title: Analysis of immunoreceptor tyrosine-based activation motif (ITAM) binding to ZAP-70 by surface plasmon resonance.
abstract: The signaling function of the T cell antigen receptor (TCR) is mediated via CD3 polypeptides, the cytoplasmic sequences of which bear conserved immunoreceptor tyrosine-based activation motifs (ITAM). ITAM are defined by two YxxL/I sequences separated by a six-eight amino acid long spacer. Upon antigen recognition, ITAM become phosphorylated on both tyrosine residues, creating a high affinity binding site for the tandem SH2 domains found in the protein tyrosine kinase ZAP-70. Using surface plasmon resonance, we further dissected the sequences required for the binding of ZAP-70 to each TCR-associated ITAM. First, we generated protein tyrosine phosphatase-resistant ITAM peptide analogs, in which difluorophosphonomethyl phenylalanyl (F2p) replaced both phosphotyrosines, and showed that those protein tyrosine phosphatase-resistant analogs bind ZAP-70 with high affinity, establishing a rational strategy for the design of novel pharmacological tools capable of interfering with TCR signaling function. Second, we substituted the five amino acids separating the two YxxL/I sequences of the CD3 zeta 1 ITAM with a non-peptidic linker made up of gamma-amino butyric acid units and demonstrated that the length of this intervening sequence rather than its chemical composition is essential for high affinity binding of phosphorylated ITAM to the ZAP-70 SH2 domains.
authors.affiliation: Centre d'Immunologie INSERM/CNRS de Marseille-Luminy, France.
authors.forename: F
authors.initials: F
authors.lastname: Vély
authors.forename: J A
authors.initials: JA
authors.lastname: Nunès
authors.forename: B
authors.initials: B
authors.lastname: Malissen
authors.forename: C J
authors.initials: CJ
authors.lastname: Hedgecock
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D018360:Crystallography, X-Ray; D006801:Humans; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017459:Receptors, Amino Acid / Q000032:analysis*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011971:Receptors, Immunologic / Q000032:analysis*; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
chemical_list: D010455:Peptides; D017459:Receptors, Amino Acid; D011948:Receptors, Antigen, T-Cell; D011971:Receptors, Immunologic; C087659:tyrosine receptor; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human