ITAMs versus ITIMs: striking a balance during cell regulation (original) (raw)
The observation that the TCR, BCR, and some Fc receptors are associated with multiple ITAM-signaling subunits suggests that these subunits support the efficient signal amplification upon receptor engagement. Alternatively, the pairing of distinct ITAM-containing subunits could allow activated receptors to link in a modular manner to specific downstream signaling pathways by binding to distinct SH2 domain–containing effector molecules.
Probably the most thoroughly studied of the ITAM containing-receptors is the TCR complex, consisting of a pair of antigen-recognizing chains (αβ or γδ), the CD3 chains (εγ and εδ), and a homodimeric pair of ζ chains (Figure 1). In total, the TCR complex contains ten ITAMs, one from each of the CD3 chains and six from the ζ dimer. By contrast, other ITAM-containing receptors have two to four ITAMs each. The role of the CD3 and ζ chains in regulating T cell development has been firmly established, and we recommend the recent review by Love and Shores (9) on this subject.
A model depicting TCR-mediated ζ chain phosphorylation. The figure depicts the TCR (α and β) along with the associated CD3 chains and the p21 form of the ζ homodimer. In the resting state, some TCRs are associated with p21ζ, which bears phosphorylated tyrosines in ITAM B (3, 4) and C (5, 6) that can potentially be bound by up to four inactive ZAP-70 molecules (depicted in upper TCR complex). The Src family PTK Lck is shown myristoylated and associated with CD4 or CD8. The Src family PTK Fyn is not shown but is known to associate with the CD3ε subunit. Upon agonist binding there is receptor clustering (not shown), and CD4/8-associated Lck (and CD3ε-associated Fyn, not shown) can then phosphorylate the four tyrosines within the membrane-proximal ITAM (ITAM-A, tyrosines 1 and 2) to produce p23ζ. In addition, active Lck phosphorylates and activates bound ZAP-70, and tyrosines within the ITAMs of other CD3 subunits, resulting in the recruitment and activation of more ZAP-70 molecules (depicted in lower left). In contrast, upon binding to a partial agonist or an antagonist there is potentially less activation of Lck, which results in neither the full phosphorylation of the p21ζ into the p23ζ form (bottom right) nor the activation and recruitment of ZAP-70 (bottom right). Because of the blunted response through the TCR, signals that would normally result in T cell activation do not occur. Whether or not monophosphorylated ITAMs are formed and whether they serve to recruit negative regulators following partial agonist- or antagonist-TCR interaction will require further experimentation.
To explore the role of these ITAMs in TCR function, several groups have transduced ζ chains lacking one or more of these sequences into TCR transgenic, ζ chain–deficient mice. In all cases, it appears that peripheral T cells from the ζ chain–reconstituted animals can still be activated through the TCR (reviewed in refs. 9, 10), suggesting that ITAMs present in the CD3 chains are sufficient for this process. Indeed, different CD3 chain ITAMs have been found to interact with distinct substrates in vitro (reviewed in ref. 6), implying that there is some potential for activating distinct signaling cascades through these various motifs. Thus, as Sommers et al. have noted, ITAM-mutant CD3ε subunits placed into CD3ε-deficient mice do not support T cell survival as the wild-type subunit does, at least in the transgenic TCR mouse line studied (11). These authors also found that T cell maturation appeared normal, suggesting that, as was seen in ζ chain ITAM-deficient animals, the loss of a functional CD3ε ITAM leads to a quantitative but not a qualitative effect on TCR signaling (11). A recent study has shown that in T cells from CD3δ-deficient mice, which fail to undergo normal positive selection, TCR-induced ζ chain phosphorylation within lipid raft fractions and extracellular signal–regulated kinase (ERK) activation are defective. Interestingly, both events can be reconstituted following introduction of a CD3δ subunit lacking not just its ITAM but its entire cytoplasmic domain (12). Consistent with this finding, mutation of a TCRα domain required for association with CD3δ blocks positive selection (7). Therefore, in addition to their contribution to TCR signaling, the CD3 subunits may also act through non-ITAM domains to detect or influence TCR conformational changes occurring during ligand binding.
