first with antibodies. Precipitated protein was recovered by centrifugation, washed three times in lysis buffer, eluted by boiling in SDS-PAGE sample buffer, and analyzed as described above, except for the filters with biotinylated proteins, which were incubated sequentially with streptavidin, biotinylated alkaline phosphatase, and CDP-Star substrate (New England Biolabs). Developed filters were exposed on X-Omat S film (Eastman Kodak Co.) several times depending on the intensity of the signal. When needed, the blots were stripped as described elsewhere (29).
Dot blots were treated as described above, except when measuring alkaline phosphatase (AP) levels. AP activity was developed directly with bromochloroindolyl phosphate/nitro blue tetrazolium substrate (BCIP/NBT, BioRad). The spots were quantitated by scanning the filters and densitometry (ImageMaster 1D, Amersham Biosciences).
Cell Proliferation and Calcium AssaysThree-day PHA-blasts were cultured in 96-well culture plates at 1 x 105 cells/well in a total volume of 100 µl of complete medium with/without 20% conditioned medium (medium in which PBMCs were activated previously for 3 days). In the preincubation assays, cells were cultured with the anti-CD45R0 and CD45RA Abs for 30 min before adding the cytokines, whereas in the postincubation assays, the Abs were added 30 min after the cytokines. Controls were cells incubated in the absence of cytokines. After 2 days of culture, 20 µl/well CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI) was added to the plate 4 h before the absorbance was recorded at 492 nm in a Labsystems Multiskan MS plate reader. All cultures were performed in triplicate.
For calcium measurements, 5 x 106/ml washed cells were resuspended in calcium-containing assay buffer (145 mM NaCl, 5 mM KCl, 0.5 mM MgSO4,5mM glucose, 1 mM CaCl2,10mM HEPES, 1 mM Na2HPO4) with 4 µg/ml fluo-3-penta-acetoxymethylester (Fluo-3 AM; Molecular Probes, Eugene, OR) in the presence of 0.025% Pluronic F-127 (Molecular Probes) and 2 mM probenecidin (Sigma) for 30-45 min at 37 °C. After washing, cells were diluted (250,000 cells/ml) and warmed prior to use (37 °C for 15 min). Lymphocyte stimulation was carried out with anti-CD3 mAb OKT3 (t = 80 s) plus GAM H+L mAb (t = 280 s). Analyses were performed by flow cytometry and MFI 3.4J2 software, a kind gift of Eric Martz (Scripps Institute), for mean fluorescence intensity data.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Because CD26 is known to associate with CD45 (69), we performed immunoprecipitation studies. Fig. 1C shows that, in the presence of IL-12 together with the expected CD26 up-regulation, anti-CD26 TP1/16 mAb also coprecipitates more CD45 (the two low Mr isoforms). In consonance with the above result, neither CD45 nor R0 Ags (which were surface biotinylated) were down-regulated by IL-12. The nature of the coprecipitated bands is confirmed later on in this article.
An attractive model to explain these results is that IL-12 is up-regulating a certain type of CD26 expression (already existing; see Fig. 1C) that induces interaction with CD45R0 and/or other molecules and masks the anti-R0 Ab epitope. Thus, as observed in Fig. 1, UCHL-1 could precipitate only the free CD45.
CD26 and CD45 Are Present in PM Microdomains, and Their Distribution Changes with T Cell Activation in the Presence or Absence of IL-12CD26 was found in GEMs (4) of mouse T cell lines (2526) and porcine lung (32). We considered a possible relationship between the CD26-CD45R0 association and GEMs/PM rafts. The well known GPI-anchored (32) AP was used as a control for microdomain purification by equilibrium density gradient centrifugation. AP was detected in the light density fractions (LDF) (Fig. 2A, lanes 46). Transferrin R (CD71) was used as control for soluble, non-raft protein (data not shown).
|
The dual distribution of CD26, with 28% of the material detected in the LDF and 51% in the heavy density fractions (HDF) (Fig. 2A, lanes 10 and 11) of the sucrose gradient, is shown in human T cells (because monocytes and B and NK cells from PBMCs are essentially CD26) (Fig. 2C). In activated T cells, in addition to a higher intensity, CD26 was redistributed to the GEMs (37% in LDF and 30% in HDF, percentages that are in agreement with data from mouse T cell line (25), indicating the activated status of T cells). IL-12 enhanced CD26 intensity and enriched the intermediate fractions (33 and 26%, respectively in LDF and HDF, n = 4; Fig. 2, C and F). Note that there is more CD26 than the well described CD4 glycoprotein (which shows a bipolar pattern) in the rafts. In fact, CD26 is present in almost all sucrose gradient fractions, including fraction 3 (AP and CD4 are not detected in this fraction) (Fig. 2B).
