Molecular Immunology 41 (2004) 615–630 The role of adaptor proteins in lymphocyte activation Mauro Togni, Jon Lindquist, Annegret Gerber, Uwe Kölsch, Andrea Hamm-Baarke, Stefanie Kliche, Burkhart Schraven∗ Institute of Immunology, Otto-von-Guericke-University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany Available online 24 May 2004 Abstract Research within the last 10 years has provided compelling evidence that adaptor proteins regulate the major pathways of lymphocyte activation. Based upon their differential subcellular localization, transmembrane adaptors and cytosolic adaptors can be distinguished. Here we review some of the most recent findings about both types of adaptor proteins which have facilitated our understanding how immunoreceptors control lymphocyte activation and differentiation. © 2004 Elsevier Ltd. All rights reserved. Keywords: T-lymphocytes; B-lymphocytes; Cell activation; Signal transduction; Cell adhesion; Adaptor proteins 1. Introduction During the last 10 years much has been learned about the molecular mechanisms regulating receptor-mediated signaling cascades in lymphocytes. One of the major breakthroughs in this regard was the finding that the first biochemical events following occupancy of the immunoreceptors (e.g. the T-cell receptor, TCR or the B-cell receptor, BCR) by antigen are activation of tyrosine kinases belonging to the Src-family which subsequently induce the tyrosine phosphorylation of so called ITAMs (Immunoreceptor Abbreviations: ADAP/SLAP-130/Fyb, adhesion and degranulation promoting adaptor protein/SLP-76-associated phosphoprotein of 130 kDa/Fyn-binding protein; AICD, activation induced cell death; ITAMs, immunoreceptor tyrosine based activation motifs; ITIM, immunoreceptor tyrosine based inhibition motif; LAT, Linker of Activation of T-cells; LAX, Linker for Activation of X; LIME, Lck Interacting Membrane Protein; NTAL/LAB, Non T-cell Activation Linker/Linker for Activation of B-cells; PAG/Cbp, protein associated with GEMs/Csk binding protein; PLC, phospholipase C; PSMACs, Peripheral Supra Molecular Activation Clusters; PTKs, protein tyrosine kinases; PTPase, protein tyrosine phosphatase; RapL, Regulator for Adhesion and Polarization enriched in Lymphocytes; RBD, rap-binding domain; SIT, SHP2 interacting transmembrane adaptor protein; SKAP-55, Src kinase associated phosphoprotein of 55 kDa; SKAP-HOM, SKAP-55 homologue; SLP-76, SH2-domain containing leucocyte protein of 76 kDa; TBSMs, tyrosine based signaling motifs; TRAPs, transmembrane adaptor proteins; TRIM, T-cell Receptor interacting molecule; XLP, X-linked proliferative syndrome ∗ Corresponding author. Tel.: +49-391-67-15800; fax: +49-391-67-15852. E-mail address: Burkhart.schraven@medizin.uni-magdeburg.de (B. Schraven). 0161-5890/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2004.04.009 Tyrosine based Activation Motifs, consensus sequence: YxxL/V(x)6–8 YxxL/V, where x stands for any amino acid) present in the cytoplasmic domains of the TCR-associated members of the CD3-complex and the ␨-chains (Chan et al., 1994; Howe and Weiss, 1995; Reth, 1989). In their phosphorylated state, ITAMs serve as docking sites for the tandem SH2-domains of Syk-related PTKs such as Syk and ZAP-70. By binding to the phosphorylated ITAMs, ZAP-70 and Syk are recruited to the activated immunoreceptors where they become tyrosine phosphorylated and activated by the Src-kinases (Iwashima et al., 1994). These initial signaling steps seem to be a prerequisite for the induction of many, if not all, downstream events that culminate in cellular activation (June et al., 1990a,b). A major focus of research during the last years has been the elucidation of intracellular signaling events occurring immediately downstream of ITAM phosphorylation and/or Syk-kinase activation. One topic in this regard was the search for proteins that are involved in coupling the immunoreceptors to intracellular effector molecules such as PLC␥ and ras (Downward et al., 1990; Weiss et al., 1991). These attempts have lead to the identification of a plethora of intracellular molecules that regulate lymphocyte activation. Among them is a group of polypeptides which have collectively been termed adaptor proteins. Adaptor proteins lack either enzymatic or transcriptional activities, but are capable of mediating noncovalent protein-protein interactions with other signal transducing molecules via tyrosine based signaling motifs (TBSMs) or modular protein-protein-interaction domains (e.g. SH2-, SH3-, PH-, 616 M. Togni et al. / Molecular Immunology 41 (2004) 615–630 WW-, PTB- and PDZ-domains). The major function of adaptor proteins is thus to facilitate the formation of multicomponent signaling complexes that allow the initial signal to be transduced from the cell surface into the intracellular environment. Many excellent reviews describing the roles of adaptor proteins in lymphocytes have been published during the last few years (Koretzky and Boerth, 1999; Koretzky and Myung, 2001; Leo and Schraven, 2001; Lindquist et al., 2003; Samelson, 1999; Zhang and Samelson, 2000). For this reason, we will focus here only upon a number of most recent advancements that have supported our understanding of how adaptor proteins regulate immune functions. 2. Positive and negative regulatory transmembrane adaptor proteins It has been known for a long period of time that a number of important intracellular effector molecules become recruited to the inner leaflet of the plasma membrane immediately after perturbation of immunoreceptors, for example phospholipases, nucleotide exchange factors, lipid kinases and cytoplasmic tyrosine phosphatases. Since most of these molecules express at least one SH2-domain, it was proposed that their recruitment to the cell membrane is facilitated by membrane-associated molecules carrying tyrosine based signaling motifs in their cytoplasmic tails. Examples of such molecules are the components of the CD3-complex, the ␨-chains, but also cell surface receptors such as CD5, CD28, CTLA-4 etc. Each of these receptors possesses at least one tyrosine based signaling motif and all of them have been demonstrated to interact with intracellular effector molecules after tyrosine phosphorylation. However, studies within the last 6 years have led to the identification of a group of specialized transmembrane proteins whose exclusive function seems to be to mediate phosphorylation dependent interactions with the SH2-domains of intracellular signaling molecules and thus to target the latter to the inner leaflet of the plasma membrane (where they come into close proximity with the immunoreceptors and/or their physiologic substrates). The common features of these proteins are the presence of only short extracellular domains (likely lacking external ligands), a single membrane spanning domain, and the expression of up to ten tyrosine based signaling motifs (different from ITAMs) within the cytoplasmic tails. We had suggested naming this novel group of polypeptides transmembrane adaptor proteins, shortly TRAPs. Until now seven transmembrane adaptor proteins have been identified, linker of activation of T-cells (LAT), T-cell receptor interacting molecule (TRIM), SHP2 Interacting Transmembrane adaptor protein (SIT), protein associated with GEMs/Csk binding protein (PAG/Cbp), and, most recently Non T-cell Activation Linker/Linker for Activation of B-cells (NTAL/LAB), Linker for Activation of X (LAX) and Lck Interacting MEmbrane protein (LIME) (Brdicka et al., 2000, 2002; Brdicková et al., 2003; Bruyns et al., 1998; Hur et al., 2003; Janssen et al., 2003; Kawabuchi et al., 2000; Marie-Cardine et al., 1999a; Zhang et al., 1998a; Zhu et al., 2002). LAT, PAG, NTAL and LIME are targeted to the lipid rafts via palmitoylation of a juxta-membrane CxxC-motif within the cytoplasmic domain, whereas TRIM, SIT and LAX are excluded from the lipid microdomains. All of the transmembrane adaptor proteins so far identified become rapidly tyrosine phosphorylated after cellular activation and then