TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts
2002; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês
10.1093/emboj/21.8.1899
ISSN1460-2075
AutoresPhilippe Drevot, Claire Langlet, Xiaojun Guo, Anne-Marie Bernard, Odile Colard, Jean‐Paul Chauvin, Rémi Lasserre, Hai‐Tao He,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoArticle15 April 2002free access TCR signal initiation machinery is pre-assembled and activated in a subset of membrane rafts Philippe Drevot Philippe Drevot Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Claire Langlet Claire Langlet Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Xiao-Jun Guo Xiao-Jun Guo LBBN, CNRS-ESA 6033, Faculté des Sciences et Techniques de Saint-Jérôme, F-13397 Marseille, Cedex 20, France Search for more papers by this author Anne-Marie Bernard Anne-Marie Bernard Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Odile Colard Odile Colard INSERM U538, CHU Saint-Antoine, 27 rue de Chaligny, F-75012 Paris, France Search for more papers by this author Jean-Paul Chauvin Jean-Paul Chauvin LGPD-IBDM, Case 907, F-13288 Marseille, Cedex 9, France Search for more papers by this author Rémi Lasserre Rémi Lasserre Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Hai-Tao He Corresponding Author Hai-Tao He Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Philippe Drevot Philippe Drevot Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Claire Langlet Claire Langlet Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Xiao-Jun Guo Xiao-Jun Guo LBBN, CNRS-ESA 6033, Faculté des Sciences et Techniques de Saint-Jérôme, F-13397 Marseille, Cedex 20, France Search for more papers by this author Anne-Marie Bernard Anne-Marie Bernard Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Odile Colard Odile Colard INSERM U538, CHU Saint-Antoine, 27 rue de Chaligny, F-75012 Paris, France Search for more papers by this author Jean-Paul Chauvin Jean-Paul Chauvin LGPD-IBDM, Case 907, F-13288 Marseille, Cedex 9, France Search for more papers by this author Rémi Lasserre Rémi Lasserre Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Hai-Tao He Corresponding Author Hai-Tao He Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France Search for more papers by this author Author Information Philippe Drevot1, Claire Langlet1, Xiao-Jun Guo2, Anne-Marie Bernard1, Odile Colard3, Jean-Paul Chauvin4, Rémi Lasserre1 and Hai-Tao He 1 1Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Mediterranee, Case 906, F-13288 Marseille, Cedex 9, France 2LBBN, CNRS-ESA 6033, Faculté des Sciences et Techniques de Saint-Jérôme, F-13397 Marseille, Cedex 20, France 3INSERM U538, CHU Saint-Antoine, 27 rue de Chaligny, F-75012 Paris, France 4LGPD-IBDM, Case 907, F-13288 Marseille, Cedex 9, France ‡P.Drevot and C.Langlet contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1899-1908https://doi.org/10.1093/emboj/21.8.1899 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent-insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37°C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of ∼70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR–CD3 complex, Lck and ZAP-70 kinases, and CD4 co-receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation. Introduction Membrane rafts are found in all mammalian cell types as well as in Drosophila, Dictyostelium and yeast (Simons and Ikonen, 1997; Brown and London, 1998; Simons and Toomre, 2000). Not only are rafts enriched in sphingolipids (sphingomyelins and glycosphingolipids) and cholesterol, but these constituents are essential for the formation of rafts (Simons and Ikonen, 1997; Brown and London, 2000). An increasing amount of data suggest that rafts play fundamental roles in diverse cellular functions, particularly in signal transduction, by promoting a segregated arrangement of membrane proteins and lipids (Brown and London, 2000; Simons and Toomre, 2000). Studies in model membranes indicate that rafts correspond to a phase of