Regulation of T-cell receptor signalling by membrane microdomains

Abstract

There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms.

The t-cell antigen receptor

T lymphocytes recognize antigenic determinants through their T-cell receptor (TCR), a multicomponent structure expressed on their cell surface. The TCR is composed of a highly polymorphic heterodimer (α/β or γ/δ) which detects antigen presented on the surface of antigen-presenting cells (APCs) in the context of appropriate major histocompatibility complex (MHC) proteins.^1,2 The α/β (or γ/δ) chains have a very small cytoplasmic tail and are unable to communicate signals generated by antigen binding. Instead, they are non-covalently associated with the non-polymorphic transmembrane proteins CD3γ, CD3δ and CD3ε (CD refers to cluster of differentiation) and a zeta homodimer (TCRζ). The stoichiometry of proteins in the complete TCR complex is an α/β (or γ/δ) dimer associated with two CD3ε, one of each of CD3γ and CD3δ, and a TCRζ homodimer.^3–5 The CD3 and TCRζ components of the receptor are responsible for transmitting the signal into the cell interior via a structurally conserved amino acid motif present in their cytoplasmic domains. This motif contains paired tyrosine residues and is known as immunoreceptor tyrosine-based activation motif (ITAM).^6–8 Other immune receptors, such as the B-cell receptor (BCR) and the Fcγ immunoglobulin receptor, also use ITAMs to signal.⁹ The consensus amino acid sequence of this motif is YXX(L/I)X₆₋₈YXX(L/I) (where Y is tyrosine, L is leucine, and X any amino acid). The TCRζ chain contains three ITAMs in tandem while each of the CD3 chains have one, resulting in 10 ITAMs per single receptor complex. Most likely, the large number of ITAMs present in the TCR has a quantitative role in signal amplification rather than a qualitative role whereby different signals originate from different ITAMs.^10,11 Signalling by the TCR is also facilitated by the CD4 and CD8 coreceptors, which interact with MHC molecules expressed on APCs during antigen presentation (12 and references within).

Productive stimulation of the TCR leads to the activation of a number of signalling pathways that involves generation of second messengers, increased transcriptional activity and production of new proteins that mediate effector functions of activated T cells.^13,14 Inherent or environmentally imposed changes in the activity of signalling pathways in T cells can lead to pathological conditions such as autoimmunity or alternatively immunodeficiency. Therefore, it is important that signalling homeostasis is maintained precisely. At the plasma membrane, as part of such regulation, is the compartmentalization of signalling proteins and receptors into distinct domains. It is now well documented that initiation and propagation of the TCR-generated signal is critically dependent on the transient assembly and spatial reorganization of such proteins.

Tyrosine kinases proximal to the tcr

Upon receptor stimulation, the first detectable biochemical event is phosphorylation of the tyrosine residues present within ITAMs. The Src-family kinases Lck and Fyn have been implicated in this phosphorylation.^14–16 Genetic evidence has suggested a more critical role for Lck, as was demonstrated in Lck-deficient cell lines where TCR stimulation failed to trigger ITAM phosphorylation and down-stream signalling^17,18 while in Lck^−/− mutant mice, T-cell development was blocked at an early stage (CD4^– CD8^–) during thymocyte development.¹⁹ Expression of an inducible Lck transgene in the Lck^−/− background revealed that when expression of the transgene was switched off in the peripheral T-cell pool the long-term survival of naïve T cells was not affected but their homeostatic proliferation was compromised.²⁰ Lck interacts with the cytoplasmic tail of the CD4 and CD8 coreceptors. These receptors bind to MHC molecules on APCs during antigen presentation, bringing Lck in proximity to the TCR and thus facilitating ITAM phosphorylation.^21–23

In contrast to Lck, Fyn seems to plays a more specialized role during TCR signalling since fyn-null mice exhibit a defect that is restricted to certain stages of T-cell development.^24,25 Fyn may regulate aspects of T-cell activation by phosphorylating key adapter molecules such as ADAP (adhesion and degranulation promoting adapter protein, also known as FYB (Fyn T-binding protein) or SLAP (SLP-76-associated protein), and SKAP55 (Src kinase-associated phosphoprotein of 55 000 MW).^26–28 These molecular scaffolds form multiprotein complexes during T-cell activation that regulate integrin clustering and adhesion.^29–31 Interestingly, upon TCR stimulation, SKAP55 translocates to lipid rafts where it interacts with Fyn implicating these membrane domains in integrin-mediated adhesion.³²