associate with SH2-domain containing intracellular signaling and effector molecules (e.g. Grb2, PLC␥, SLP-76, PI3-kinase and SHP2). However, a quick look at the cytoplasmic tails of the various transmembrane adaptor proteins shows that many of the tyrosine based signaling motifs are similar if not identical (Fig. 1). For example, the number of potential binding sites for the adaptor protein Grb2 (YxN) that are present in the transmembrane adaptor proteins is enormous. In addition, LAT, LAX and TRIM have been reported to bind the regulatory subunit of PI3-kinase (Bruyns et al., 1998; Zhang et al., 1998a; Zhu et al., 2002) and SIT, PAG and LIME seem to be capable of binding the SH2-domain of the tyrosine kinase Csk (Brdicka et al., 2000; Brdicková et al., 2003; Hur et al., 2003; Kawabuchi et al., 2000; Pfrepper et al., 2001). Why do the transmembrane adaptor proteins share so many TBSMs within their cytoplasmic domains? One possibility could be that this redundancy is a simple safety mechanism which guarantees that loss of one particular TRAP does not lead to a complete failure of the immune system. A striking argument against this point of view is given by the phenotype of LAT-deficient mice and LAT−/− T-cell lines, which clearly shows that the loss of LAT has severe effects upon both the development and the function of T-lymphocytes. Moreover, these defects cannot be compensated by any other transmembrane adaptor protein (Zhang et al., 1999). Structure function analysis of the cytoplasmic domain of LAT showed that its essential role within the T-cell compartment results from its ability to recruit two key players of lymphocyte activation, the cytosolic adaptor protein SLP-76 (SH2-domain containing leucocyte protein of 76 kDa) and the ␥1 isoform of Phospholipase C (PLC) to the plasma membrane. The ability to simultaneously bind both SLP-76 and PLC␥1 has not been described for any of the other TRAPs known so far. Studies in T-cell lines suggest that the functions of other transmembrane adaptor proteins, (e.g. TRIM and SIT) is less essential within the immune system compared to LAT. Thus, it seems as if the majority of transmembrane adaptor proteins rather mediate subtle signals that are required to optimize and fine tune the cellular response by organizing signaling scaffolds either at the right place within the cell (e.g. in rafts versus non-rafts) or the right time after TCR engagement. Further, as shown for LIME, particular TRAPs seem to preferentially or exclusively integrate signals that are delivered by accessory receptors rather than by the M. Togni et al. / Molecular Immunology 41 (2004) 615–630 617 Fig. 1. Transmembrane adaptor proteins (TRAPs) and their cytosolic tyrosine based signaling motifs (TBSMs). Lipid raft and non-raft associated TRAPs are depicted and (potential) binding partner(s) for each TRAP are indicated. Amino acids are presented in single letter code. TCR (see further). Finally, it seems reasonable to propose that some transmembrane adaptor proteins possess negative regulatory or dual functions during T-cell activation, making their functional analysis difficult. A primarily negative regulatory role for T-cell activation has been proposed for the lipid raft associated transmembrane adaptor protein PAG/Cbp (Brdicka et al., 2000; Kawabuchi et al., 2000; Ohtake et al., 2002; Takeuchi et al., 2000; Yasuda et al., 2002). In cell lines and non-transformed human peripheral blood T-cells, PAG recruits the major negative regulator of Src-protein tyrosine kinases, the cytosolic PTK p50csk to the plasma membrane via a YSSV-motif (Brdicka et al., 2000; Kawabuchi et al., 2000). The observation that PAG is capable of recruiting Csk was of interest because it provided an explanation as to how a cytosolic PTK (Csk) is recruited to the plasma membrane and brought into proximity with its physiologic substrates (the Src tyrosine kinases) to regulate their activity (Cary and Cooper, 2000). Initial studies on PAG yielded the surprising result that in resting human T-cells PAG is expressed as a constitutively phosphorylated protein that binds Csk (Brdicka et al., 2000). Moreover, it was demonstrated that immediately after T-cell activation, PAG becomes dephosphorylated and releases Csk. The model emerging from these observations was that in resting T-lymphocytes the activity of Src-kinases is kept low because the negative regulatory tyrosine residue (e.g. Y505 of Lck) is phosphorylated by Csk that is bound to phosphorylated PAG. After perturbation of the TCR, PAG becomes dephosphorylated, thus releasing Csk. As a consequence, Y505 of Lck gets dephosphorylated by the membrane associated protein tyrosine phosphatase CD45 (which is abundantly expressed on the surface of T-cells). Dephosphorylation of Y505 allows activation of the Src-kinase and facilitates initiation of TCR-mediated signaling. An elegant study in which lipid rafts from primary T-cells before and shortly after TCR-triggering were analysed for the presence of tyrosine phosphorylated proteins confirmed this model by showing that concomitantly to TCR-mediated PAG-dephosphorylation (and loss of Csk from the lipid rafts) LAT becomes phosphorylated (Torgersen et al., 2001). In this scenario PAG was considered to act as a counterplayer of LAT. The above model of PAG-function in T-cells has been discussed controversially during the past 2 years. However, a most recent study in which transgenic mice, overexpressing either wild type PAG or PAG-mutants lacking the Csk bind- 618 M. Togni et al. / Molecular Immunology 41 (2004) 615–630 ing site, were investigated for their ability to signal via the TCR is in complete agreement with the model (Davidson et al., 2003). Still, the study using PAG-transgenic mice cannot answer the question as to whether PAG plays such an essential role as LAT in T-lymphocytes and whether additional transmembrane adaptors exist in T-cells that serve PAG-like functions. These questions can only be answered by the generation of PAG-deficient mice, whose description is eagerly awaited. Another question that needs to be addressed relates to the nature of the protein tyrosine phosphatase responsible for PAG-dephosphorylation. Davidson et al. suggest that CD45, the major membrane associated PTPase expressed in T-lymphocytes (and a counterplayer of Csk with regard to the regulation of Src-kinase activity), might be responsible for PAG-dephosphorylation (Davidson et al., 2003). Whether or not this is the case requires further studies. Another interesting aspect of PAG-function comes from the observation that PAG constitutively associates with the Src protein tyrosine kinase Fyn and that PAG apparently represents a specific substrate for Fyn in T-lymphocytes (Brdicka et al., 2000; Yasuda et al., 2002). This observation is interesting in light of “old” data indicating that Fyn-activity is strongly upregulated in anergic T-lymphocytes (Boussitis et al., 1997). One question emerging from these observations is whether PAG is involved in maintaining or inducing anergy in T-lymphocytes. Indeed, PAG-transgenic T-cells seem to behave like anergic T-cells (Davidson et al., 2003). Thus, it will be interesting to determine both the phosphorylation state of PAG as well as its molecular interactions in anergic T-lymphocytes. However, since PAG not only carries a binding site for Csk within its cytoplasmic domain, but eight additional potential tyrosine phosphorylation sites (see Fig. 1), its function may be more complex than so far appreciated. A major challenge will be to assess which of the non-Csk binding sites within the cytoplasmic domain of PAG become phosphorylated (or dephosphorylated) during T-cell activation. It is reasonable to propose that not all of these sites serve negative regulatory roles, making it likely that PAG also possesses positive regulatory properties in T-cells. Further studies are required to answer this question. A dual signaling function has also been proposed for the transmembrane adaptor protein SIT. Overexpression studies in Jurkat