the lipid bilayer, namely the liquid-ordered (lo) phase (Brown and London, 2000; Simons and Toomre, 2000). The formation of this lo phase is promoted by sphingolipids, the long saturated acyl chains of which allow tight molecular packing (Brown and London, 2000), and further facilitated by the presence of cholesterol (Simons and Ikonen, 1997; Brown and London, 2000). The use of GPI-anchored proteins and other raft markers to investigate the existence of rafts in living cells has revealed that they are usually very small in size (Simons and Toomre, 2000). Engagement of the T cell receptor (TCR) by its specific peptide-MHC (pMHC) ligand triggers intracellular signaling cascades that are required for T lymphocyte development and functions (for a review see Weiss and Littman, 1994). Such cascades are initiated by the activation of a signal transduction machinery, the main components of which include the TCRαβ heterodimer and the tightly associated CD3 ϵ, γ, δ and ζ polypeptides, Lck and ZAP-70 kinases, and the CD4 (or CD8) co-receptor. Recognition of pMHC by TCR results in phosphorylation of the cytoplasmic domains of the CD3 complex by the src kinase Lck, which permits the recruitment and activation of the syk kinase ZAP-70 (Weiss and Littman, 1994). During pMHC recognition, CD4 (also CD8) is believed to bind to the same pMHC molecule as the TCR, thus contributing to the recruitment of the Lck via its cytoplasmic tail to the TCR–CD3 complex engaged by the class II (I) pMHC (Janeway, 1992). This tyrosine kinase recruitment strongly ‘boosts’ TCR recognition (Janeway, 1992; Krummel et al., 2000). Recent reports have ascribed a crucial role for rafts in the activation of the TCR signaling cascades (Montixi et al., 1998; Moran and Miceli, 1998; Xavier et al., 1998; Zhang et al., 1998; Janes et al., 1999; for reviews see Langlet et al., 2000; Harder, 2001). In particular, upon TCR engagement the activated TCR–CD3–ZAP-70 complexes are found within rafts where they phosphorylate the raft resident protein LAT (linker for the activation of T cells), the major substrate of ZAP-70 and a central signaling scaffolder (for a review see Zhang and Samelson, 2000). Although substantial evidence supports the implication of rafts in TCR-dependent signaling cascades, a key issue remains unclear: how is TCR ligation coupled to the activation events in rafts? Initial data (Montixi et al., 1998; Xavier et al., 1998) led to the proposition that TCR moves into rafts upon ligation, perhaps after an initial phosphorylation of the TCR–CD3 (Montixi et al., 1998). ZAP-70 is then recruited to rafts by the phosphorylated TCR–CD3 and activated to phosphorylate LAT. However, some observations have suggested that the TCR–CD3 complex could already associate weakly with rafts prior to ligand binding (Montixi et al., 1998; Janes et al., 1999), which might be inefficiently detected by the cold Triton X-100-based raft isolation method (Janes et al., 1999; Langlet et al., 2000). In this paper, we have demonstrated the existence of detergent-insoluble microdomains exhibiting the expected biochemical characteristics of rafts at 37°C. These findings strongly support the existence of rafts at physiological temperature. They also are the basis for the development of a new raft-isolation method that has enabled us to investigate further the role of rafts in TCR signal transduction. We showed that a fraction of TCR–CD3 and ZAP-70 are constitutively associated to rafts. Moreover, using a CD4+ T cell line, we uncovered the existence a raft subset that localizes essentially all the raft-associated TCR–CD3 complex, enriched in the CD4 co-receptor and containing Lck and ZAP-70 kinases. Our results further indicated that TCR initiates signaling upon ligation in this raft subset, which is accompanied by rapid propagation to other raft populations. Taken together, these data provide new insights into how rafts take part in TCR signal transduction. Results Isolation