Phosphorylated ITAMs form docking sites for the tandem Src homology (SH) 2 domains of the cytosolic tyrosine kinase ZAP-70.³³ Upon recruitment to the TCR, ZAP-70 is phosphorylated, most likely by Lck, and activated.^16,34–36 In humans, absence of ZAP-70 protein, as seen in certain patients, leads to severe immunodeficiency characterized by lack of CD8⁺ cells while mature CD4⁺ cells are unresponsive to TCR stimulation.^37–39 In mice, ZAP-70-null mutants reveal a role for this kinase in both positive and negative selection of thymocytes.⁴⁰ A substrate for ZAP-70 activity is the adapter molecule LAT (linker for activation of T cells).⁴¹ It contains multiple tyrosine residues in its cytoplasmic domain and when phosphorylated it nucleates multiprotein signalling complexes at the plasma membrane.^41–44 Studies with LAT-negative cell lines showed that while initial tyrosine phosphorylation, including phosphorylation of ITAMs, remained intact downstream signalling events were blocked.⁴⁵ Also, thymocyte development was blocked within the double negative stage (CD4^– CD8^–) in lat-null mutant mice.⁴⁶ Proteins that directly bind phospho-LAT include phospholipase Cγ1 (PLCγ1), and the adapter molecules Grb2 and Gads.^41,47 The Tec-family tyrosine kinases Itk/Emt and Txk/Rlk are also involved in PLCγ1 regulation following TCR stimulation.^48,49 Another adapter molecule that participates in these ‘signalosomes’ through its interaction with Gads is SLP-76, which is also critical in linking early to distal TCR signalling events.⁵⁰ Formation of such signalling complexes directs downstream events such as mobilization of intracellular Ca²⁺ and stimulation of the Ras/MAP (mitogen-activated protein) kinase pathway.^51,52 The co-ordinated action of enzymes, adapter molecules and second messengers leads to increased activity of transcription factors like nuclear factor (NF)-AT, AP-1 and NF-κB and expression of new proteins such as CD69, CD25 and interleukin-2 (IL-2).¹³ A schematic representation of major participants during TCR signalling is illustrated in Fig. 1.

Figure 1.

Signalling cascades stimulated by the TCR. Schematic depiction of key protein components of major signalling cascades that are stimulated following recognition of antigen by the TCR. Differential colouring identifies biochemical events and proteins with distinct function.

Regulation of lck

The Src-family of kinases have a conserved structure characterized by distinct functional domains (reviewed in,^53,54). It includes an amino terminal (N-terminal) motif containing signals for attachment of lipid moieties,^55–57 which in the case of Lck are a myristic acid added cotranslationally to glycine at position 2, and palmytic acid attached to juxtapositioned cysteine residues 3 and 5.^58–65 This dual acylation is sufficient for membrane localization of the protein.^66,67 Following the membrane-targeting motif is a region unique to individual members, which may have specific functions, although such functions remain unclear at present. In the case of Lck, serine and threonine phosphorylation sites have been identified in the unique domain. In particular phosphorylation of serine 59, catalysed by the extracellular regulated kinase (ERK) MAP kinase (MAPK) cascade, is shown to have a role in determining binding of Lck to protein partners.^68–73 Downstream of the unique region is an SH3 domain followed by an SH2 domain. These are involved in protein–protein interactions, with the SH3 domain interacting with sequences containing the core P-X-X-P motif (where P is proline and X any amino acid) while SH2 binds to phosphorylated tyrosine residues (reviewed in ⁷⁴). Through a linker sequence, the SH2 domain is connected to the catalytic (SH1) region. Two tyrosine (Y) residues one within the ‘activation loop’ of the catalytic domain and the other at the carboxy-terminus (C-terminus) of the protein play critical regulatory roles (Y394 and Y505 in murine Lck protein).^75–78 When phosphorylated the C-terminal tyrosine interacts with the SH2 domain in the same molecule promoting the folding of the enzyme into a low activity state (‘tail-bite’ structure, Fig. 2).^79,80 In this configuration the SH3 domain of the protein interacts with the linker segment connecting the SH2 and SH1 modules, thus stabilizing the ‘closed’ structure.⁷⁸ In contrast, autophosphorylation of the tyrosine residue within the ‘activation loop’ induces the molecule to adopt an ‘open’ conformation which has significantly elevated enzymatic activity.^76,81

Figure 2.