Although the various CD3 subunits appear to have similar activities, other ITAM-containing subunits may engage different signaling pathways. For instance, the FcεRIβ and FcεRIγ, which are both required for efficient activation of mast cells through FcεRI (reviewed in ref. 13), act in concert to stimulate the PTK activity of Syk. FcεRIβ associates with Lyn and, upon activation, phosphorylates FcεRIγ, which then recruits and activates Syk. The biological activities of the Ig-associated Igα and Igβ subunits, which also bear ITAMs in their cytoplasmic domains, remain somewhat controversial, apparently because they bind distinct sets of kinases (14). Chimeric receptors containing the Igα and Igβ signaling domains have been used to identify the sequences that mediate receptor coupling with different intracellular signaling cascades (reviewed in refs. 6, 15). Surprisingly, early transgenic experiments in mice suggest that the two subunits are functionally redundant (16, 17). In an attempt to resolve this discrepancy, Reichlin et al. (18) have tested B cell maturation in mice lacking the Igβ cytoplasmic tail (IgβΔC) and have found that these cells indeed differ from their Igα cytoplasmic tail (IgαΔC) deletion counterparts. In the former animals, B cell development occurs normally up to the immature B cell stage, and the B cells show normal calcium signaling upon BCR cross-linking, but they die by apoptosis prior to leaving the bone marrow (18). In homozygous IgαΔC mice, by contrast, pre-B cell development and allelic exclusion occur normally, but there is a progressive and quite substantial loss of B cells throughout all stages of development after the pre-B cell stage (19). Taken together, these data imply that the presence of at least one functional ITAM-containing signaling subunit within the BCR is enough to drive early B cell development but is inadequate for the maintenance of B cell homeostasis. Significantly, IgβΔC/ΔC mice carrying homozygous Y→F ITAM-inactivating mutations at the Igα loci (IgαFF/FF) demonstrate a complete block in B cell development at the pro–B cell stage (20). Thus, although there appears to be significant functional redundancy between the Igα and Igβ ITAM, there is a clear requirement for at least one functional ITAM in the pre-BCR at the pro- to pre-B cell transition.
The data above are consistent with the observed differences in signaling potential between chimeric Igα and Igβ receptors transfected into T and B cell lines. They fit a model in which phosphorylation of the Igα and Igβ subunits occur in a set sequence, with the proximal tyrosine in the Igα ITAM being the first residue to be modified (15). In addition, the presence of additional non-ITAM tyrosines within Igα (1) and the serine and threonine phosphorylation of Igβ (15) suggest that these two subunits play other roles in B cell development and activation. This in part appears to be true, in that the IgαFF/FF mouse, which still retains non-ITAM tyrosines within the Igα cytoplasmic tail, shows a less severe block in B cell development than does the IgαΔC/ΔC mouse. IgαFF/FF mice exhibit a nearly normal level of BCR-induced tyrosine phosphorylation of Igβ and Syk, but impaired Lyn tyrosine phosphorylation. Igα is also tyrosine-phosphorylated in this strain, indicating that other tyrosines within the cytoplasmic tail are targets of BCR-activated kinases and may play an important role during B cell development and activation. Indeed, the B cell adapter protein BLNK (SLP-65) has been found to associate through its SH2 domain with a non-ITAM phosphorylated tyrosine within the Igα cytoplasmic tail (21). Whether this interaction represents an important event in BCR-stimulated maturation or activation of B cells awaits the generation of the appropriate mutant mice. In addition, mice harboring Y→F mutations within the ITAMs of Igβ will be important in determining the contribution of Igβ cytoplasmic tail–associated serine/threonine phosphorylation to B cell development and activation.