and characterization of detergent-insoluble membrane microdomains using Brij 98 Lo and liquid-disordered (ld) phases display different behaviors when treated with non-ionic detergents. Resistance to solubilization by Triton X-100 at 4°C has been widely utilized as a basis for isolating raft membranes (Simons and Ikonen, 1997; Brown and London, 1998). However, Triton X-100-resistant membrane complexes could only be found at low temperatures. This raises the question of how well these membrane complexes reflect raft domains in cell membranes under physiological conditions, since chilling is believed to strongly modify the phase behavior of membranes (Brown and London, 2000). In addition, Triton X-100 has a limited capability to distinguish ordered from disordered membrane domains, since the lo phase is substantially solubilized by this detergent even at 4°C (Brown and London, 2000). To find a detergent that could discriminate more ordered domains from their phospholipid environments at 37°C through selective solubilization of the latter, we screened members of the polyoxyethylene ether (Brij) series. We selected Brij 98, characterized by its relatively bulky polyoxyethylene headgroup and mono-unsaturated ether moieties, which predicted a preferential partitioning in (and therefore solubilization of) the loosely packed, fluid phase, rather than the tightly packed, ordered phase of the lipid bilayer. Figure 1 shows that following incubation of a post-nuclear supernatant (PNS) of mouse thymocytes with 1% Brij 98 for 5 min at 37°C, low-density detergent-insoluble membranes (DIMs) are separated from the solubilized material by ultracentifugation in a sucrose density gradient. The DIMs floated to the buoyant fractions, whereas the soluble counterpart remained in the heavy (H) fractions of the gradient. These DIMs contained only ∼2.5% of total proteins found in the PNS (Figure 1A), yet were markedly enriched in membrane proteins modified by saturated-chain lipids such as (GPI-anchored) Thy-1, (dual palmitoylated) LAT and (myristoylated and palmitoylated) Lck, as well as GM1 glycosphingolipid (Figure 1B). In contrast, membrane proteins modified by prenyl groups, which have a branched and bulky structure, such as Rab-5 and Gβ that should be partitioning to disordered phases (Melkonian et al., 1999) were fully solubilized (Figure 1B). Figure 1.(A) PNS of thymocytes (2 × 108) was treated with Brij 98 at 37°C and fractionated on a sucrose gradient as described in Materials and methods. A 50 μl aliquot of each fraction was resolved on SDS–PAGE and stained with Coomassie Blue. The gel was scanned and protein bands were quantitated using the NIH image version 1.42 software system. (B) A 50 μl aliquot of each fraction of the gradient was blotted with the specific probes, as indicated. For the analysis of Gβ, the PNS was treated with 100 mM GTPγS in order to promote the dissociation between Gβγ and Gα. (C) Lipids were extracted from thymocyte DIM fraction and from total membranes in the PNS and subjected to high-performance thin-layer chromatography (HPTLC) as described in the Supplementary data. Lipids extracted from DIMs (lanes DIM, each from 9 × 107 cells) or from total membranes (lanes TM, each from 3 × 107 cells) were revealed by primulin. Migration standards (lanes S) included: CL, cholesterol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, sphingomyelin. Download figure Download PowerPoint Lipid analysis (Figure 1C) showed that, in comparison with the total membranes present in the PNS, DIMs were rich in sphingomyelins and cholesterol but poor in phospholipids. Indeed, quantitative studies indicated that the recovery of sphingomyelin, cholesterol and total phospholipids in DIMs were 43 ± 12.3%, 32.9 ± 3% and 9.4 ± 2.2%, respectively, of those found in total membranes. Therefore, both protein and lipid composition of DIMs correspond to those expected for raft domains (Simons and Ikonen, 1997; Brown and London, 