T-lymphocytes initially suggested a primarily negative regulatory role of SIT (Marie-Cardine et al., 1999a). However, expression of SIT mutants in Jurkat T-cells in which all but one tyrosine were eliminated later revealed that the negative regulation of TCR signaling by SIT is exclusively mediated via a C-terminal YASV-motif that binds a still unknown ligand (Pfrepper et al., 2001). Studies performed in pervanadate treated T-cells suggested that the YASV motif (which is very similar to the YSSV-motif of PAG) binds Csk, but this assumption could not be confirmed using more physiologic stimuli (e.g. anti-CD3 antibodies). Equally interesting was the finding that mutation of the YASV-motif of SIT not only eliminated its negative regulatory properties of SIT, but even reverted the molecule into a positive regulator of T-cell activation. Further analysis showed that the positive regulatory function of the YASV-mutant depends on the integrity of the membrane proximal YGNL-motif that represents a binding site for Grb2 (Pfrepper et al., 2001). The intracellular signaling pathway controlled by the YGNL-motif has yet to be elucidated. PAG and SIT may not be the only transmembrane adaptor proteins with dual functions. Several recent reports suggested that such a property might even be true for LAT. This assumption emerges from the analysis of knock-in mice expressing a LAT mutant in which only the binding site for PLC␥1 was eliminated (Y136F mice, Aguado et al., 2002; Sommers et al., 2002). Thymic development of the LATY136F mice is impaired (albeit not as strongly as in mice lacking the complete molecule) with a block in development at DN3 (CD25+ /CD44− ). However, in contrast to LAT-deficient thymi, low numbers of double positive thymocytes are detectable in the LATY136F animals. Surprisingly, after 4 weeks of age a high number of CD4+ T-cells appear in the periphery of the LATY136F mice which show an activated phenotype and secrete large amounts of TH2 cytokines (e.g. IL-4) upon stimulation with phorbol ester and ionomycin. Moreover, the serum of LATY136F animals contains high levels of IgG1 , IgM and IgE. Elevated numbers of B-lymphocytes, macrophages and eosinophils are also detectable in these animals. The mice spontaneously produce autoantibodies and develop autoimmunity later in life. The majority of LATY136F mice die from a massive infiltration of hematopoietic cells into tissues and in vitro, T-cells show a failure to undergo programmed cell death after stimulation. When analysed biochemically, both thymocytes and peripheral blood T-cells from LATY136F mice do not flux calcium after TCR-stimulation. However, in at least one of the two strains that have been published, the induction of ras-dependent responses (CD5 expression) and Erk activation after stimulation appear to be normal (Sommers et al., 2002). This rather surprising result might be due to the use of a combination of CD3 and CD4 antibodies (rather than either CD3 mAb alone or CD3 and CD28 mAbs) for stimulation and thus could reflect LAT independent activation of ras/Erk by CD4-mediated signaling (e.g. via LIME, see also further). The molecular basis for the striking phenotype of the LATY136F mice is unclear at present. However, most recent reports describing the phenotype of mice lacking the expression of rasGRP1, a recently discovered nucleotide exchange factor for ras, that binds to the PLC␥1 generated second messenger DAG via a C1-domain (Ebinu et al., 1998) might suggest that the phenotype of the animals is at least partially caused by the impaired activity of rasGRP1 (Dower et al., 2000; Ebinu et al., 2000; Hogquist, 2001; Layer et al., 2003; Priatel et al., 2002). M. Togni et al. / Molecular Immunology 41 (2004) 615–630 619 Fig. 2. Model of the LAT-PLC␥1-DAG-Ras and the LAT-Grb2-SOS-Ras pathway. Note that other TRAPs might contribute to Grb2/SOS-mediated activation of Ras. Also note that induction of Ca++ -flux after generation of IP3 and PKC-activation by DAG are not indicated. According to the currently accepted model (for reviews see e.g. Jordan et al., 2003; Leo et al., 2002), engagement of the TCR by antigen or monoclonal antibodies activates the protein tyrosine kinase ZAP-70 which subsequently phosphorylates LAT. Next, PLC␥1 binds to Y136 of LAT via its SH2-domain and simultaneously the cytosolic adaptor protein SLP-76 binds to LAT via the small adaptor protein Gads (Fig. 2). Binding of SLP-76 to LAT allows phosphorylation of SLP-76 by ZAP-70 thus creating a binding site for the SH2-domain of the Tec-family protein tyrosine kinase Itk. ZAP-70 and Itk then phosphorylate PLC␥1, leading to activation of the enzyme. This event is further facilitated by a direct interaction between LAT bound PLC␥1 and SLP-76 (Yablonski et al., 2001). Activated PLC␥1 hydrolyses phosphatidyl-inositol-4,5 bisphosphate (PIP2 ) thereby generating the second messengers diacyloglycerol (DAG) and inositol trisphosphate (IP3 ). While IP3 mediates Ca++ -flux, DAG was for a long time believed to be mainly responsible for the activation of protein kinase C. However, several recent reports demonstrated that DAG not only recruits PKC isoenzymes to the plasma membrane, but also the nucleotide exchange factor rasGRP, an activator of ras. Thus, it seems as if in T-cells two TCR-initiated signaling cascades would regulate ras activation, the LAT-PLC␥1-DAG-rasGRP-ras connection and the TCR-LAT-Grb2-SOS-ras pathway (Fig. 2). Which T-functions are controlled by these two different pathways is not completely clear at present. However, a most recent report described the phenotype of mice that carry a spontaneous mutation in the gene encoding rasgrp (Layer et al., 2003). Although there are some differences between the LATY136F mice and the rasGRP1−/− mice, the phenotype of the latter to a large extend recapitulates the phenotype of the former. Thus, thymic development of rasGRP1-deficient animals is affected resulting in lower numbers of thymocytes and an impaired production of single positive mature T-cells. Moreover, the mice accumulate large numbers of activated CD4+ T-cells in the periphery which display an impaired ability to mount in vitro responses upon stimulation with anti-CD3 mAbs. Further, B-cells from rasGRP−/− mice produce large amounts of autoantibodies, and peripheral T-cells secrete the TH2 cytokine IL4. Finally, similar to the LATY136F mice, rasGRP-mice die from massive infiltration of the tissues by activated lymphocytes. Interestingly, the authors report that conventional rasGRP knock-out mice also develop autoimmunity, a phenotype that had not been described before (Dower et al., 2000; Ebinu et al., 2000; Priatel et al., 2002). In summary, both the LATY136F mice and the rasGRP1−/− mice show similar alterations of thymic development on the one hand and peripheral autoimmunity that is mediated by activated CD4+ T-cells on the other hand. It is unclear at present whether the phenotype of the two mouse strains is mediated by particular T-cells that escape thymic selection and migrate to the periphery where they expand in an uncontrolled fashion. However, functional analysis of the peripheral T-cells of rasGRP−/− and LATY136F mice showed that CD3-mediated AICD (Activation Induced Cell Death) is severely impaired. Whether this is due to altered signaling via the TCR or to an intrinsic defect in AICD has not yet been investigated. Nevertheless, these data might suggest that the loss of rasGRP not only affects thymic development and selection processes, but also has an impact on the maintenance of peripheral tolerance. Moreover, the striking similarities between the Y136F mice and the rasGRP1-deficient mice imply that the phenotype of the Y136F mice is at least in part due to a failure to activate the LAT/PLC␥1/DAG/rasGRP1/ras pathway. Recently the role of rasGRP1 during thymic development was assessed with much care using transgenic mice that express TCRs with low or high affinity for antigen, respectively (Priatel et al., 2002). These experiments suggested that the TCR/rasGRP1/ras/Erk