Abstract
SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 μM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 μM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.
Highlights
The balance between activation and inhibition in an immune response is critically dependent on the specificity of phosphorylated tyrosine motifs in the cytoplasmic regions of leukocyte surface receptors and associated adaptors for recruitment of intracellular enzymes
In our previous studies we focused on comparison between human and mouse CD244 Immunoreceptor Tyrosine-based ‘‘Switch’’ Motifs (ITSMs) showing that SAP and EAT-2 had similar specificity for phosphorylated ITSMs in both species [22]
Signalling through the SLAM family receptor, CRACC has been reported to be independent of SAP [9,30], suggesting a difference in specificity of the CRACC ITSMs for SAP and EAT-2
Summary
The balance between activation and inhibition in an immune response is critically dependent on the specificity of phosphorylated tyrosine motifs in the cytoplasmic regions of leukocyte surface receptors and associated adaptors for recruitment of intracellular enzymes. An alternative strategy to regulating the balance between activation and inhibition through tyrosine phosphorylation is in the specificity of receptor motifs for the intracellular binding partners. The SLAM family receptors within the CD2 family of leukocyte surface receptors contain multiple Immunoreceptor Tyrosine-based ‘‘Switch’’ Motifs (ITSMs) (Figure 1). These motifs carry the consensus sequence TxYxxV/I/L and biochemical and functional analysis indicate they have overlapping specificity for activating and inhibitory binding partners. Among the defects in lymphocyte function are impairment of cytotoxic activity in both NK cells and CD8+ T cells [13,14,15,16,17,18]
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