2000). Cholesterol is known to be critically involved in the formation of ordered raft domains (Simons and Ikonen, 1997; Brown and London, 2000). Therefore, we evaluated the effect of saponin on Brij 98 DIMs. Figure 2A indicates that cholesterol sequestration by saponin completely disrupts Brij 98 DIMs. Moreover, we observed a significant reduction of DIM recovery when Brij 78 (C18.0E20) was used instead of Brij 98 (C18.1E20) to solubilize membranes (Figure 2B). The two detergents have the same headgroup and only differ by one double bond present in the alkyl moiety of Brij 98, but not Brij 78. Although the two detergents display the same hydrophilic–lipophilic balance (HLB = 15.3), Brij 78 is expected to have higher partitioning into the ordered domains and promotes their solubilization. Together, these results suggest that Brij 98 DIMs might exhibit properties of ordered domains. However, further investigation will be required to address this issue directly. Figure 2.(A) Thymocyte PNS was treated with Brij 98 at 37°C in the presence or absence of 0.2% saponin (Sp). The presence of DIMs was monitored by the distribution of Lck and GM1 over the sucrose gradient. (B) Thymocyte PNS was treated with Brij 98 or Brij 78 at 37°C, and fractionated in the sucrose density gradient before being blotted with the indicated antibodies (Abs). A schema of the structure of Brij 98 and Brij 78 is included. (C) Thymocyte DIMs were concentrated and subjected to EM analysis as described in the Supplementary data. DIM size was analyzed using Scion Image Beta 4.02 software and a representative result from a measurement of 195 vesicles is shown. Inset shows parts of an electron micrograph. Bar, 100 nm. (D) DIMs from Thy-1.2-expressing (lanes 1, 2, 7 and 8) or Thy-1.1-expressing (lanes 3, 4, 9 and 10) AKR1 thymomas, or a mixture (1:1) of both (lanes 5, 6, 11 and 12) were immunoisolated and blotted with the indicated mAbs. DIMs from 3 × 107 (lanes 1, 3, 5, 7, 9 and 11) and 6 × 107 (2, 4, 6, 8, 10 and 12) thymoma cells were analyzed. (E) Thymocyte PNS was either solubilized for 5 min with 1% Brij 98 at 37°C, diluted by the cold sucrose containing buffer A and boiling in SDS sample buffer (lanes 1 and 3), or immediately boiling in SDS sample buffer (lanes 2 and 4). The proteins were resolved on SDS–PAGE and revealed by Coomassie Blue staining (lanes 1 and 2) or immunoblotting with 4G10 anti-phosphotyrosine (PY) mAb (lanes 3 and 4). Download figure Download PowerPoint Electron microscopy examination shows that Brij 98 DIMs appear as membrane vesicles exhibiting a broad range of sizes with a mean diameter of 67 ± 39 nm (Figure 2C). Fusion among heterologous membrane fragments did not take place during Brij 98 detergent extraction at 37°C, since from a mixture of cells expressing either Thy-1.1 or Thy-1.2 the isolated DIMs were positive for only one, but not both Thy-1 alleles (Figure 2D). These results also rule out de novo DIM formation in vitro during PNS chilling following detergent treatment at 37°C. Another concern related to the membrane solubilization at 37°C is the possible increase in enzymatic degradation. However, we found no significant difference in the profile of total and tyrosine phosphorylated (Ptyr) proteins in PNS subjected or not to the 37°C incubation step (Figure 2E). Besides the inclusion of enzymatic inhibitors, the relatively short solubilization period (5 min) seemed to be important in this context. Our data thus suggest that Brij 98 DIMs correspond to the raft membrane microdomains that are present at physiological temperature. Constitutive partitioning and activation upon receptor ligation of TCR–CD3 and ZAP-70 in rafts Using the new raft isolation procedure, we analyzed the initiation of TCR signaling pathways in these membrane domains. The first signaling events following TCR engagement include a series of protein tyrosine phosphorylations, particularly by Lck and ZAP-70 kinases. To examine these events, we used