Abstract
Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys(68)Ala-Glu(70)Ala h2B4 further demonstrated that Lys(68) and Glu(70) in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys(68) and Glu(70) in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Alanine / genetics
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Amino Acid Sequence
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Animals
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Antigens, CD / chemistry
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Antigens, CD / genetics*
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Antigens, CD / metabolism*
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Antigens, Surface / chemistry
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Antigens, Surface / metabolism*
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Antigens, Surface / physiology
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CD48 Antigen
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Cytotoxicity, Immunologic / genetics*
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DNA Mutational Analysis
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Dimerization
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Down-Regulation / genetics
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Down-Regulation / immunology
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Glutamic Acid / genetics*
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Glutamic Acid / metabolism
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Humans
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Immunosuppressive Agents / antagonists & inhibitors
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Immunosuppressive Agents / chemistry
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Immunosuppressive Agents / metabolism
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Killer Cells, Natural / immunology*
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Killer Cells, Natural / metabolism
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Lymphocyte Activation / genetics*
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Lymphocyte Activation / immunology
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Lysine / genetics*
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Lysine / metabolism
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Melanoma, Experimental / genetics
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Melanoma, Experimental / metabolism
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Membrane Glycoproteins / antagonists & inhibitors
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Membrane Glycoproteins / chemistry
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Membrane Glycoproteins / genetics*
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Membrane Glycoproteins / metabolism*
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Membrane Glycoproteins / physiology
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Mice
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Molecular Sequence Data
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Protein Binding / genetics
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Protein Binding / immunology
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Protein Structure, Tertiary / genetics
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Receptors, Immunologic / antagonists & inhibitors
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Receptors, Immunologic / chemistry
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Receptors, Immunologic / genetics*
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Receptors, Immunologic / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Signaling Lymphocytic Activation Molecule Family
Substances
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Antigens, CD
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Antigens, Surface
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CD244 protein, human
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CD48 Antigen
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CD48 protein, human
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Cd48 protein, mouse
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Immunosuppressive Agents
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Membrane Glycoproteins
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Receptors, Immunologic
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Recombinant Fusion Proteins
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Signaling Lymphocytic Activation Molecule Family
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Glutamic Acid
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Lysine
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Alanine