Regulation of Lck at the plasma membrane. In T cells, Lck is localized to both detergent-insoluble lipid rafts and detergent-soluble non-raft fractions. The raft-associated Lck is phosphorylated at the C-terminal regulatory tyrosine, which then interacts with the SH2 domain promoting a folded-low activity structure. Phosphorylation of Lck at the C-terminal tyrosine is mediated by the PAG/Cbp-Csk protein complex and it may persist in lipid rafts because of the exclusion of the CD45 tyrosine phosphatase from these domains. In contrast, the pool of Lck present in the bulk of the plasma membrane preferentially adopts an ‘open’ conformation with higher catalytic activity due to the positive action of CD45, which dephosphorylates the C-terminal inhibitory tyrosine.

The interconversion of Lck, and of other Src-family members, is dependent on the enzymatic activity of other proteins. Thus the cytosolic tyrosine kinase Csk can phosphorylate Y505 and down-regulate Lck (and other Src-family members) activity by inducing the ‘tail-bite’ structure (Fig. 2).^82–86 In contrast, the receptor-type tyrosine phosphatase CD45 is the principal phosphatase in T cells able to disrupt the SH2–pY505 intramolecular interaction and to cleave the phosphate group on Y505 (Fig. 2).^87–90 Interestingly, in addition to its positive role, CD45 can also downregulate the activity of Lck by dephosphorylating Y394 and possibly downstream substrates of Lck.^91,92 It is unclear how the positive and negative actions of CD45 are balanced during the early stages of TCR signalling. It is possible that the kinetics of Y505 dephosphorylation is faster providing a time window for activated Lck to phosphorylate protein substrates. Alternatively, Y505 could be more accessible to CD45 than Y394, in which case fewer phosphatase molecules located in proximity could dephosphorylate Y505 and activate Lck, while at high local concentrations of CD45 this advantage may be lost resulting in signal inhibition.

Lipid raft domains

Attachment of myristate and palmitate groups at the N-terminus not only promotes membrane anchorage of Lck but also governs its partitioning into lipid rafts.^58,60,63 Lipid rafts are considered as specialized microdomains within the plane of the plasma membrane with a lipid composition that is different from the glycerophospholipid-rich bilayer of the surrounding membrane. They are instead rich in glycosphingolipids, sphingomyelin and cholesterol.^93–97 One of their properties, widely used for purification purposes, is their insolubility during extraction of cells with cold non-ionic detergents, albeit different detergents may vary in their ability to solubilise lipid raft membranes.⁹⁸ Because of this property and their distinct lipid composition, other names given to these insoluble membranous preparations are detergent-resistant membranes (DRMs), glycosphingolipid-enriched membranes (GEMs), and detergent-insoluble glycolipid-enriched membranes (DIGs). Raft formation (or disassembly) in cell membranes could, in part, be regulated by the type and concentration of lipids present in the bilayer and in a manner similar to raft formation seen in model membranes where sphingolipids assemble to form distinct areas that are resistant to non-ionic detergent extraction.⁹⁹ Inclusion of cholesterol in these artificial lipid bilayers stabilizes the sphingolipid-formed structures.⁹⁹ Similarly, in living cells, pharmacological extraction of cholesterol from the plasma membrane results in disruption of lipid rafts, indicating that cholesterol is a critical structural component.^100–102 In the past few years, sophisticated techniques such as fluorescence resonance energy transfer (FRET),¹⁰³ fluorescence recovery after photobleaching (FRAP),¹⁰⁴ single particle tracking (SPT)^105,106 and chemical cross-linking,^107,108 among others, have provided support for the existence of plasma membrane domains in unperturbed cells. These techniques, by providing resolution at the nanometre scale, have suggested that lipid raft domains could be rather small structures, possibly up to 25 nm in diameter, a size much smaller from what was initially measured in detergent-insoluble preparations.¹⁰⁵ Therefore, detergent extraction almost certainly induces coalescence of rafts into bigger conglomerates. In addition, in intact cells it is generally assumed that lipid raft domains are not rigid structures but instead they are dynamic with lipid molecules rapidly exchanging between raft and non-raft membrane. It is unknown if de novo formation of lipid rafts takes place at the plasma membrane by the spontaneous assembly of resident lipids. In mammalian cells, the study of proteins which are known to target to lipid rafts revealed that incorporation of newly synthesized proteins into DRMs is first visible in the Golgi. Raft-containing vesicles subsequently move to the plasma membrane,¹⁰⁹ a process which may involve the actin cytoskeleton.¹¹⁰ One report suggests that in T cells, lipid raft domains could be constitutively assembled by the actin cytoskeleton into larger patches, which can function as carriers for ferrying molecules to the T-cell/APC contact site during antigen presentation.¹¹¹