mature T lymphocytes rather than thymocytes, which display a marked heterogeneity in terms of cell populations and TCR responses. Figure 3A shows that a stimulation of 30 s with anti-CD3ϵ monoclonal antibody (mAb) triggered an immediate appearance of Ptyr substrates in both the raft and non-raft fractions. Nonetheless, each fraction displays a specific pattern. In the raft fraction, in addition to phospho-LAT, we also detected the phospho-CD3ζ chain p23ζ and phospho-ZAP-70. p23ζ is fully phosphorylated at all of its three immunoreceptor tyrosine-based activation motifs (ITAMs) (van Oers et al., 2000) and ZAP-70 phosphorylation indicates its catalytically active form (Weiss and Littman, 1994). Both events depend on Lck, and the appearance of p23ζ has been correlated with the activation of ZAP-70 (van Oers et al., 2000). The data in Figure 3A indicate that upon TCR stimulation the ratio of p23ζ to either the constitutively phosphorylated p21ζ or the non-phosphorylated np16ζ were much higher in raft than non-raft compartments. This finding suggests that during TCR signal initiation the TCR–CD3 complex becomes highly activated in raft microdomains. Moreover, although a much stronger ZAP-70 signal was detected in the non-raft fraction, phospho-ZAP-70 was seen readily only in the raft fraction (also see Figure 3B). Taken together, these results strongly corroborate the suggestion that raft membrane microdomains play an important role in the activation of the earliest TCR signaling steps (Montixi et al., 1998; Xavier et al., 1998; Janes et al., 1999). Figure 3.(A) Nylon wool-purified splenic T cells (2 × 108) were stimulated or not with 15 μg/ml of 145-2C11 anti-CD3ϵ mAb at 37°C for 30 s. Proteins in raft (DIM) and one-quarter of non-raft (H) fractions were concentrated and blotted with anti-PY mAb. The positions of phospho-CD3ζ p23 and p21, phospho-LAT and phospho-ZAP-70 are indicated. The blot was then stripped and reprobed with anti-CD3ζ (that reveals non-phosphorylated CD3ζ np16) and anti-ZAP-70 Abs, respectively. (B) 3A9 T cell hybridomas (2 × 107) were incubated for 30 s at 37°C with the paraformaldehyde-fixed LK35.2 B cells (1 × 107) pulsed with or without the HEL peptide, as described in Materials and methods. Raft (DIM) and non-raft (H) fractions were prepared and treated for raft solubilization with 60 mM N-octylglucoside for 1 h at 4°C, before being subjected to immunoprecipitation with 145-2C11 anti-CD3ϵ mAb (Montixi et al., 1998). Immunoprecipitates were blotted with anti-PY mAb. The blot was then stripped and reprobed with the indicated Abs. Download figure Download PowerPoint In contrast to previous studies, we found that both the p21ζ and np16ζ, which account for the quasi-totality of the CD3ζ chains, and ZAP-70 constitutively partitioned in rafts, and this distribution was not significantly modified following the TCR–CD3 engagement (Figure 3A). Semi-quantitative analysis using an anti-TCR mAb revealed that at steady state ∼10% of the total TCR is associated with rafts in splenic T lymphocytes (Supplementary figure 1 available at The EMBO Journal Online). We also conducted experiments in 3A9 T cell hybridomas harboring a TCR that recognizes a peptide (residues 48–63) of hen egg lysozyme (HEL) in the context of H-2Ak (Liu et al., 2000) to examine TCR signal transduction triggered directly by pMHC. To this end, 3A9 cells were incubated at 37°C for 30 s with LK35.2 antigen presenting cells prior to raft isolation. Supplementary figure 2 shows that addition of HEL peptide induced an immediate accumulation of Ptyr proteins, including the phosphorylated forms of CD3ϵ, CD3ζ, ZAP-70 and LAT in rafts of 3A9 cells. In addition, we again observed a constitutive raft-partitioning of TCR–CD3 and ZAP-70. However, in contrast to primary T cells (Figure 3A), TCR stimulation in 3A9 cells resulted in an increase of ZAP-70 in rafts, which paralleled an augmentation of p21ζ. Finally, the amount of TCR–CD3 in rafts remained