pathway primarily plays a role in positive selection driven by low affinity TCR/antigen interactions whereas positive selection of T-cells expressing high affinity TCRs are less dependent (although not com- 620 M. Togni et al. / Molecular Immunology 41 (2004) 615–630 pletely independent) of rasGRP1. Thus, failure of activating rasGRP1 in rasGRP1-deficient T-cells likely favors development of T-cells expressing high affinity TCRs. Many or some of these cells could represent autoreactive T-cells. The failure to eliminate them within the thymus (again due to a blunted activation of ras in the absence of rasGRP1) could explain the appearance of autoreactive T-cells in the periphery of rasGRP1-deficient (and LAT Y136F ) animals. LAT still bears surprises. A recent report described the phenotype of knock in mice carrying a LAT mutant (LATY7/8/9F ), in which the three distal tyrosine residues (excluding the PLC␥-binding site) were mutated to phenylalanine (Nunez-Cruz et al., 2003). Mutation of the three distal tyrosine residues affects binding of the Grb2/Sos complex that links the TCR to ras activation as described above (Zhang et al., 2000). In addition it impairs binding of the Gads/SLP-76 complex (Zhang et al., 2000) that is required to activate PLC␥1 and to link the TCR to other downstream signaling pathways via the protein-protein interaction domains of SLP-76 (Myung et al., 2001). Thymic development in the LATY7/8/9F animals was found to be similarly impaired as in conventional LAT-knockouts and in LAT knock-ins in which all four distal tyrosine residues were mutated to phenylalanine (Sommers et al., 2001; Zhang et al., 1999). Thus, the LATY7/8/9F mice do not produce appreciable numbers of DP thymocytes and thymic development is arrested at the DN3 stage (CD25+ /CD44− ). In this respect the LATY7/8/9F mice differ from the LATY136F animals. However, stimulation of LATY7/8/9F thymocytes with CD3 mAbs still allowed some TCR-mediated signaling (likely due to residual activation of PLC␥1). These data collectively suggest that the three distal tyrosine residues of LAT are mandatory for the development of ␣/␤ T-cells. Further they might explain why retroviral expression of the transmembrane adaptor protein NTAL/LAB (which will be described in the following paragraph of this review) in the bone marrow of LAT-deficient animals is able to rescue thymic development (Janssen et al., 2003). Interestingly, LATY7/8/9F thymi contain significant numbers of CD5low/ CD25+ ␥/␦ T-cells and more importantly, in contrast to conventional LAT-deficient mice and LATY6/7/8/9F mice, ␥/␦ T-cells are able to reach the periphery where they expand (similar to ␣/␤ T-cells in LATY136 mice) and finally give rise to a polyclonal lymphoproliferative disorder. Thus, elder LATY7/8/9F mice have enlarged spleens and lymph nodes which contain strongly enhanced numbers of ␥/␦ T-cells. Moreover, similar to the LATY136F mice, the ␥/␦ T-cells of LATY7/8/9F animals possess an activated phenotype, but cannot be activated in vitro to proliferate or to express either CD25 or CD69 after TCR-stimulation. This suggests that TCR-mediated signaling is strongly impeded in these cells. However, when TCR-mediated signaling is bypassed by applying a combination of PMA and Ionomycin the LATY7/8/9F ␥/␦ T-cells cells produce large amounts of the TH2 cytokines. This resembles to a large extent the TH2-phenotype of the CD4+ ␣/␤-T-cells that reach the periphery in LATY136F mice (see above). The expansion of ␥/␦ T-cells possessing a TH2-phenotype also likely explains the finding that spleens of the LATY7/8/9F mice contain enhanced numbers of activated and antibody secreting B-lymphocytes and that the serum levels of IgG1 and IgE are strongly enhanced. Another interesting observation emerging for the analysis of the LATY7/8/9F mice should be noted as well. These animals lack dendritic epithelial cells (a thymus dependent ␥/␦ T-cell population that resides in the epidermis) and CD8␣/␣+ ␥/␦ intraepithelial gut T-cells (IELs) which develop independently of the thymus. Thus, LAT controls development of these two types of ␥/␦ T-cells as well. In summary, it seems as if Y136 of LAT is primarily important to mediate homeostasis of the ␣/␤ T-cell compartment whereas the three distal tyrosine residues are also required for homeostasis of ␥/␦ T-cells. It will be important in the future to assess the phenotype of knock-in mice in which the three distal tyrosine residues are mutated individually. Such experiments will help to clarify the question of whether the binding of either the Gads- or the Grb2-modules is primarily responsible for maintaining homeostasis within the ␥/␦-lineage. 3. NTAL/LAB As already mentioned above, LAT is presently the best characterized transmembrane adaptor. Since there are numerous recent reviews describing its general function in T-lymphocyte signaling, we will not focus on this issue here further. However, it is important to reconcile that one of the major functions of LAT in T-lymphocytes is to provide the docking site for the small adaptor protein Gads, which brings another central player in lymphocyte activation, the cytosolic adaptor protein SLP-76, to the membrane and thus facilitates formation of a multimolecular complex that allows the induction of calcium signaling after TCR triggering (Fig. 2). As described above, this complex comprises LAT, Gads, SLP-76, the Tec kinase Itk and PLC␥1. In contrast to T-cells, mature B-lymphocytes do not express LAT or SLP-76 although a recent report has demonstrated a role for LAT during pre-B cell development (Su and Jumaa, 2003). However, similar to T-lymphocytes, perturbation of the B-cell receptor complex on mature B-lymphocytes results in the activation of PLC␥2 and in the elevation of intracellular calcium. Therefore, one of the major questions since the discovery of LAT in 1998 was whether B-cells express a LAT-like molecule that organizes a “calcium-initiation-complex” leading to the activation of PLC␥2. In 2002 and 2003, two groups independently reported the identification of a novel transmembrane adaptor protein that could represent B-cell LAT. This protein has been termed M. Togni et al. / Molecular Immunology 41 (2004) 615–630 NTAL (Non T-cell Activation Linker) by one group and LAB (Linker for Activation of B-lymphocytes) by the other (Brdicka et al., 2002; Janssen et al., 2003). The strategies allowing isolation of NTAL/LAB were quite distinct, but both took advantage of the particular molecular properties of LAT. LAT is targeted to the lipid rafts (also called GEMs) via palmitoylation of a di-cysteine motif that is located directly adjacent to the transmembrane domain (CxxC-motif) (Zhang et al., 1998b). Mutation of the two cysteins within this motif does not ablate membrane insertion of LAT, but impairs its ability to facilitate TCR-mediated signaling (Lin et al., 1999). Therefore, Brdicka et al. postulated that a LAT-like molecule in B-lymphocytes should be a component of the lipid rafts and attempted to identify raft associated phosphoproteins in non-T-cells (e.g. B-lymphocytes and/or macrophages) using standard biochemical approaches, including raft preparation, immunoprecipitation, 2-dimensional gel electrophoresis and MALDI-analysis of the obtained silver stained protein spots. This approach allowed the identification of NTAL (Brdicka et al., 2002). Another feature of LAT is the presence of five TBSMs within the cytoplasmic domain that represent potential binding sites for the SH2-domain of Grb2 (YxN, whereby x stands for any amino acid, see Fig. 1) (Zhang et al., 1998a). To identify B-cell LAT, Janssen et al. searched public data bases for proteins possessing multiple Grb2 binding sites. In addition to another novel transmembrane adaptor protein termed Linker for Activation of X (LAX, see further) this approached yielded the identification of LAB (Janssen et al., 2003). Human NTAL/LAB possesses a 6 AA extracellular domain, an 18 AA transmembrane region and a 219 AA cytoplasmic tail (the cytoplasmic domain