Detergent insolubility and low buoyancy, which allows flotation on dense gradients, have been exploited in order to purify DRMs and to study their protein content in a variety of cell types including T lymphocytes. Initial limited analysis of proteins copurifying with the low-density, detergent-insoluble fraction indicated enrichment of signalling proteins particularly those modified by addition of lipids. Such proteins were members of the Src-family of kinases, heterotrimeric GTP-binding proteins and small GTPases, and glycosylphosphatidylinositol (GPI)-anchored receptors.^93,96 Lately, it has become apparent that a new group of signalling proteins also localises to membrane rafts. These are transmembrane adapters, which form signalling complexes at the plasma membrane. They contain two cysteine residues as part of a C-X-X-C motif (C is cysteine and X any amino acid) that immediately follows the transmembrane segment. These membrane-proximal cysteines become palmitylated and are critical for targeting the protein to lipid rafts. Members of this group identified so far include LAT,¹¹² PAG/Cbp (protein associated with GEMs/Csk binding protein),^113,114 NTAL/LAB (non-T-cell activation linker/linker for activation of B cells)^115,116 and LIME (Lck-interacting molecule).^117,118 In the case of LAT, structure/function experiments have documented the importance of the C-X-X-C motif since a LAT mutant, where the two membrane-proximal cysteines were substituted, did not partition into DRMs and failed to support downstream signalling in response to TCR stimulation.^112,119

In recent studies, proteomic analysis of purified DRM fractions was employed to produce a map of protein components associated with these domains. In the most detailed study so far published by Foster et al.¹²⁰ quantitative proteomics was used to specifically identify proteins whose association with the DRM from HeLa cells was sensitive to cholesterol-depleting agents. Because depletion of cholesterol disrupts lipid rafts, the authors reasoned that in contrast to contaminants resulting from the purification protocol, association of authentic raft components would be susceptible to treatment of cells with cholesterol extracting agents. Using this methodology, they identified 241 polypeptides, the majority of which were signalling proteins, but a number of structural proteins were found as well. Association of cytoskeletal proteins was also detected in detergent-insoluble rafts isolated from neutrophils.¹²¹ Thus, this analysis supports the supposition made by earlier studies that lipid rafts may preferentially concentrate signalling molecules.

