Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys 68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells

Stephen O. Mathew, Pappanaicken R. Kumaresan, Jae Kyung Lee, Van T. Huynh, Porunelloor A. Mathew

Research output: Contribution to journalArticlepeer-review

21 Scopus citations

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 Lys68Ala-Glu70Ala h2B4 further demonstrated that Lys68 and Glu70 in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys68 and Glu70 in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.

Original languageEnglish
Pages (from-to)1005-1013
Number of pages9
JournalJournal of Immunology
Volume175
Issue number2
DOIs
StatePublished - 15 Jul 2005

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