of the mouse homologue is 36 AA shorter). The cytoplasmic domain contains nine potential tyrosine based signaling motifs, five of which represent potential binding sites for Grb2 (Fig. 1). In contrast to LAT, NTAL/LAB does not have a consensus binding site for the SH2-domain of PLC␥1. In addition to the TBSMs, the cytoplasmic domain or NTAL/LAB carries the membrane proximal CxxC-motif that is a hallmark of all transmembrane adaptor proteins that are components of the lipid rafts. The genomic organization of NTAL/LAB is very similar to that of LAT which could suggest that both proteins have a common evolutionary origin (Brdicka et al., 2002). Within the hematopoetic system, NTAL/LAB is expressed in B-lymphocytes, mast cells and macrophages, but not in resting T-lymphocytes or thymocytes. Upon stimulation of B-lymphocytes via the BCR or after crosslinking of Fc-receptors on macrophages or mast cells (FcεRIII, Fc␥RI), NTAL/LAB becomes phosphorylated by protein tyrosine kinases of the Syk-family (ZAP-70 or Syk). Furthermore, and consistent with the presence of multiple Grb2 binding motifs, NTAL is capable of recruiting the 621 Grb2/SOS complex after phosphorylation. However, which of the five potential binding sites is responsible for mediating the binding of Grb2/SOS is unclear at present. Besides Grb2/SOS, phospho-NTAL binds Gab1 and, at least in the monocytic cell line THP-1, the ubiquitin ligase cbl. The latter observation is consistent with the finding that NTAL is detectable as a polyubiquitinated protein in lysates of activated B-lymphocytes (Brdicka et al., 2002). Whether or not NTAL/LAB represents the B-cell LAT is unclear at present, but a number of points argue against this possibility. For example, when expressed in the LAT-deficient Jurkat T-cell line JCaM2.5, NTAL/LAB is capable of partially rescuing Erk-activation (probably because of its association with Grb2/SOS), but not the calcium signaling defect that has been described in this cell line (Brdicka et al., 2002). These data are in line with biochemical studies which so far failed to convincingly demonstrate an association between NTAL/LAB and PLC␥1/2 or SLP-76/SLP-65 in various cell lines. However, they are contrasted by the finding that, when expressed in LAT-deficient mice, LAB reconstitutes thymic development, although the LAB reconstituted LAT−/− T-cells that find their way to the periphery are not capable of producing IL-2 in response to TCR stimulation (Janssen et al., 2003). Moreover, the downregulation of LAB expression in the B-cell A20 line by siRNA impairs BCR-mediated calcium flux and also activation of Erk (Janssen et al., 2003). The latter data suggests a role of NTAL/LAB in regulating Ca++ -flux in B-lymphocytes. These conflicting data indicate that the elucidation of NTAL/LAB function requires additional analyses in particular the production of NTAL/LAB-deficient mice or NTAL−/− DT40 cells. What if NTAL/LAB does not represent B-cell LAT, but rather serves a different role in lymphocyte activation? This possibility immediately leads to the question how triggering of the BCR can mediate activation of PLC␥1 and how the BCR is coupled to the calcium pathway in mature B-lymphocytes. As reported above, one of the major functions of LAT in T-cells is to recruit Gads/SLP-76 to the plasma membrane, a prerequisite for allowing activation of PLC␥1. Mature B-lymphocytes do not express SLP-76, but rather a related molecule called SLP-65/BLNK/Bash. SLP-65 seems to be of a similar importance as SLP-76 in T-cells, as evidenced from the findings that SLP-65-deficient mice or patients show a major defect in B-lymphocyte development and that SLP-65-deficient DT40 cells fail to flux calcium after BCR-triggering (Jumaa et al., 2001, 1999; Wollscheid et al., 1999). Importantly, these defects cannot be rescued by simply replacing SLP-65 with SLP-76 in DT40 cells unless LAT is also co-expressed. Conversely, SLP-65 alone cannot rescue the signaling defect of SLP-76 deficient Jurkat T-lymphocytes suggesting that SLP-65 is incapable of binding to LAT (either directly via its SH2-domain or indirectly via Gads). What then brings SLP-65 to the plasma membrane in B-cells? Does SLP-65 require a B-cell Gads to bind to a still undiscovered B-cell LAT or does 622 M. Togni et al. / Molecular Immunology 41 (2004) 615–630 it function independently of such adaptor proteins, e.g. by directly binding to Ig␣/Ig␤ as recently suggested (Engels et al., 2001; Kabak et al., 2002) and by directly interacting with PLC␥2? These questions require elucidation to allow understanding of the fundamental signaling events in mature B-lymphocytes. true also for primary T-lymphocytes or B-cells (which also express LAX) requires further studies. An alternate possibility might be that LAX binds a negative regulatory protein that impairs p38 activation. The elucidation of this question will require a detailed structure function analysis of LAX and/or the generation of LAX-deficient mice. 4. ReLAXing T-cell signaling? 5. The discovery goes on: LIME The search for proteins possessing multiple Grb2 binding sites not only yielded the identification of LAB, but also of an additional adaptor protein that was termed LAX (Linker for Activation of X) (Zhu et al., 2002). LAX does not share all classical properties of the transmembrane adaptor proteins as the extracellular domain of LAX is considerably longer (40 AA). However, the cytoplasmic domain of LAX contains 10 tyrosine residues, four of which represent potential binding sites for Grb2 (Fig. 1). Among these, there is one site (Y193 VNV) that is identical to the Gads binding site of LAT. Moreover, a Y268 VNM-motif present in LAX represents a consensus binding site for the 85 kDa regulatory subunit of PI3-kinase. Consistent with the presence of these motifs, tyrosine phosphorylated LAX binds Grb2, Gads and PI3K after overexpression in Jurkat cells. At least the binding of Grb2 has been confirmed using the endogenous protein whereas it has not yet been reported whether Gads and PI3K can also associate with endogenous LAX in activated cells. No association could be found between LAX and PLC␥1/2 or SLP-76 making it likely that LAX serves a different function as LAT in T-cells. Similar to the other transmembrane adaptor proteins, LAX becomes phosphorylated by membrane proximal tyrosine kinases of the Src and Syk family. The expression of LAX seems to be restricted to the hematopoietic system although a detailed expression study has not yet been performed. Unlike NTAL/LAB, LAX does not possess the typical palmitoylation motif and therefore is not detectable in lipid rafts. Moreover, LAX is not capable of rescuing the signaling defect of LAT deficient Jurkat T-cells. Rather, transient overexpression of LAX has been shown to impair TCR-mediated activation of the transcription factor NF-AT and AP-1 and when stably expressed in Jurakt cells, LAX seems to selectively impair the activation of p38 MAP kinase. The molecular mechanisms underlying the functional effect of LAX need to be further elucidated. It is however important to note that neither TCR-mediated tyrosine phosphorylation nor Ca++ -fluxes seem to be affected in cells overexpressing LAX. The major question emerging from the functional studies performed with LAX is how this transmembrane adaptor protein impairs TCR-mediated signaling. The presence of multiple Grb2 binding sites could indicate that phosphorylated LAX sequesters Grb2 from LAT and thus limits activation of the MAPK-pathway. In line with these considerations is the finding that expression of LAX becomes rapidly upregulated after stimulation of Jurkat T-cells. Whether this holds It seems as if the phase of discovering transmembrane adaptor proteins is not yet over. Screening public databases for proteins possessing a stretch of hydrophobic amino acids, a CxxC-motif and a putative consensus phosphorylation site for Src protein tyrosine kinases, Brdickova et al. recently identified a novel transmembrane adaptor protein termed LIME (standing for