Since a functional role for lipid rafts during TCR signalling has been suggested (see section below), groups including our own have sought to identify proteins resident in DRM preparations from T cells (Fig. 3 and Table 1).^122,123 For this purpose, low density, detergent-resistant preparations from the human leukaemic T-cell line Jurkat, were resolved by one- or two-dimensional (D) gel electrophoresis and individual protein bands (or spots in the case of 2D-gels) were analysed by mass spectrometry. We resolved detergent-resistant lipid rafts isolated from 50 × 10⁶ Jurkat cells by 2D-gel electrophoresis and proteins were visualized by silver staining (Fig. 3). Individual spots, indicated by arrows, were excised, digested with trypsin, and analysed by mass spectrometry. A list of protein spots identified by their peptide ‘fingerprint’ is shown in Table 1. Taken together, the results from the above studies on Jurkat lipid rafts (122,123 and our own results) reveal an enrichment of signalling and cytoskeletal proteins in these preparations. However, the presence of mitochondrial and nuclear proteins shows that unrelated polypeptides can copurify with this method of raft preparation as there is no evidence today that nuclear and mitochondrial membranes contain microdomains. Therefore, caution should be exercised when proteins are assigned as raft-associated. On the other hand, while proteins with high affinity for raft domains are resistant to detergent extraction, molecules that are loosely associated with these domains may be sensitive to detergent extraction and therefore lost during purification. An example of weakly associated proteins whose partition to lipid rafts under certain conditions is sensitive to detergent extraction could be the TCR (see discussion below). Therefore, although detergent insolubility has been, and will continue to be, a valuable tool to study rafts and their content, more sophisticated methodologies for raft purification must be developed for the field to move forward.

Figure 3.

2D-gel electrophoresis of T-cell lipid rafts. 50 × 10⁶ Jurkat T cells were extracted in ice-cold lysis buffer containing 1% Triton-X-100 detergent plus protease and phosphatase inhibitors. Lipid rafts were purified by flotation on sucrose gradient and resuspended in rehydration buffer appropriate for isoelectric focusing of proteins on 3–10 pH strips. The rehydration buffer also contained 20 mm MβCD, which assists in the complete disruption of lipid rafts. Following 2D-gel electrophoresis, proteins were visualized with a silver stain compatible for analysis with mass spectrometry. Discernible protein spots were excised, digested with trypsin and the resulting peptides were recorded by mass spectrometry. The peptide ‘fingerprint’ obtained from this analysis was used to search available protein databases. Arrows indicate protein spots for which a positive identification was made and their identity is summarized on Table 1.

Table 1.

Proteins copurifying with detergent-insoluble membranes from T cells

Spot no.	Identified protein	MW '000)	pI	Swiss-Prot/TrEMBL identification no.
1	Rho GDP dissociation inhibitor 2 (Rho-GDI beta)	23	5·1	P52566
2	Lymphocyte specific protein LSP1	37	4·7	P33241
5	ZAP-70 kinase (fragment)	70	7·8	P43403
6	Enolase 1α	47	7	P06733
7	Flotillin 2	42	5·2	Q14254
8	Protein disulphide isomerase ER60	57	5·9	P30101
9	Sorbin & SH3 containing protein (fragment)	100	7	Q9BX64
10	Heat-shock protein 60 (HSP60)	60	5·5	P10809
11	Transformation up-regulated nuclear protein	51	5·2	Q07244-2
12	Similar to ATP synthase, H + transporting itochondrial F1 complex	56	5·3	P06576
14	Actin	44	5·7	P02570
15	UV excision repair protein RAD23 homologue B	43	4·8	P54727
17	ATP synthase β chain mitochondrial precursor	56	5·4	P06576
18	Dynactin 2	44	5·1	Q13561
19	Heterogenous nuclear ribonucleoprotein F	46	5·4	P52597
20	Heterogeneous nuclear ribonucleoprotein F	46	5·4	P52597
21	Capping protein	33	5·4	P47756
22	Nucleophosmin	32	4·7	P06748
23	Urokinase-type plasminogen activator receptor	32	5·8	Q03405
24	F1F0-type ATP synthase D chain	18	5·2	O75947
25	Similar to protease (prosome, macropain) 26S subunit	25	5·4	Q81V79
26	Heat shock protein (HSP60) (fragment)	60	5·5	P10809
27	Ribosomal protein S14	12	11	P06366
30	Endoplasmic reticulum lumenal protein ERp29	29	6·8	P30040
31	Telomerase reverse transcriptase (fragment)	42	5·3	Q8NG38
34	Triose phosphate isomerase	27	6·5	Q8WWDO
35	Triose phosphate isomerase	27	6·5	Q8WWDO
36	Cyclophilin B	18	8·2	P23284
37	Heterogeneous nuclear ribonucleoprotein A1	34	9·2	AAH02355
38	Cyclophilin A	18	7·7	P05092
41	Glyceraldehyde-3-phasphate dehydrogenase	36	8·3	P00354
42	Nebulette protein	82	8·5	O76041
45	C2H2 type zinc finger protein	68	8·8	O75820
48	P32/inhibitor of growth family member 1 like	33	5·1	O95698
50	Tropomyosin	30	5·1	P09493
51	Chloride intracellular channel protein 1	27	5·1	O00299
53	Aldehyde dehydrogenase 1	31	5·5	P00352
54	Haematopoietic lineage-specific protein HS1	54	4·7	P14317
55	Glucose regulated protein	72	5·1	P38646

