title: Fine specificity and molecular competition in SLAM family receptor signalling.
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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Wilson
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Lee I
authors.initials: LI
authors.lastname: Garner
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Clive
authors.initials: C
authors.lastname: Metcalfe
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Elliott
authors.initials: E
authors.lastname: King
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Margraf
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
authors.forename: Marion H
authors.initials: MH
authors.lastname: Brown
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D007113:Immunity, Innate; D061625:Immunoreceptor Tyrosine-Based Activation Motif; D061626:Immunoreceptor Tyrosine-Based Inhibition Motif; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D051966:Phospholipase C gamma / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics / Q000378:metabolism; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3 / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D000071176:Signaling Lymphocytic Activation Molecule Family; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C084093:CD244 protein, human; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C103572:SH2D1B protein, human; C000604060:SLAMF6 protein, human; C448987:SLAMF7 protein, human; D000071176:Signaling Lymphocytic Activation Molecule Family; D014157:Transcription Factors; D000071177:Signaling Lymphocytic Activation Molecule Family Member 1; C516724:PTPN11 protein, human; C431695:PTPRH protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D054631:Receptor-Like Protein Tyrosine Phosphatases, Class 3; D051966:Phospholipase C gamma