Lck Interacting MEmbrane protein) (Brdicková et al., 2003). In parallel, Hur et al. identified the same molecule in the yeast two hybrid system using parts of Lck (the SH2-domain and the kinase domain) as bait (Hur et al., 2003). Like other transmembrane adaptors, LIME possesses a short extracellular domain of only 4 AA, a classical 22 AA transmembrane region that is followed by the CxxC-motif required for raft targeting and a 295 AA cytoplasmic domain that contains five potential tyrosine phosphorylation sites, one representing a potential ITIM (Immunoreceptor Tyrosine based Inhibition Motif). Although the two papers describing the identification and cloning of LIME agree in many aspects, there are also conflicting results which we will briefly summarize and discuss. The first discordance between the two reports relates to the expression pattern of LIME. Whereas one group found that LIME is expressed preferentially in T-cells (including DN, DP and SP thymocytes) and NK-cells, but not outside of the hematopoietic system (with the exception of liver), the other group showed that LIME is expressed also in B-lymphocytes, lung, spleen, but not in thymocytes. Further, one group observed expression in Jurkat T-cells, whereas the other group reported that Jurkat T-cells do not express the molecule. In addition to the expression data, conflicting data were reported regarding the levels of expression in resting versus activated T-lymphocytes. Thus, Brdickova et al. reported that LIME expression is strong in resting T-cells and declines after prolonged TCR-stimulation using CD3 mAbs and IL-2, whereas Hur et al. suggest that LIME expression is low in resting T-cells, but becomes rapidly upregulated after CD3/CD28 co-crosslinking. Finally, another discrepancy relates to the question as to how LIME becomes phosphorylated. Hur et al. reported phosphorylation after CD3 stimulation whereas Brdickova suggested that LIME exclusively becomes phosphorylated after triggering of the CD4 and CD8 co-receptors. In addition, they claimed that CD3-stimulation induces a decrease in LIME-phosphorylation rather than leading to its phosphorylation. M. Togni et al. / Molecular Immunology 41 (2004) 615–630 The reasons for these diverging results are unclear at present. One possibility could be that different types of cells express different isoforms of LIME which are only detectable by particular anti-LIME reagents. Another possibility would be that the expression pattern differs markedly between human and mouse. Regarding the up/downregulation of LIME expression in resting versus activated T-cells it is important to note that different types of cells have been investigated. For example, Bridckova et al. analysed primary human peripheral blood T-cells, whereas Hur et al. investigated LIME-expression in cells from murine lymph nodes. Both types of cells might regulate LIME expression in a different way upon stimulation. Moreover, the possibility has to be considered that the various LIME-specific antisera and antibodies that were used for detection of LIME in Western-blotting show differential reactivities in dependency upon the phosphorylation status of LIME. Finally, the two studies used different modes of stimulation to activate T-cells (CD3+IL-2 versus CD3 and CD28) which could explain the diverging findings. Clearly, further studies are necessary to clarify this point. The same applies to the question whether or not LIME becomes phosphorylated after TCR-stimulation or after engagement of the CD4 and CD8 co-receptors. If the latter would be confirmed, it would be an exciting finding 623 since it might help to further understand the physiology of CD4-mediated signal transduction and/or the pathophysiology of AIDS. One of the two reports describing LIME showed that LIME binds both Csk and Src kinases (Lck and Fyn) after CD4-triggering (Brdicková et al., 2003). At first, this might appear to suggest that LIME represents a negative regulatory molecule involved in the inhibition of Src-kinase activity by recruiting Csk to the membrane/lipid rafts as described above for PAG. Indeed, it was found that the LIME associated fraction of Lck is strongly phosphorylated on the negative regulatory tyrosine residue, Y505 . However, the situation seems to be more complex because mutation of the individual tyrosine residues within the cytoplasmic domain of LIME revealed that both Csk and the Src-kinases bind to LIME via their SH2-domains. This means that, even when the negative regulatory tyrosine residues of the LIME-associated Src kinases are phosphorylated, their SH2-domain is blocked because it interacts with a tyrosine residue of LIME. The consequence of this scenario would be that despite phosphorylation of the negative regulatory tyrosine, the Src-kinases cannot acquire the closed conformation that leads to their inhibition (Fig. 3). Thus, LIME-associated Src-kinases should be enzymatically active rather than being inhibited. Data obtained from Jurkat Fig. 3. Differential regulation of Src-kinase activity by LIME and PAG. Lck and Csk both bind to LIME via their SH2 domains. Despite phosphorylation of Y505 by LIME-associated Csk, Lck remains enzymatically active because the SH2-domain is not available for generation of the “closed” conformation of Lck. Potentially, the phosphorylated negative regulatory tyrosine of Lck could even serve as a docking site for the SH2 domain of other signaling molecules (including Src-kinases such as Fyn), thus amplifying TCR/CD4-mediated signals. In contrast to LIME, PAG only provides a docking site for the SH2 domain of Csk. Therefore, Lck phosphorylated on the negative regulatory tyrosine by PAG bound Csk can refold to the SH2-domain, leading to inactivation of the enzyme. Thus, a single TBSM determines whether or not a particular TRAP primarily acts as an inhibitor or amplifier of T-cell activation. Note that both transmembrane adaptors could have additional functions which are not depicted here. 624 M. Togni et al. / Molecular Immunology 41 (2004) 615–630 T-cells stably overexpressing LIME are in accordance with this hypothesis (Brdicková et al., 2003). Indeed, in such transfectants many molecules were found to be constitutively tyrosine phosphorylated. The enhanced phosphorylation of the proteins could be reverted by partially inhibiting Src kinases using the Src-specific inhibitor PP2. The latter finding suggests that enhanced tyrosine phosphorylation of proteins in LIME transfectants is dependent on Src-kinase activity. Moreover, when analysed more carefully it was found that the lipid rafts of LIME-expressing Jurkat T-cells are strongly enriched in Csk (note that LIME is a raft associated protein). In addition, although LIME-associated Lck was found to be strongly phosphorylated on Y505 in these cells its enzymatic activity was not impaired. Indeed, there was no difference in the ability to phosphorylate an exogenous substrate between LIME-associated Lck and equal amounts of Lck isolated from the pool of total cellular enzyme. Finally, when transiently overexpressed in Jurkat T-cells, LIME enhanced TCR-mediated rise in intracellular calcium (Brdicková et al., 2003) and amplified the transcriptional activity of the IL-2-promotor (Hur et al., 2003). In summary, the published data are compatible with the idea that LIME serves as a positive regulator of T-cell activation rather than inhibiting TCR-mediated signaling. The surprising result thus would be that a molecule that is believed to primarily be a negative regulator of Src-kinase activity (Csk) rather facilitates activation of T-cells by phosphorylating Src-kinases at the negative regulatory C-terminal tyrosine residue (Fig. 3). Provided that these conclusions are correct, the major question is whether e.g. phospho-Y505 of LIME associated Lck is capable of recruiting other SH2-domain containing molecules to the complex, and if so what consequences emerge from these interactions. One possibility could be that via phosphorylated Y505 , Lime-associated Lck could recruit