Another question addressed by Bini et al. in their study using 2D-gel analysis was how the protein composition of DRMs changed following stimulation of the TCR. Comparison of 2D-gel protein maps corresponding to different time points of stimulation up to 15 min, showed that TCR stimulation induces substantial changes in their protein composition.¹²² Intensity of some protein spots was reduced over the stimulation period, possibly indicating their exit from lipid rafts, while the silver stain signal of another group of proteins intensified indicating an increase in their affinity for raft domains.¹²² These changes could reflect biological processes initiated by the stimulated TCR, which take place in membrane microdomains.

Lipid rafts and tcr signalling

As mentioned above the tyrosine kinase Lck and the adapter molecule LAT constitutively reside in raft domains, a process that requires S-acylation of two membrane-proximal cysteines.^58,60,124 Mutant versions of the proteins that lack these cysteines but which remain attached to the membrane, in the case of Lck by fusion to a transmembrane domain, fail to partition into DRMs and lose their capacity to couple the TCR to downstream signalling cascades indicating that lipid raft localization is crucial for the signalling function of Lck and LAT.^63,112 Also, recently it has been suggested that following TCR stimulation Lck-containing microdomains¹²⁵ and LAT-containing microdomains¹²⁶ are recruited to the site of TCR engagement.

An ever-growing list of signalling molecules, apart from Lck and LAT, are shown to transiently translocate to membrane microdomains after stimulation of the TCR.^127–134 The CD4 coreceptor is targeted to lipid rafts through its interaction with Lck and its palmitylation on two membrane-proximal cysteine residues.^135,136 CD4 stimulation is shown to induce lipid raft aggregation and to enhance TCR signalling partly through the induction of molecular clustering at the immunological synapse.^136,137 The affinity of the TCR itself for lipid rafts seems to increase following its stimulation, as components of the TCR complex such as the ζ and ε chains and their phosphorylated/activated forms copurify with DRM fractions isolated from stimulated cells.^128,138 Recently, it was shown that T-cell activation by super-antigens is mediated by signalling events occurring in membrane microdomains.¹³⁹ In addition, confocal microscopy has revealed colocalization of TCR molecules with GPI-anchored receptors or with the ganglioside GM1, both of which are used as markers of membrane rafts.¹⁴⁰ GM1 is the target of cholera toxin B subunit (CTB) and has been extensively used in visualizing rafts and their potential colocalization with surface molecules using microscopy. However, in a recent report the authors using FRET analysis of GPI-linked proteins and CTB in Jurkat T cells were unable to detect accumulation of lipid rafts in the area of stimulated TCR complexes.¹⁴¹ Furthermore, it is possible that polarization of lipid rafts during activation is T-cell subset specific since unlike CD4⁺ T cells, primary human CD8⁺ cells did not show polarization of lipid rafts when stimulated via their TCR and CD28 receptors.¹⁴² How the affinity of the TCR for lipid raft domains increases upon its stimulation remains enigmatic and as of today there is no direct evidence linking induction of signalling pathways with increased affinity of the TCR for detergent-insoluble lipid rafts. Some experiments have suggested that the TCR could constitutively associate with raft domains albeit with reduced affinity. This interaction is sensitive to extraction with strong non-ionic detergents like Triton-X-100 but more resistant to mild detergents such as Brij 98.^128,140,143 Cross-linking may result in the TCR becoming more resistant to detergent extraction by increasing its affinity for lipid rafts. The cell cytoskeleton could be involved in this process.¹⁴⁴

