Fig. 4.

Mutagenesis of four codons that differ between two KIR2DS5 alleles identifies residues altering cell surface expression. NKL cells were transfected with constructs encoding V5-tagged nonexpressed KIR2DS5 (*001 with L-20, P111, S164, A174), expressed KIR2DS5 (*002 with S-20, S111, F164, T174) and mutants of KIR2DS5 altering codons shown in Table 1. The mutants are labeled with the four polymorphic amino acids present; residues found in the expressed KIR2DS5 (*002) are underlined. a Lysates from each transfectant were electrophoresed under denaturing conditions and proteins analyzed by Western blotting using a V5-specific Ab. Two isoforms were exhibited by KIR2DS5 (*002) and positive control KIR2DS1; only the lower molecular weight isoform was present in the lane containing lysate from KIR2DS5 (*001). Mutants 5, 8, and 11 which carry S111 and F164 of KIR2DS5 (*002) exhibited two isoforms with molecular weights identical to KIR2DS5 (*002) although the upper molecular weight bands were faint in mutants 8 and 11. The remaining mutants, like KIR2DS5 (*001), only exhibited the lower molecular weight isoform. Cells transfected with empty vector served as a negative control. b Representative flow cytometric analysis of transfectants expressing a subset of the mutant constructs. Mutants with both S111 and F164 (mutants 5, 8, 11) were expressed on the cell surface as is KIR2DS5 (*002). The levels of expression of KIR2DS5 (*002) and mutant 5 which shares T174 were similar while the surface expression of mutants 8 and 11 with A174 were lower than KIR2DS5 (*002). The levels of mutants 8 and 11 were not significantly different from one another. Mutants 6 and 7, lacking S111 or F164, were not expressed. A vector carrying a KIR insert without the external HA tag served as a negative control. By Student’s t-test compared to wild type KIR2DS5 (*002) for a representative experiment performed in triplicate: *, 0.05–0.01; **, p-value 0.01–0.001