title: A single amino acid change in inhibitory killer cell Ig-like receptor results in constitutive receptor self-association and phosphorylation.
abstract: Signaling by immunoreceptors is often initiated by phosphorylation of cytosolic tyrosines, which then recruit effector molecules. In the case of MHC class I-specific inhibitory receptors, phosphorylation of cytosolic tyrosine residues within ITIMs results in recruitment of a protein tyrosine phosphatase that blocks activation signals. Recent work showed that signaling by an HLA-C-specific killer cell Ig-like receptor (KIR) is independent of signaling by activation receptors. It is not known how ITIM phosphorylation is initiated and regulated. In this article, we show that substitution of His-36 in the first Ig domain of KIR2DL1 with alanine (KIR2DL1-H36A) resulted in constitutive KIR2DL1 self-association and phosphorylation, as well as recruitment of tyrosine phosphatase SHP-1. Furthermore, substitution of His-36 with a similar bulky amino acid, phenylalanine, maintained the receptor in its unphosphorylated state, suggesting that steric hindrance by the His-36 side chain prevents constitutive KIR2DL1 self-association and ITIM phosphorylation. The equally strong phosphorylation of KIR2DL1 and KIR2DL1-H36A after inhibition of tyrosine phosphatase by pervanadate suggested that KIR2DL1-H36A is selectively protected from dephosphorylation. We propose that KIR phosphorylation is controlled by the accessibility of ITIM to tyrosine phosphatases and that KIR binding to HLA-C must override the hindrance that His-36 puts on KIR2DL1 self-association. Expression of KIR2DL1-H36A on NK cells led to stronger inhibition of lysis of HLA-C(+) target cells than did expression of wild-type KIR2DL1. These results revealed that ITIM phosphorylation is controlled by self-association of KIR and that His-36 serves as a gatekeeper to prevent unregulated signaling through KIR2DL1.
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852;
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20892; and.
authors.forename: Pabak
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; Lung Immunology Group, Infectious Diseases and Immunity, Department of Medicine, Imperial College, Hammersmith Hospital, London W12 0NN, United Kingdom.
authors.forename: Malcolm J W
authors.initials: MJ
authors.lastname: Sim
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852;
authors.forename: Sumati
authors.initials: S
authors.lastname: Rajagopalan
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20892; and.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Vogel
authors.affiliation: Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; elong@nih.gov.
authors.forename: Eric O
authors.initials: EO
authors.lastname: Long
mesh_terms: D019943:Amino Acid Substitution / Q000276:immunology*; D002460:Cell Line; D015236:HLA-C Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology*; D010766:Phosphorylation / Q000235:genetics / Q000276:immunology; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000235:genetics / Q000276:immunology; D054342:Receptors, KIR2DL1 / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology
chemical_list: D015236:HLA-C Antigens; C093100:KIR2DL1 protein, human; D054342:Receptors, KIR2DL1; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6