PERSPECTIVE SERIES Lymphocyte signal transduction Gary Koretzky, Series Editor Adapters in lymphocyte signaling Albrecht Leo,1 Jürgen Wienands,2 Gottfried Baier,3 Vaclav Horejsi,4 and Burkhart Schraven5 1Institute for Immunology, Ruprecht-Karls-University Heidelberg, Heidelberg, Germany 2Institute for Biochemistry, University of Bielefeld, Bielefeld, Germany 3Institute for Medical Biology and Human Genetics, University of Innsbruck, Innsbruck, Austria 4Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 5Institute for Immunology, Otto-von-Guericke-University, Magdeburg, Germany Address correspondence to: Burkhart Schraven, Institute for Immunology, Otto-von-Guericke-University Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany. Phone: 49-391-67-15800; Fax: 49-391-67-15852; E-mail: [email protected]. J. Clin. Invest. 109:301–309 (2002). DOI:10.1172/JCI200214942. In order to exert their effector functions, lymphocytes Two groups of adapter proteins can be defined: need to be activated. This process requires at least two adapter proteins that represent integral membrane stimuli, a primary stimulus, which is mediated via an proteins (transmembrane adapter proteins, or TRAPs) immunoreceptor (e.g., the T cell receptor [TCR], the B cell and adapter proteins that are preferentially located in receptor [BCR], or the Fc receptors [FcRs]), and a costim- the cytoplasm (cytosolic adapter proteins, or CAPs). ulatory signal (see Frauwirth and Thompson, this Per- Four TRAPs have been identified and cloned: LAT spective series, ref. 1), which is mediated via so-called (linker for activation of T cells), TRIM (T cell recep- accessory receptors (e.g., CD28 on T cells and CD19 on B tor–interacting molecule), SIT (SHP-2–interacting cells). These two types of signals, as well many others that TRAP), and PAG/Cbp (protein associated with gly- are generated by triggering of regulatory receptors cosphingolipid-enriched microdomains/Csk-binding expressed on the lymphocyte surface, are transmitted to protein). The known TRAPs all have short extracellu- the intracellular compartment, where they initiate cas- lar domains, which (except perhaps for SIT) probably cades of biochemical events that finally produce a cellu- do not bind specific external ligands. In contrast to lar response. How, then, are these externally applied sig- the CAPs, most of which possess both modular bind- nals integrated to yield an appropriate immune response? ing domains and TBSMs, the cytoplasmic domains of Here we review the current knowledge about the the TRAPs lack SH2 domains, SH3 domains, and membrane-adjacent signaling events that are generat- other modular protein-protein interaction domains ed immediately after triggering of immunoreceptors, but contain multiple (up to ten) TBSMs. These focusing in particular on the functions of one group of sequences become phosphorylated by Src and/or Syk signaling proteins, collectively termed “adapter” or protein tyrosine kinases (PTKs) after triggering of “linker” proteins. In addition, we offer a (very subjec- antigen receptors (Figure 1). tive) discussion of key unanswered questions related to The main function of TRAPs can be deduced based the connection of signal-transducing receptors with on these structural properties. After phosphorylation, the cellular environment. TRAPs serve as anchors for the SH2 domains of intra- cellular signaling and effector molecules (e.g., phos- TRAPs and CAPs pholipases, lipid kinases, protein tyrosine kinases, pro- By definition, adapter proteins lack both enzymatic and tein tyrosine phosphatases, or CAPs such as Grb2, transcriptional activities but control lymphocyte acti- Gads, and SLP-76), thus targeting the latter to the plas- vation by mediating constitutive or inducible protein- ma membrane. This allows the formation and nucle- protein or protein-lipid interactions via modular inter- ation of membrane-associated signaling scaffolds, action domains. Such domains include, for example, Src which are required for the propagation of receptor- homology 2 (SH2), Src homology 3 (SH3), phosphoty- mediated signals to the intracellular environment (for rosine-binding (PTB), and pleckstrin-homology (PH) examples, see Figures 2 and 3). domains, as well as tyrosine-based signaling motifs (TBSMs; for details regarding the binding specificities Adapters in signaling through the TCR of these domains, see Figure 1). TBSMs comprise short Among the best-characterized membrane-associated peptide sequences containing a core tyrosine residue, signaling scaffolds in T cells is the Ca2+-initiation com- which, upon phosphorylation, mediates a high-affinity plex, which comprises LAT (a TRAP), Gads and SLP-76 interaction with SH2 or PTB domains. The