title: DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells.
abstract: KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.
authors.affiliation: Georgetown University Medical Center, Research Building, Room E404, Georgetown University Medical Center, 3970 Reservoir Rd., N.W., Washington, DC 20057, USA.
authors.forename: Tiernan J
authors.initials: TJ
authors.lastname: Mulrooney
authors.forename: Phillip E
authors.initials: PE
authors.lastname: Posch
authors.forename: Carolyn Katovich
authors.initials: CK
authors.lastname: Hurley
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism*; D008247:Lysosomes / Q000502:physiology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D054340:Receptors, KIR / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C496212:KIR2DS1 protein, human; C433932:KIR2DS2 protein, human; C473953:KIR2DS4 protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D054340:Receptors, KIR; D011993:Recombinant Fusion Proteins; C110546:TYROBP protein, human
keywords: innate immunity; receptor internalization; receptor maturation