unchanged following treatment with PP2, an inhibitor of src family kinases, indicating that the constitutive raft-association of TCR–CD3 occurs in a manner that is independent of src kinase activity (Supplementary figure 3). Immunoprecipitation analysis shows that upon stimulation by the HEL peptide, TCR–CD3 complexes in the raft fraction are enriched in phospho-CD3 chains compared with the non-raft fraction (Figure 3B). It is of note that relative to the amount of CD3ϵ, much less CD3ζ was co-precipitated in the non-raft fraction. The reason for this is presently unknown. The most significant finding in this experiment was that although ZAP-70 could be co-immunoprecipitated from both raft and non-raft fractions by anti-CD3ϵ mAb after TCR stimulation, phosphorylated (activated) ZAP-70 was only seen in the immunoprecipitate from the raft fraction (Figure 3B). Thus, these results demonstrate that raft microdomains are privileged membrane sites where TCR signaling initiation pathways are organized and activated. Rafts are required for the initiation of TCR signal transduction The requirement for rafts in early TCR signaling events such as induction of tyrosine phosphorylation and Ca2+ flux has previously been studied by using methyl-β-cyclodextrin as well as filipin and nystatin (Xavier et al., 1998), all of which affect raft integrity via cholesterol depletion or sequestration (Simons and Toomre, 2000). However, some concerns have recently been raised about the use of these compounds as they seem to induce on their own transient tyrosine phosphorylation of the TCR signaling proteins, e.g. CD3ζ, ZAP-70 and LAT in Jurkat T cells (Kabouridis et al., 2000). To clearly establish the role of rafts in TCR signal transduction, we altered raft microdomains through membrane cholesterol modification by cholesterol oxidase. Xu and London (2000) have recently reported that cholesterol, once oxidized, is incapable of promoting the formation of lo phases in model membranes. Moreover, cholesterol oxidase has been found to selectively inhibit the function of caveolae (a special type of raft) (Okamoto et al., 2000). Conditions of cholesterol oxidase treatment in 3A9 cells were first calibrated so that neither cell viability nor TCR surface expression was affected (not shown). The cholesterol oxidase treatment was found to strongly reduce the quantity of rafts detected in sucrose gradient (Figure 4A), and severely impaired the TCR–CD3-induced phosphorylation of CD3ζ, ZAP-70 and LAT in the total cell lysate (Figure 4B). Of note, cholesterol oxidase alone did not induce these phosphorylation events (Figure 4B). Hence, these results strongly indicate that rafts are critically required in the initiation of TCR signaling. Figure 4.(A) 3A9 cells (3 × 107) were treated, or not, with 4 U/ml of cholesterol oxidase at 37°C for 1 h. Brij 98 solubilized PNS was fractionated on the sucrose gradient and blotted with anti-CD3ζ mAb. (B) 3A9 cells were treated or not with cholesterol oxidase as in (A). They were then stimulated with or without 15 μg/ml of anti-CD3ϵ mAb at 37°C for 30 s. The proteins in the total cell lysate were blotted with anti-PY mAb. The blot was then stripped and reprobed with anti-CD3ϵ Ab. Download figure Download PowerPoint TCR–CD3 is concentrated in a subset of rafts Recent studies suggest that different raft populations may exist within the same cell membrane (Madore et al., 1999; Roper et al., 2000; Gomez-Mouton et al., 2001). Therefore, we were interested in determining whether TCR is homogenously distributed in T cell rafts. We have compared raft microdomains immunoprecipitated via TCR or Thy-1. To this end, raft membrane vesicles isolated from 3A9 cells were precipitated with either the CD3ϵ- or Thy-1-specific mAb. Figure 5A (upper panels) shows that regardless of TCR stimulation by the HEL peptide, more CD3 chains were seen in the rafts immunoisolated with anti-CD3 than with anti-Thy-1. In stark contrast, a very