Rhesus Macaque Activating Killer Immunoglobulin-Like Receptors Associate With Fc Receptor Gamma (FCER1G) and Not With DAP12 Adaptor Proteins Resulting in Stabilized Expression and Enabling Signal Transduction

Abstract

Activating killer cell immunoglobulin-like receptors (KIR) in macaques are thought to be derived by genetic recombination of the region encoding the transmembrane and intracellular part of KIR2DL4 and a KIR3D gene. As a result, all macaque activating KIR possess a positively charged arginine residue in the transmembrane region. As human KIR2DL4 associates with the FCER1G (also called Fc receptor-gamma, FcRγ) adaptor, we hypothesized that in contrast to human and great ape the activating KIRs of macaques associate with FcRγ instead of DAP12. By applying co-immunoprecipitation of transfected as well as primary cells, we demonstrate that rhesus macaque KIR3DS05 indeed associates with FcRγ and not with DAP12. This association with FcRγ results in increased and substantially stabilized surface expression of KIR3DS05. In addition, we demonstrate that binding of specific ligands of KIR3DS05, Mamu-A1*001 and A1*011, resulted in signal transduction in the presence of FcRγ in contrast to DAP12.

Keywords: Activating killer cell immunoglobulin-like receptors; Co-immunoprecipitation (co-IP); DAP12; FCER1G; NK cell; activating KIR; adaptor association; rhesus macaque (Macaca mulatta).

Figure 1

Co-immunoprecipitation of adaptor proteins. (A) Immunoprecipitation of KIR3DS05 was performed with anti-rhesus macaque pan-KIR3D antibody 1C7 (26) as indicated (IP). As positive control we used cell lysates of KIR and adaptor protein-expressing cells. Anti-myc antibody was used in blots to detect myc-tagged FcRγ and DAP12 adaptor proteins. A representative figure is shown from five independent experiments. The molecular weight of rhesus macaque FcRγ and DAP12 is 13.2 and 10.8 kDa, respectively, and the size difference is hardly detectable in the western blot analyses. (B) Lysates of rhesus macaque PBMCs were used directly (pos. control) or were subjected to immunoprecipitation with antibody 1C7 (IP). Detection of adaptor proteins in western blots was performed with anti-FcRγ and anti-DAP12 polyclonal antibodies. To control for unspecific binding of the polyclonal anti-adapter protein antibodies in western blot, we used FcRγ and DAP12 negative cells (untransfected HEK-293 cells; neg. control). The results from one out of two independent experiments are shown.

Figure 2

Flow cytometry analysis of rhesus macaque KIR3DS05 cell surface expression in the presence of myc-tagged FcRγ or DAP12 adaptor proteins. (A) Histograms of a representative experiment of HEK-293 and HeLa cells transiently transfected with AcGFP-tagged KIR3DS05, either alone or in combination with either DAP12 or FcRγ (thick lines). Monoclonal anti-rhesus macaque KIR antibody 1C7 was used to detect KIR3DS05 expression indicated as percent positive cells. Untransfected HEK-293 and HeLa cells were used as respective negative controls of 1C7 staining (thin lines). (B) Mean fluorescence intensity of KIR3DS05 expression in transfected cells among independently performed experiments (HEK-293 n=5; HeLa n=3). (C) HEK-293 cells after transfection with AcGFP-tagged KIR3DS05 were gated on the singlet cells and then 1C7-stained cells were measured within the fraction of AcGFP-positive cells population. A representative experiment is shown. (D) 1C7-stained cells within the AcGFP-positive fraction were measured in individual experiments (n=3). ns, statistically not significant.

Figure 3

Confocal laser microscopy analysis of rhesus macaque AcGFP-tagged KIR3DS05 cell surface expression in the presence of myc-tagged FcRγ or DAP12 adaptor proteins. (A) Cells were stained as indicated. In the merged figures, yellow color indicates co-localization of KIR3DS05 (green) and adaptor (red). DAPI staining (blue) shows cell nuclei. Scale bar represents 10.20 µm. (B) Single confocal sections of 11 randomly chosen individual cells were also used to analyze mean fluorescence intensity values using the ZEN software (version 2.3). The means of columns were calculated and corresponding experiments were compared. Statistical significance of differences is shown. ns, not significant. (C) Flow cytometric analysis of correlation of cell surface expressed KIR3DS05 in combination with adaptor proteins in cells stained with 1C7 and anti-myc antibodies. The gates indicate: a = 1C7 and myc-positive; b = only myc-positive.

Figure 4

Stability of AcGFP-tagged KIR3DS05 expression in the presence of myc-tagged FcRγ and DAP12 adaptor proteins. HEK-293 cells stably transfected with AcGFP-tagged KIR3DS05 either alone or in combination with either myc-tagged FcRγ or DAP12 adaptor proteins were followed for KIR3DS05 expression over time (one cell passage corresponds to 2-3 days). (A) Flow cytometric analysis of KIR3DS05 cell surface expression measured with antibody 1C7 after each passage of the cells. (B) Identical percentages of KIR3DS05-positive proteins were used as starting point (passage 0) and expression of KIR3DS05 (% positive cells) was measured after each passage of the cells. The results of three independent experiments and of unpaired t tests are shown (ns, not significant).

Figure 5

Signal transduction via adaptor protein resulting from interaction of KIR3DS05 with its cognate ligands. Transfected (bold lines) and untransfected control (thin lines) cells are shown. (A) Jurkat cells were transfected with plasmids carrying AcGFP-tagged KIR3DS05 and either myc-tagged DAP12 or FcRγ. Cell surface expression of KIR3DS05 was analyzed in FACS using antibody 1C7. (B) Flow cytometric analysis of AcGFP expression in HEK-293 cells stably transfected with constructs encoding AcGFp-tagged versions of Mamu class I that were described previously (25, 27). (C) The transfected Jurkat cells were incubated for 14 h with the HEK-293 cells expressing the KIR3DS05 ligands Mamu-A1*001 and A1*011 or the non-interacting Mamu-B*030 as negative control. Recognition of the ligands and signal transduction in the transfected Jurkat cells was monitored by CD69 expression in flow cytometry.

Figure 6

Functional consequences resulting from presence of different adaptor proteins in rhesus macaque NK cells expressing stimulatory KIR receptors.