other SH2-domain containing signaling molecules (including Lck or Fyn) to the lipid rafts, thus amplifying CD4-mediated signals. In this regard it is important to note that a recent report demonstrated that TCRxCD4-mediated translocation to and activation of Fyn in the lipid rafts is preceded by activation of Lck (Filipp et al., 2003). It is tempting to speculate that LIME-associated Lck that is phosphorylated on Y505 recruits Fyn to the lipid rafts following CD4-engagement thus allowing its activation. On the other hand it cannot be excluded that LIME serves a negative regulatory role for T-cell activation under particular conditions. If this would be the case, the discovery of LIME might provide a molecular basis for the well known phenomenon that engagement of CD4 in the absence of TCR triggering delivers a negative regulatory signal for T-cell activation or induces tolerance under particular conditions (Bank and Chess, 1985; Benjamin and Waldmann, 1986; Eichmann et al., 1987; Harding et al., 2002; Laub et al., 2002, 2001; Owens et al., 1987). Moreover, it might also explain recent data showing that CD4 triggering alone causes activation of Csk (Marinari et al., 2003). Clearly, additional studies are required to solve the puzzles emerging from the identification of LIME. 6. The TCR SLPs via SLAP-130/ADAP, SKAP-55 and potentially Rap1 to intergrins Resting circulating lymphocytes are not very strongly adherent. This phenotype dramatically changes after the encounter of antigen. The molecules that are primarily responsible for the increase in adherence of activated lymphocytes are integrins. Integrins are widely expressed heterodimeric molecules that consist of an ␣- and a ␤-chain. They are expressed on the cellular surface in two different conformations. In the inactive conformation the affinity of integrins for ligands is low. However, shortly after cellular activation, integrins undergo a conformational change that increases their affinity for external ligands. Moreover, besides enhancing the affinity, the avidity of integrins becomes enhanced in activated lymphocytes by a process called “clustering”. Already in the 90s Tim Springer’s group provided experimental evidence for the dramatic functional changes that integrins undergo in activated T-lymphocytes (Dustin and Springer, 1989). However, the molecular mechanisms underlying the crosstalk between the TCR and integrins is still unclear and one of the major interests these days is the elucidation of the intracellular signaling pathways that are involved in the process of “inside-out” signaling which leads to activation of the integrins. Several recent reports have shed new light into these questions and we will review some of the most interesting data dealing with this issue here. In 1997 two cytosolic adaptor proteins were discovered by Marie-Cardine and co-workers which were termed SKAP-55 (Src kinase associated phosphoprotein of 55 kDa) and SLAP-130/Fyb (standing for SLP-76-associated phosphoprotein of 130 kDa or Fyn-binding protein, respectively) (da Silva et al., 1997; Marie-Cardine et al., 1997; Musci et al., 1997). Meanwhile SLAP-130 was renamed ADAP, standing for Adhesion and Degranulation promoting Adaptor Protein. Thus, we will use the name ADAP throughout this review. Both SKAP55 and ADAP comprise several protein-protein interaction domains, such as proline rich regions and tyrosine based signaling motifs. The function of the two proteins remained unknown until recently. Initially, overexpression studies in various T-cell lines yielded conflicting functional effects of ADAP with regard to TCR-mediated activation of the IL-2 gene (da Silva et al., 1997; Musci et al., 1997). Similarly, overexpression of SKAP-55 or mutants thereof in Jurkat T-cells did not shed any light onto the function of this adaptor protein. However, Marie-Cardine et al. reported in 1998 that in T-lymphocytes ADAP and SKAP-55 are expressed as tightly M. Togni et al. / Molecular Immunology 41 (2004) 615–630 associated proteins which likely from a functional complex (Marie-Cardine et al., 1998). Both biochemical experiments as well as two-hybrid screens further indicated that the association between SKAP-55 and ADAP is mediated via the SH3-domain of SKAP-55 and a proline-rich region within ADAP (Liu et al., 1998; Marie-Cardine et al., 1998). More recently it was further suggested that ADAP and SKAP-55 also associate via a SH3-like domain of ADAP and the YxRR motif within the interdomain of SKAP-55 that connects the PH-domain with the C-terminal SH-3 domain (Kang et al., 2000). The first evidence that SKAP-55/SKAP-HOM might be involved in regulating adhesion in T-lymphocytes came from the functional analysis of another adaptor protein, SKAP-HOM, which represents the non-T cell homologue of SKAP-55 (Liu et al., 1998; Marie-Cardine et al., 1998). Thus, it was found that the adhesion of macrophages to fibronectin (a ligand for the ␣4/␤1 integrin VLA-4) induced rapid tyrosine phosphorylation of SKAP-HOM (Timms et al., 1999). Moreover, in macrophages prepared from mice lacking the protein tyrosine phosphatase SHP1 (which are hyperadhesive), SKAP-HOM was found to be expressed as a constitutively phosphorylated protein. Finally, expression of the tyrosine phosphatase YopH, a major virulence factor of Yersinia enterocolitica that (besides other mechanisms of pathogenesis) paralyses macrophages by altering their adhesiveness, in macrophages induced dephosphorylation of SKAP-HOM (Black et al., 2000). All these data indicated that the ability of macrophages to adhere or to not adhere to intergin ligands correlate with the tyrosine phosphorylation status of SKAP-HOM. In parallel to the SKAP-HOM data, Geng et al. and Hunter et al. reported that ADAP regulates integrin-mediated adhesion in mast cells and T-cells (Geng et al., 2001; Hunter et al., 2000). Final proof that ADAP regulates TCR-mediated inside-out signaling was provided by the analysis of ADAP deficient mice. Two groups independently reported the phenotype of these animals in 2001 (Griffiths et al., 2001; Peterson et al., 2001). ADAP deficient mice develop normally and do not show major alterations in lymphocyte development. Moreover, when analysed biochemically, it appears as if the proximal signaling pathways in ADAP−/− T-cells are intact. This applies for TCR-mediated tyrosine phosphorylation and Ca++ -flux, Erk-activation, etc. However, purified ADAP deficient T-cells fail to upregulate activation markers such as CD25 or CD69 after activation and in vitro proliferation was found to be strongly impaired. Moreover, immunization of ADAP−/− animals with T-dependent antigens fails to induce a B-cell response. Further analysis revealed that ADAP-deficient T-cells do not adhere to fibronectin, ICAM or VCAM after TCR-triggering, while PMA-mediated adhesion and adhesion induced by divalent cations is unimpaired. The latter finding indicated that the intergrins per se are intact. Rather, confocal laser scanning analysis showed that TCR-mediated clustering of integrins does not occur in ADAP−/− T-cells. 