The importance of lipid rafts in TCR signalling has been suggested from experiments where T cells were treated with cholesterol-depleting agents such as methyl-β-cyclodextrin (MβCD). Such agents disrupt raft domains and although initial reports showed inhibition of all TCR-generated signals in treated cells¹³⁸ more detailed studies subsequently revealed that such agents have more complex effects on cells by inhibiting certain signalling pathways but stimulating others.¹⁰² Furthermore, recent work indicated that MβCD depletes intracellular Ca²⁺ stores independently of its effects on lipid raft integrity.¹⁴⁵ Therefore, results obtained using cholesterol-depleting agents should not be the sole supportive evidence when arguing for a role for lipid raft domains in a particular biological process.¹⁴⁶

Other molecules known to participate in TCR signalling, that are transiently recruited to lipid rafts after stimulation, are ZAP-70^128,138,140 and PLCγ1.^128,138,147 Interestingly, phosphatidyl inositol 4,5 bisphosphate (PIP₂), the substrate for PLC, is enriched in DRMs suggesting that these microdomains may represent the major sites of PLC action.^148,149 Activation of PLCγ1 in lipid rafts may be facilitated by the recruitment of SLP-76¹⁵⁰ another adapter molecule shown to have a critical role in the regulation of downstream signalling cascades.¹⁵¹ Grb2 and SOS proteins which regulate Ras activity are also recruited⁴⁵ as is the theta isoform of protein kinase C (PKCθ), which plays a critical role in T-cell activation by stimulating the NF-κB pathway.^152,153 A role for lipid rafts during costimulation has been demonstrated in T cells where CD28 engagement resulted in the redistribution of rafts to the site of TCR engagement thus amplifying and/or prolonging the TCR-generated signal.^154,155 Based on these results a model of activation has been proposed where TCR stimulation induces aggregation of rafts and phosphorylation of the receptor by resident Lck molecules, consequentially leading to the assembly of functional ‘signalosomes’. In addition to their role in costimulation, lipid raft function is regulated by the expression of negative regulators of TCR signalling, as shown for the cytotoxic T lymphocyte antigen-4 (CTLA-4) receptor. Coligation of CTLA-4 strongly inhibited the upregulation in lipid raft expression following stimulation of cells via the TCR and CD28 receptors.¹⁵⁵ Furthermore, a pool of CTLA-4 expressed on the surface of activated T cells is concentrated in DRM preparations where it was found to associate with the TCRζ chain, suggesting that CTLA-4 possibly functions by controlling TCR accumulation/retention in raft domains.^156,157 Collectively, these results suggest that negative regulators may limit T-cell activation by, at least in part, modifying lipid raft function. Interestingly, LAT was found to selectively associate with the open form of Lck in lipid rafts, an interaction that might have functional consequences during TCR signal transduction.¹⁵⁸

Exclusion of CD45 from lipid rafts may favour tyrosine phosphorylation of protein substrates in these domains.^130,159 However, some reports have suggested that a small fraction of the phosphatase is present in DRMs and that the ectodomain of the molecule has a role in determining its membrane distribution.^160,161 Therefore, the levels of CD45 present in raft microdomains, and possibly its redistribution in and out of these domains during T-cell activation, may regulate the strength of the TCR signal by determining the levels of active Lck. Interestingly, studies on peripheral blood T cells isolated from patients with the autoimmune disease systemic lupus erythematosus, revealed that a higher proportion of CD45 associates with GM1-containing raft domains in these cells, which may be linked to their ‘hyperactive’ phenotype.^162–164 On the other hand, strong accumulation of CD45 in lipid rafts, as achieved experimentally by expression of a raft-targeted mutant, could have the opposite effect by inhibiting TCR signalling.¹⁶⁵

