DAP12 impacts trafficking and surface stability of killer cell immunoglobulin-like receptors on natural killer cells

Abstract

Killer cell immunoglobulin-like receptors (KIR) aid in the regulation of natural killer (NK) cell activity. In this study, the effect of the interaction between the two domain stimulatory KIR (KIR2DS) and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when co-transfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12 specific siRNA. Treatment of the KIR2DS and DAP12 transfected cells with either cycloheximide or brefeldin A repressed KIR2DS surface expression revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS indicating the interaction between these proteins initiates early in the maturation process, likely within the endoplasmic reticulum. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.