625 All these data strongly suggested that the loss of ADAP results in a failure to connect the TCR with the upregulation of affinity and avidity of the integrins resulting in a loss of adhesiveness. The phenotyope of SKAP-55 knockout mice has not yet been reported, but preliminary evidence suggests that the loss of SKAP-HOM has a similar effect on the adhesiveness of B-lymphocytes as the loss of ADAP in T-cells. Moreover, a recent report suggested that overexpression of SKAP-55 in T-cell lines facilitates adhesion of T-cells and, perhaps more importantly, supports cluster formation between T-cells and antigen presenting cells (Wang et al., 2003). Thus, it seems as if SKAP-55 (probably in concert with ADAP) is involved in the crosstalk between the TCR and integrins. A major question emerging from these findings relates to the factors/molecules that act upstream and/or downstream of the ADAP/SKAP-55 complex. Both issues are still black boxes. With regard to the upstream mediators, it is important to note that ADAP has been reported to interact with the SH2-domain of the cytoplasmic adaptor protein SLP-76 (Boerth et al., 2000; da Silva et al., 1997). Indeed, ADAP was originally purified by affinity chromatography using the SH2 domain of SLP-76 (Musci et al., 1997). However, so far there is no report convincingly describing a SLP-76/ADAP/SKAP-55 complex in T-lymphocytes. Thus, it is unclear whether the SLP-76 associated portion of ADAP is involved TCR-mediated in inside out signaling or whether this complex regulates a distinct pathway of T-cell activation. As reported above ADAP was also termed Fyb because of its ability to bind to the SH2-domain of the Src protein tyrosine kinase Fyn (da Silva et al., 1997). Moreover, SKAP-55 and ADAP/Fyb were shown to represent components of the “Fyn-complex” which can be isolated from peripheral blood T-lymphocytes by anti-Fyn reagents (Marie-Cardine et al., 1999b). Interestingly, this complex not only comprises Fyn, ADAP, SKAP-55 and PAG, but also the tyrosine kinase Pyk2 another prominent player in cellular adhesion processes (Marie-Cardine et al., 1999b; Timms et al., 1999). These data could indicate that it is not the SLP-76 associated fraction of ADAP that regulates T-cell adhesion after antigen recognition but rather the Fyn/ADAP/SKAP-55 complex. In this regard it would be important to revisit Fyn deficient T-lymphocytes and to re-assess these cells for their ability to mediate inside-out signaling and to adhere after antigenic challenge. 7. A role for Rap1 and RAPL in TCR-mediated activation of integrins The downstream targets of ADAP and SKAP-55 are largely unknown. A promising candidate however, could be the small GTPase Rap1 (Abraham, 2003; Christian et al., 2003; Katagiri et al., 2000, 2002; McLeod et al., 1998, 2002; Sebzda et al., 2002; Shimonaka et al., 2003, Fig. 4). Rap1 626 M. Togni et al. / Molecular Immunology 41 (2004) 615–630 Fig. 4. Possible “inside-out” connection between the TCR and integrins such as LFA-1. ADAP regulates integrin clustering and interacts with the cytosolic adaptor proteins SLP-76 and/or SKAP-55/SKAP-HOM. The factors/molecules acting upstream and/or downstream of ADAP and/or SKAP-55/SKAP-HOM are largely unknown. A potential downstream effector could be the GTPase Rap1 and RapL which is apparently capable of mediating an interaction between active Rap1 and intergins. The protein tyrosine kinase Fyn might play an essential role in TCR mediated inside-out signaling via its association with SKAP55/SKAP-HOM. was originally believed to be involved in the maintenance of anergy in T-lymphocytes (Boussitis et al., 1997). The model was that in anergic T-lymphocytes Rap1 becomes activated in a Fyn-dependent fashion and sequesters the serine-threonine kinase Raf from Ras, thus impairing activation of the transcription factor AP-1. However, rather than being anergic transgenic mice expressing a constitutively active form of Rap1 (V12Rap1A) seem to be hyperreactive towards TCR-mediated stimuli. Further, by crossing the V12Rap1A mice with TCR-transgenic animals (expressing either the HY- or P14 T-cell receptor) is was shown that positive selection (in the HY system) of thymocytes and anti-peptide responses of peripheral T-cells are enhanced (Sebzda et al., 2002). The enhanced activity of V12Rap transgenic mice apparently emerges from a higher capability of the cells to adhere to fibronectin or ICAM-1. This results from a higher avidity rather than from an increase in affinity of ␤1 and ␤2-intregrins. Thus, while binding of soluble ICAM-1 to LFA-1 seems to be similar in V12Rap1A transgenic T-cells (indicating that affinity is not altered) TCR-mediated clustering of LFA-1 and conjugate formation between T-cells and APCs is strongly enhanced. Independently of the underlying mechanism, the V12Rap1 data collectively indicate that Rap1 exerts an important function during TCR-mediated regulation of integrins. Most recently, the picture became a bit clearer with the identification of a novel cytosolic protein, RapL (Regulator for Adhesion and Polarization enriched in Lymphocytes), that apparently acts downstream of Rap1 and potentially connects Rap1 with integrins (Katagiri et al., 2003, Fig. 4). RapL was identified in the yeast two hybrid system using the above described constitutive active form of Rap1 (V12Rap1) as bait. RapL represents a 30 kDa protein comprising a RBD (rap-binding domain) and a C-terminal coiled coil. The expression of RapL is mainly restricted to the lymphatic system. Pulldown experiments indicated that RapL only interacts with GTP-bound active Rap, but not with RapGDP. Perhaps more important was the finding that overexpression of RapL in T-cell lines enhanced the ability of these cells to adhere to ICAM-1 and induced clustering of LFA-1 in fashion similar to constitutive V12Rap1 (with the difference that overexpression of RapL not only enhanced the avidity of the intergrins, but also the affinity for soluble ligands). Furthermore, a dominant negative mutant of RapL (DN) abolished the ability of V12Rap1 to facilitate integrin clustering. This suggests that RapL acts upstream of the integrins, but M. Togni et al. / Molecular Immunology 41 (2004) 615–630 downstream of Rap1. This assumption is further supported by the finding that RapL inducibly associates with LFA-1 upon activation of T-lymphocytes and that the molecule localizes to the pSMACs (Peripheral Supra Molecular Activation Clusters), the site of LFA-1 localization, during contact formation between T-cells and peptide loaded APCs. Thus, all data suggest that the TCR mediates inside out signaling via the connection Rap1-RapL-LFA-1 (Fig. 4). Still there are many questions to be answered. For example, it is not clear whether activation of Rap1 after TCR-triggering requires expression of either SLP-76, ADAP or SKAP-55/SKAP-HOM or whether its activity is regulated by other factors (Fig. 4). Also, it is unclear at present whether RapL only regulates the avidity and affinity of LFA-1 towards ICAM-1 or also other integrins that play a prominent role in T-cell activation. The elucidation of these questions requires additional studies, for example crossing V12Rap1 transgenic mice with ADAP−/− mice. This animal system could allow answering the question whether constitutive active Rap1 can overcome the adhesion defect of ADAP−/− T-cells. Similar experiments could be performed with SKAP-55-deficient mice once these animals are available. 8. Concluding remarks The past 10 years have led to the identification of a plethora of adaptor proteins which are expressed in cells of the lymphatic system. Due to space limitations, we have focused here on a few aspects regarding this interesting and important group of signaling molecules. We apologize that we have omitted some new insights into the role of other adaptor proteins that regulate signaling pathways in lymphocytes. For example we have not discussed the exciting recent data on the function of the adaptor protein Carma-1 which seems to be required to connect immunoreceptors with the NF␬B-pathway (Abbas and Sen, 2003; Egawa et al., 2003; Gaide et al., 2002; Hara et al., 2003; Jun et al., 2003; Newton and Dixit, 2003; Thome and Tschopp, 2003; Wang et al., 2002). Also, we have not reviewed recent findings on TSAd, an adaptor protein that regulates homoeostasis within the immune system (Choi et al., 1999; Drappa et al., 2003; Greene et al., 2003; Spurkland et al., 1998; Sundvold et al., 2000) or on SAP that plays a major role in coupling SLAM and 2B4 receptors to downstream signaling pathways and which is involved in a disease called XLP (X-linked proliferative syndrome, Benoit et al., 2000; Chan et al., 2003; Howie et al., 2000; Latour et al., 2001, 2003; Latour and Veillette, 2003; Morra et al., 2001; Nakajima et al., 2000; Nichols et al., 2001; Sayos et al., 1998; Tangye et al., 2000; Veillette, 2002, 2003; Veillette and Latour, 2003; Wu et al., 2001). 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