low level of Thy-1 was found in the CD3-immunoisolated rafts compared with the Thy-1-immunoisolated ones. The distribution pattern of LAT in the immunoisolates was very similar to that of Thy-1, while that of Lck was somewhat intermediate: its reduction in the CD3-immunoisolated rafts relative to the Thy-1-immunoisolated rafts appeared to be less important than those of Thy-1 and LAT. [14C]cholesterol and [3H]phosphatidylcholine labeling experiments (Figure 5B) indicated a similar cholesterol enrichment, relative to the unfractioned membrane fraction in the PNS, for both immunoisolates. However, there was probably at least 10-fold less of the membrane precipitated by anti-CD3 than anti-Thy-1 under our experimental conditions. These observations suggest that TCR–CD3 raft distribution is not homogenous and that a subset of rafts exhibit a particularly high TCR:Thy-1 ratio (‘TCR rafts’) than do others. The lower level of Thy-1 in the TCR rafts may well be apparent due to a lower membrane content in the CD3 precipitates. We next carried out sequential immunoisolation experiments. Figure 5C shows that although a pre-depletion of TCR-containing rafts did not significantly change the amount of Thy-1 that can be precipitated (upper panels), a pre-depletion of Thy-1-containing rafts quantitatively removed the TCR in raft preparations (lower panels). These findings support the idea that the TCR raft subset represents a very minor fraction of Thy-1-containing rafts. Figure 5.(A) 3A9 cells were stimulated or not with the HEL peptide for 30 s at 37°C, as in Figure 3B. Isolated rafts were precipitated with 20 μl of protein A–Eupergit beads pre-bound with the same amount (1 μg) of anti-CD3ϵ or anti-Thy-1 mAb. Immunoprecipitates were blotted with anti-PY mAb before being stripped and reprobed with the indicated Abs. (B) 3A9 cells were labeled with [14C]cholesterol and [3H]choline-chloride as described in the Supplementary data. Isolated rafts were precipitated as in (A). [14C]cholesterol and [3H]phosphatidylcholine present in the total PNS membranes and the immunoprecipitates were determined as described in the Supplementary data. The index of [14C]cholesterol to [3H]phosphatidylcholine ratio for the immunoprecipitates was calculated by considering that for the corresponding total PNS membranes as 1 unit. Values are means ± SE of the duplicates. Two individual experiments that are representative of four independent ones are shown. In experiment 1, the yields of [14C]cholesterol are 6301 ± 401 d.p.m. and 72 069 ± 4730 d.p.m. for the CD3 and Thy-1 precipitates, respectively; while those of [3H]phosphatidylcholine are 20 137 ± 1985 d.p.m. and 202 511 ± 5641 d.p.m. for the CD3 and Thy-1 precipitates, respectively. In experiment 2, the yields of [14C]cholesterol are 4380 ± 675 d.p.m. and 58 945 ± 6667 d.p.m. for the CD3 and Thy-1 precipitates, respectively; while those of [3H]phosphatidylcholine are 31 817 ± 4048 d.p.m. and 379 795 ± 25 434 d.p.m. for the CD3 and Thy-1 precipitates, respectively. (C) Isolated rafts were submitted to three rounds of precipitation with 20 μl of protein A–Eupergit pre-bound without (Ep), or with 1 μg anti-TCRβ mAb (upper panels) or 1 μg anti-Thy-1 mAb (lower panels). The resulting supernatants were then precipitated with 20 μl of protein A–Eupergit pre-bound with 1 μg anti-TCRβ or anti-Thy-1 mAb. The precipitates were then blotted with the indicated Abs. In each series of precipitation, only results of the first and the last precipitation are shown. Download figure Download PowerPoint TCR stimulation did not significantly modify the distribution pattern of all the molecules examined in the immunoisolates (the slight increase of Thy-1 and LAT in the CD3 isolates after TCR stimulation was not consistantly observed). As expect, the CD3-precipitated rafts contained more phospho-CD3 chains than the Thy-1-precipitated ones (Figure 5A, lower panel). To our surprise, a comparable level of phosp
Referência(s)