Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells.
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.
Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA. pmathew@hsc.unt.edu
Stephen O
SO
Mathew
Pappanaicken R
PR
Kumaresan
Jae Kyung
JK
Lee
Van T
VT
Huynh
Porunelloor A
PA
Mathew
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000954:Antigens, Surface / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000071178:CD48 Antigen; D003602:Cytotoxicity, Immunologic / Q000235:genetics*; D004252:DNA Mutational Analysis; D019281:Dimerization; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000235:genetics* / Q000276:immunology; D008239:Lysine / Q000235:genetics* / Q000378:metabolism; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000071176:Signaling Lymphocytic Activation Molecule Family
D015703:Antigens, CD; D000954:Antigens, Surface; C084093:CD244 protein, human; D000071178:CD48 Antigen; C000604065:CD48 protein, human; C000604066:Cd48 protein, mouse; D007166:Immunosuppressive Agents; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D000071176:Signaling Lymphocytic Activation Molecule Family; D018698:Glutamic Acid; D008239:Lysine; D000409:Alanine