specificity (both CAPs), the effector molecules PLCγ1 and Itk (a of this interaction is determined by the amino acids PTK of the Tec family), and the Grb2/SOS complex, flanking the tyrosine residue. which activates p21ras (2). The current model for Ca2+- The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 301 Figure 1 (a–c) Schematic illustration of the binding characteris- tics of the protein-protein or protein-lipid interaction modules expressed by adapter proteins. (a) In general, SH2 and PTB domains bind to phosphorylated TBSMs, whose binding specificities are determined by the amino acids surrounding the core tyrosine (C-terminal in case of PTB domains, N-terminal in case of SH2 domains). (b) SH3 domains constitutively interact with proline- rich regions. (c) PH domains bind to phospholipids. (d) Schematic presentation of a selection of adapter pro- teins involved in proximal TCR signaling. plasma membrane after phosphorylation (4). For example, the three C-terminal TBSMs of LAT (Y171, Y191, and Y226) can mediate binding of Grb2 and its associ- ated nucleotide exchange factor SOS (5). Which of these three tyrosines actually represents the major binding site for Grb2 in vivo is as yet unclear. Two of these TBSMs (Y171 and Y191) can also facilitate binding of Gads (5), a CAP that associates constitutively with SLP-76. Binding of Gads to LAT therefore brings SLP-76 to the membrane (6, 7). Initially, it was thought that, by binding to LAT, SLP-76 might be brought into the vicin- ity of ZAP-70, thus allowing its phos- phorylation (8). However, the most recent analysis of Gads-deficient mice suggests that phosphorylation of SLP-76 may be independent of binding to LAT (9). In any case, phosphorylated SLP-76 provides the binding site for the SH2 initiation complex formation and activation in T and domain of the tyrosine kinase Itk, to generate a mem- B lymphocytes provides a good example of the mecha- brane-associated complex consisting of LAT, Gads, nisms by which TRAPs operate in signal transduction SLP-76, and Itk (10). (Figures 2 and 3). Recruitment of Itk into this complex occurs in paral- Lymphocyte activation is initiated by triggering of lel with the binding of another key effector molecule, immunoreceptors (TCR, BCR, FcRs) by their natural PLCγ1, to one of the remaining TBSMs in LAT (5). ligands (e.g., antigen/MHC in the case of T cells). Thus, PLCγ1 is also integrated into the Ca2+-initiation Immediately thereafter, Src PTKs (Lck, Fyn, and Lyn) complex, where it becomes activated by dual phospho- become activated by a mechanism that is still not rylation, mediated by ZAP-70 and Itk (Figure 3; see also completely understood, as discussed below, and in ref. 11). Activated PLCγ1 cleaves the membrane-associ- turn phosphorylate tandem tyrosine residues within ated phosphoinositide PIP2, thus generating the sec- specialized signaling motifs termed ITAMs (im- ond messengers IP3 and DAG. IP3 causes mobilization munoreceptor tyrosine-based activation motifs; Bil- of Ca2+ from intra- and extracellular stores, thus rais- ladeau and Leibson, this Perspective series, ref. 3). ing intracellular Ca2+ levels, whereas DAG represents a ITAMs are present in the cytoplasmic domains of the classical activator of conventional and novel protein immunoreceptor-associated signal–transducing sub- kinase C (PKC) isotypes (Figure 2). units such as CD3γ, -δ, and -ε, the TCRζ-chains, and Among the PKC family members that are expressed the BCR-associated signaling molecules Igα and Igβ in T cells, PKCθ stands out, since it seems to exert (CD79a and CD79b). Dually phosphorylated ITAMs nonredundant functions that are essential for T cell provide the docking sites for the tandem SH2 activation. For example, PKCθ is required for activation domains of the Syk family PTKs ZAP-70 (in T cells) of the transcription factors NF-κB and AP-1 (12, 13). and Syk (in B cells), which are thereby targeted to the Moreover, PKCθ appears to localize selectively into the cell membrane and subsequently become activated center of the mature immunological synapse (IS; see through phosphorylation by Src PTKs. Dustin, this Perspective series, ref. 14) during antigen In T cells, one major substrate for ZAP-70 is LAT, stimulation (15). The elucidation of the molecular which recruits several key signaling molecules to the events underlying targeting of PKCθ to the IS is of par- 302 The Journal of Clinical Investigation | February 2002 | Volume 109 | Number 3 ticular interest, especially because DAG is known to served TBSM in the cytoplasmic domain of Igα. recruit all PKC isotypes to the plasma membrane. Remarkably, this TBSM is located outside the ITAM Most recently it has been demonstrated that DAG (21). These data