As described previously in a murine T cell lymphoma (25) but not in Jurkat or B cells (22, 28, 33, 34), CD45 also associates with GEMs in human lymphocytes (Fig. 2D). The ratio of LDF/HDF was higher for total CD45 and R0 than for CD26. The differences in intensity between PBMC and PHA-activated lymphocyte fractions again reflect total and CD45R0 up-regulation. In comparison with CD26, PHA hardly affects CD45 redistribution (34% in LDF and 37% in HDF, n = 4; 37 and 32%, respectively, for CD45R0). In contrast to CD26, where the ratio LDF/HDF was constant, the presence of IL-12 in T cell activation reduced the percentages of CD45 in the rafts (or in fractions near the rafts). This redistribution can be attributed to the R0 isoform (Fig. 2E) (23% in LDF and a spectacular 51% in HDF, n = 5).
CD26-CD45R0 Association Is Near but Outside of RaftsDo CD26 and CD45R0 interact in a particular PM microdomain? A new approach was used, based on the resistance of rafts to solubilization by nonionic detergents and flow cytometry. As stated previously, a fraction of CD4, CD8, and CD26 molecules is present, and CD3, CD71, CD45, and HLA-DR are absent or weakly associated with lipid rafts (3, 5) (TCR cross-linking stabilizes CD3 against detergent) (22). After treating effector T cells with TX-100, only CD8, CD26, and interestingly CD45R0 staining was partially maintained (Fig. 3A). CD4 staining was lost, suggesting that our treatment was harsh enough to deplete CD4 from lipid rafts. The different solubility of CD8 may be explained by its preferential interaction with raft-associated LAT protein (35). Additional controls for AP activity in the extracted fraction and the remaining PM proteins showed very restrictive conditions for this approach (data not shown). As observed in Fig. 3B, the loss of CD45R0 staining in IL-12 cultures was maintained after Triton X-100 treatment. Cells were also treated with MCD, which disrupts rafts by depleting cellular cholesterol (22, 3436). The treatment alone did not avoid the loss of CD45R0 staining. Even after M
CD plus TX-100 treatment, CD26 and CD45R0 were detected, suggesting that a fraction of both molecules is tightly attached to the lipid microdomain core. Note that in IL-12 blasts, the most R0 was extracted with TX-100 alone, i.e. it is present in the soluble fraction. These results support those reported in the previous section. In addition, IL-12-dependent loss of CD45 staining occurred only under M
CD plus Triton X-100 treatment. It is interesting to note that CD26+ cells present weaker CD45R0 staining than CD26 cells even in the absence of IL-12 (Fig. 3B, control PHA), which could represent the CD26/CD45R0 association observed by immunoprecipitation.
|
The specificity of these results was confirmed by coprecipitation assays following two different experimental approaches. In the first one (Fig. 4A), HDF and LDF pooled samples from PHA or PHA+IL-12-stimulated blasts containing the same amount of protein were incubated with TP1/16 mAb, and immunoprecipitates were revealed with UCHL-1 mAb. Although coprecipitation could not be detected in pooled insoluble fractions from the density gradient (possibly because of sample manipulation), IL-12 enhanced CD45R0 coprecipitation. In the second approach, the selective elimination of raft-associated proteins by the HRP-33'-diaminobenzidine reaction (28) did not affect coprecipitation between CD26 and CD45R0 Ags in PHA-blasts and diminished CD45R0 coprecipitation (still higher) in IL-12-treated cells (Fig. 4B). The results altogether demonstrate that IL-12 induces a stronger CD26-CD45R0 association near (or on the outer edge of) PM raft regions. However, if loss of raft-associated CD45R0 after the HRP-33'-diaminobenzidine reaction correlates with the R0 Ag remaining after TX-100 treatment (Fig. 3B, IL-12 dot plots), it is implicit that part of the striking IL-12-dependent CD45R0 microdomain redistribution described in the previous section could be explained by CD26 masking the UCHL-1 epitope (in rafts or next to rafts).