Partitioning of PAG/Cbp (and possibly of LIME) to raft domains could maintain the raft-associated Lck pool in a folded inactive state in unstimulated cells (Figs 2 and 4).^{102,113,158,166,167} The adapter protein PAG/Cbp is tyrosine phosphorylated in unstimulated T cells and recruits the cytosolic kinase Csk, a negative regulator of Src-family kinase activity (Fig. 2).^113,114 PAG/Cbp phosphorylation is most likely caused by the action of Fyn,¹⁶⁸ which is active in lipid rafts¹⁰² and of Lck molecules that first enter raft domains from the surrounding membrane.¹⁵⁸ Therefore, TCR phosphorylation in lipid rafts may not only require raft aggregation but also a transient increase in the activity of raft-associated Lck. In this scenario, a tyrosine phosphatase must be involved capable of de-phosphorylating PAG/Cbp and shedding Csk from lipid rafts, and/or dephosphorylation of the inhibitory C-terminal tyrosine of Lck. Identifying this phosphatase will undoubtedly shed new light into the mechanisms of TCR signalling. Rephosphorylation of PAG/Cbp by active Lck may cause reattachment of Csk and termination of signal transduction (Fig. 4). In support of this hypothesis, it was shown that in human T cells stimulation of the TCR induces the transient dephosphorylation of PAG/Cbp and exit of Csk from raft domains.^113,169 Also, TCRζ phosphorylation and NF-AT production was increased in Jurkat T cells expressing dominant-negative Csk mutants.^169,170 Interestingly, in murine CD4⁺ T cells, it was shown that cross-linking of the TCR with CD4 rapidly induces the activity and subsequent translocation of a small fraction of Lck from detergent-soluble to detergent-resistant membrane. This was followed by an increase in the activity of Fyn residing in DRMs, suggesting cross-regulation of these two kinases in raft domains.^171,172 This transient increase of Src activity in raft membrane could facilitate activation of the TCR but in addition, increased Fyn activity may assist in reformation of the PAG/Cbp-Csk inhibitory complex.

Figure 4.

A two-step model for activation of the TCR in lipid rafts. In resting T cells, the TCR has low affinity for lipid raft membrane and Lck in lipid rafts is in its folded-inactive conformation due the action of the PAG/Cbp-Csk molecular complex. Antigenic stimulation of the TCR may increase its affinity for lipid rafts, a step which by itself may not be sufficient to initiate signalling. A second step may be required in which the activity of Lck in lipid rafts is transiently elevated, possibly after dephosphorylation of PAG/Cbp and dissociation of Csk, and/or dephosphorylation of Lck by a tyrosine phosphatase. Active Lck would then be able to phosphorylate the ITAMs and initiate signal transmission. Lck may also rephosphorylate PAG/Cbp leading to new recruitment of Csk and termination of the signalling cycle.

Conclusions and future considerations

In the past few years, membrane microdomains have become a popular subject of study across many disciplines. A substantial volume of work, which includes functional experiments and proteomics analysis, points to an important role for these domains as regulators of signal transduction pathways in lymphocytes. Their importance in signalling most likely reflects their ability to compartmentalise proteins at the plasma membrane and upon receptor stimulation to facilitate the assembly of signalling complexes (‘signalosomes’). However, despite the substantial progress made so far, critical questions remain unanswered. Hence, the structure of lipid rafts remains elusive, as is potential changes in their size and protein/lipid composition during stimulation or through the different stages of cell differentiation. One approach that can potentially provide useful information could be the systematic analysis of detergent-resistant membrane preparations using proteomics. Such an analysis could reveal which proteins and when move in and out of rafts during receptor signalling, and in the case of T cells during TCR stimulation. Also, such analysis could potentially identify post-translational modifications (i.e. phosphorylation, lipidation, ubiquitination) of DRM-associated proteins induced by receptor stimulation, which in certain cases may be indicative of the signalling activity of the protein. Information assimilated from the proteomics analysis can form the basis for constructing a ‘map of events’ taking place in lipid rafts after TCR stimulation.

It is also unknown if lipid rafts represent a homogeneous population or whether different types of rafts exist, potentially performing distinct tasks. Studies in leucocytes suggest that structurally and functionally diverse membrane domains may exist with a role in determining rear-front polarity during cell movement.^173–176 Further progress in this area will critically depend upon the development of new methods, as well as in the identification of specific raft markers which will allow us to visualize and track lipid rafts in living cells and possibly discriminate between different subtypes of microdomains. Understanding in detail how lipid rafts operate in T cells will not only refine our current theories of how TCR transduces signals, but will undoubtedly have implications in other fields of biology as well.