Abstract
In the human killer cell immunoglobulin-like receptors, KIR2DL2, and KIR2DL3, a triad of amino acids in the D1 domain interact to stabilize protein structure. Substitution of any one of these residues caused significant loss of cell surface expression. Although KIR2DS4 and KIR2DS5, two homologous receptors, differ for this triad, flow cytometry analysis of NK and T cell lines transfected with stimulatory KIR genes KIR2DS4 (allele *001) and KIR2DS5 (allele *002) demonstrated cell surface expression. For KIR2DS5, restoration of the triad sequence increased surface expression. Further studies of the receptor encoded by KIR2DS5*002 showed both mature and immature protein isoforms upon gel electrophoresis coupled with surface biotinylation or deglycosylation. In contrast, the KIR2DS5*001 allelic product was not expressed on the cell surface of either NK or T cells and exhibited only a single immature isoform upon gel electrophoresis. Site-directed mutagenesis demonstrated that absence of the KIR2DS5*001-encoded protein at the cell surface was imparted primarily by two amino acid polymorphisms in the D2 domain. Analysis using molecular dynamics simulations suggested that the substitution of a proline for a serine at residue 111 or the substitution of a serine for a phenylalanine at residue 164 caused destabilization of the domain structure and intracellular retention. A third polymorphism at residue 174 impacted the level of KIR2DS5 surface expression. This is the first description at a stimulatory KIR locus of the impact of specific amino acid variations on receptor maturation and the level of surface expression.
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Abbreviations
- KIR:
-
killer cell Ig-like receptor
- 2DL:
-
two extracellular domains, long cytoplasmic tail
- 3DL:
-
three extracellular domains, long cytoplasmic tail
- 2DS:
-
two extracellular domains, short cytoplasmic tail
- MD:
-
molecular dynamics
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Acknowledgements
Funding from the Office of Naval Research N00014-05-1-0784 and N00014-06-1-0726 (C.K.H.) supported this research. The views expressed in this article are those of the authors and do not reflect the official policy of the Department of Navy, the Department of Defense, or the United States government. These studies were conducted using the Tissue Culture and Flow Cytometry and Cell Sorting Shared Resources of Lombardi Comprehensive Cancer Center supported by National Cancer Institute Cancer Center Support Grant CA51008. Computing time and staff support was in part provided by the Advanced Biomedical Computing Center at the National Cancer Institute (Frederick, MD). We thank Karen Creswell and Michelle Lombard for providing excellent technical assistance and Tiernan Mulrooney, William Frazier, and Phillip Posch for helpful discussions.
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Steiner, N.K., Dakshanamurthy, S., VandenBussche, C.J. et al. Extracellular domain alterations impact surface expression of stimulatory natural killer cell receptor KIR2DS5. Immunogenetics 60, 655–667 (2008). https://doi.org/10.1007/s00251-008-0322-2
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DOI: https://doi.org/10.1007/s00251-008-0322-2