|
CD26 Is Involved in Pulling Out CD45R0 from RaftsA related question is whether both events enhanced by IL-12, the association and the membrane compartment redistribution, are connected. With the addition of antisense oligos for the CD26 mRNAs to the above described culture conditions, which resulted in a drop of the cell surface CD26 expression, it was demonstrated that loss of R0 staining in IL-12-cultured cells is associated specifically (oligonucleotide controls did not show this effect; data not shown) with a de novo expression of certain CD26 protein (Fig. 5A, right). This expression can be observed even with PHA stimulation alone (Fig. 5A, left). Experiments with brefeldin A, which avoids surface protein expression, corroborated this finding (data not shown).
|
Inhibition of CD26 biosynthesis in these cells scarcely affected CD26 distribution or R0 detection (Fig. 5B); however, IL-12-dependent redistribution of CD45R0 from GEMs to soluble membrane is avoided in cells treated with antisense, but not control, oligonucleotides. in consonance with flow cytometry results, the presence of more R0 in rafts is observed even in PHA blasts.
Is the IL-12-dependent CD26-CD45R0 Association Functionally Relevant?To verify whether incubations with soluble UCHL-1 Ab could block CD26-CD45R0 association, proliferation assays of 3-day PHA-blasts cultured for 2 additional days with UCHL-1 Ab or RP1/11 (anti-CD45RA) (data not shown), added to the cultures 30 min before or after the addition of cytokines, were carried out. A small Ab concentration was able to induce proliferation. In IL-12 cultures, the enhancing effect of UCHL-1 Ab was lost at the lowest doses of preincubation (Fig. 6A). The behavior was totally different when the Ab was added after the IL-12. If UCHL-1 effectively blocked the CD26-CD45R0 association, at least that induced by IL-12, these results suggest that the interaction regulates T cell proliferation. However, the results of postincubation imply a process of fast kinetics.
|
Another approach was to stimulate 5-day PHA or PHA+IL-12 lymphoblasts (the latter with less CD45R0 in rafts) through the TCR and measure changes in [Ca2+]. A reduced calcium response was detected in PHA+IL-12 blasts (Fig. 6B), although an identical percentage (64%) of responding cells in both blast types was observed. These results were reinforced by those from antiphosphotyrosine-Western blotting (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
However, a role in this process for a CD26-CD45 association (3739, 44) cannot be discarded, because a percentage of the PTP CD45 is associated to GEMs in human fresh T cells (B lymphocytes from PBMCs constitutively lack CD45 in GEMs (28)) and PHA-blasts. This result is not unexpected because CD45 is associated with the GPI-protein Thy-1 (CD90) and with raft-associated TCR in cell lines, probably resembling an effector state (38); nevertheless, models of membrane compartmentation after B- or T-response to antigen have been proposed showing that CD45 may be excluded from the signaling complex raft (6, 33). It is interesting to note for later discussion that these experiments have used B or Jurkat cells and both do not express CD26.
This CD45, present in rafts of effector/memory cells in proportions similar to CD26, can be ascribed to the R0 isoform. From our data on cells treated with Triton X-100 and MCD, it is easily deduced that a fraction of CD26 and CD45R0 is more resistant to the treatment than is CD4 or CD8, suggesting that this fraction is inside of the raft core. A recent report confirms that CD26 and CD45R0 can be found in membrane rafts of CD26-transfected Jurkat cells under certain conditions (44).
These data do not invalidate the model of compartmentation, which implies increased stability of phosphorylation (3, 5, 33), when considering naive T cells (they do not express the CD45R0 isoform). However, our findings appear to play a role in the far more efficient in responding to stimulation effector/memory CD45R0+ cells. In these T cells, in which the rafts are bigger than in naive cells where CD45 can be exposed at the edges of smaller rafts (5, 46), low Mr CD45 PTP activity may be necessary to maintain the activation of Src family kinases in rafts and to prime them to triggered TCR. Early observations of cocapping (4648) have shown that CD4, low Mr CD45, and the CD3-TCR complex behave as independent entities on the surface of naive T cells, whereas they are stably associated on memory T cells and TH1 clones. These data, taken together, fit into the idea that low Mr isoforms of CD45 could be associated with rafts, whereas the large CD45 isoforms would be excluded (6), and support a more complex model of compartmentation depending on the stage of T cell maturation (49).
