Abstract
NKG2D is a stimulatory receptor expressed by NK cells and some T cell subsets. Expression of the self-encoded ligands for NKG2D is presumably tightly regulated to prevent autoimmune disorders while allowing detection of infected cells and developing tumors. The NKG2D ligand Mult1 is regulated at multiple levels, with a final layer of regulation controlling protein stability. In this article, we report that Mult1 cell-surface expression was prevented by two closely related E3 ubiquitin ligases membrane-associated RING-CH (MARCH)4 and MARCH9, members of an E3 family that regulates other immunologically active proteins. Lysines within the cytoplasmic domain of Mult1 were essential for this repression by MARCH4 or MARCH9. Downregulation of Mult1 by MARCH9 was reversed by heat-shock treatment, which resulted in the dissociation of the two proteins and increased the amount of Mult1 at the cell surface. These results identify Mult1 as a target for the MARCH family of E3 ligases and show that induction of Mult1 in response to heat shock is due to regulated association with its E3 ligases.
MeSH terms
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Animals
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Blotting, Western
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Carrier Proteins / biosynthesis
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Carrier Proteins / genetics
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Carrier Proteins / immunology*
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Cell Separation
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Cells, Cultured
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Flow Cytometry
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GPI-Linked Proteins
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Gene Expression
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Gene Expression Regulation / immunology*
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Histocompatibility Antigens Class I / biosynthesis
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Histocompatibility Antigens Class I / genetics
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Histocompatibility Antigens Class I / immunology*
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Humans
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Immunoprecipitation
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / immunology*
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Killer Cells, Lymphokine-Activated / immunology
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Killer Cells, Lymphokine-Activated / metabolism
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Killer Cells, Natural / immunology
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Killer Cells, Natural / metabolism
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Membrane Proteins / biosynthesis
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Membrane Proteins / genetics
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Membrane Proteins / immunology*
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Membrane Proteins / metabolism
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Mice
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Mice, Knockout
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NK Cell Lectin-Like Receptor Subfamily K / immunology
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NK Cell Lectin-Like Receptor Subfamily K / metabolism
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Reverse Transcriptase Polymerase Chain Reaction
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Transfection
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Ubiquitin-Protein Ligases / immunology*
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Ubiquitin-Protein Ligases / metabolism
Substances
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Carrier Proteins
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GPI-Linked Proteins
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Histocompatibility Antigens Class I
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Intracellular Signaling Peptides and Proteins
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KLRK1 protein, human
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Membrane Proteins
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NK Cell Lectin-Like Receptor Subfamily K
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UL16 binding protein 1, mouse
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ULBP1 protein, human
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MARCHF9 protein, human
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Ubiquitin-Protein Ligases