The present work shows that IL-12 drives CD26 and CD45R0 to particular PM regions. It has been reported that IL-2 pulls out the chain from rafts to bind the
and
chain of the IL-2 receptor complex in soluble fractions (50). Hence, interleukins are involved in the rules governing the inclusion and exclusion of proteins into rafts. The finding that IL-12 dramatically changes the distribution of CD45R0 from rafts to soluble fraction may explain the impaired Ca2+ responses observed upon activation via TCR in IL-12-cultured cells. R0 redistributes from rafts perhaps to control IL-12 receptor-dependent signal transduction through suppression of the JAK-STAT (Janus kinase/signal transducers and activators of transcription) pathway as described for other cytokine receptors (12, 51).
Physical association between CD26 and CD45 (particularly the R0 form) has long been reported (27). The results presented here show that in cells triggered by IL-12, even more CD26 and CD45R0 proteins are associated. Although some interleukins can regulate CD45 isoforms (52, 53), the close CD26-CD45R0 association was seen only with IL-12. Taking these results together with those of in vivo elimination of rafts and CD26 expression inhibition, it can be concluded that the new surface CD26 molecules are directed to the soluble fraction near the lipid raft core and anchor CD45R0. As IL-12-dependent R0 membrane compartment redistribution is avoided in the absence of de novo CD26 expression, CD26 may be part of a shuttling mechanism for CD45. It has been reported recently that raft CD45 exchanges with the larger pool of freely diffusing CD45 after the formation of the immunological synapse (54).
Only a fraction of CD26 molecules associate with CD45R0, and IL-12 did not change the percentage (obtained from coprecipitation experiments). This finding suggests that the extracellular domains of both proteins control the interaction, although a direct binding of CD26 to the cytosolic domain of CD45 in an internalization model cannot be excluded (44). The fact that the extracellular domain of low Mr CD45 controls the association with the CD4-TCR complex (4648) and the differential homodimerization of isoforms (a way of negative regulation of PTP CD45) (9) supports our results. In this sense, one should be aware that recombinant proteins can mask certain post-translational modifications necessary for the binding and modulation of protein function. CD45R0 itself is an example (55).
PTP activity is also important in the regulation of adhesion-triggered tyrosine kinase cascades (note that several of these studies were done with CD26 Jurkat cells (3, 6, 10, 56), and CD26 can be a functional adhesion receptor (14, 39, 5659), and thus microdomain redistribution could regulate their presence at integrin-dependent adhesion sites. One can speculate on the importance of this mechanism for normal T cell behavior and, further, on the possibility that it could be blocked by particles or molecules from pathogens. Both CD26 and CD45 molecules are involved in the regulation of lymphocyte death (2, 17, 60) and in the pathophysiology of AIDS (8, 23, 39, 61). At least HIV-1 gp-120 (62), but perhaps also Tat (63, 64) and Gag (34), modulate CD4 lateral interactions with them.
![]() |
FOOTNOTES |
---|
|| To whom correspondence should be addressed: Dept. de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela, Facultade de Bioloxía, Campus Sur, 15782 Santiago de Compostela, Galicia, Spain. Tel.: 34-981-56-31-00 (ext. 13301); Fax: 34-981-59-69-04; E-mail: bnlmna{at}usc.es.
1 The abbreviations used are: Ag, antigen; Ab, antibody; mAb, monoclonal antibody; PTP, protein-tyrosine phosphatase; TCR, T cell receptor; PM, plasma membrane; GEM, ganglioside-enriched membrane; GPI, glycosylphosphatidylinositol; AP, alkaline phosphatase; PBMC, peripheral blood mononuclear cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; HDF, heavy density fraction; LDF, light density fraction; MCD, methyl-
-cyclodextrin; IL, interleukin; TH1 cell, T helper-1 cell; GAM, goat anti-mouse; HRP, horseradish peroxidase; PHA, phytohemagglutinin; EPEI, ethoxylated polyethileneimine; oligos, oligonucleotides; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|