Fig 1.
Overview of the CD200R signaling pathway.
p-Akt is inhibited by Dok2, which also reqruits RasGAP. RasGAP inhibits p-Erk, resulting in inhibition of IL-8. Inhibition of p-rpS6 by CD200R is a result of the inhibition of both p-Akt and p-Erk, although CD200R-mediated p-rpS6 inhibition might also be the result of another yet unknown pathway. This figure was adapted from Van der Vlist et al. [17].
Table 1.
Schematic representation of primers used for mutagenesis.
Table 2.
Primer sequences for mutagenesis and sub-cloning.
Table 3.
Overview of materials.
Fig 2.
The high conservation of the intracellular domain of CD200R among species is not limited to the NPxY-motif.
The NCBI protein BLAST tool identified 179 human CD200R1 homologues amongst all 5 animal classes. A) Phylogenetic analysis of a balanced selection of full CD200R1 homologues. Mouse CD200 sequence was used as an outgroup to root the tree. B) All CD200R sequences identified in A) were plotted in a conservation plot using WebLogo [35]. C-E) Conservation logo’s created by WebLogo [35] per animal class: mammals (C); birds (D); and bony fish (E). Maximal conservation represents 4.2 bits. The height of the amino acid letters shows the frequency of the amino acid on that particular position in the sequence alignment. The amino acids are colored according to the physio-chemical properties of their side chains: polar (green), neutral (purple), basic (blue), acidic (red), or hydrophobic (black).
Fig 3.
U937-CD200R mutants express CD200R on their surface.
A) Conserved residues of CD200R in mammals were mutated as indicated. B) U937 cells were transduced with different CD200R mutants and tested for their CD200R surface expression by flow cytometry. Representative figures of the gating strategy are shown. First the population of live cells was selected. From these live cells, single cells were selected and from these single cells CD200R expressing cells were selected for phosflow analysis. SSC-A: side scatter area; FSC-A: forward scatter area; FSC-W: forward scatter width. Control: staining with the secondary antibody only.
Fig 4.
EEDE279, P285 and K292 are important for CD200R function.
A-E) U937-CD200R mutants were stimulated with an agonistic CD200R antibody (OX108). p-Erk (B), p-Akt308 (C), p-Akt473 (D) and p-rpS6 (E) of CD200R+ cells were assessed by phosflow analysis. IL-8 secretion after 24 hours of LPS stimulation was measured by ELISA (F). The percentage of inhibition is depicted as the relative difference between isotype and anti-CD200R stimulated U937 cells. Error bars represent the mean with SEM. All mutants were tested for significant differences compared to WT (*) and Truncated (□) using a Kruskal-Wallis test. N≥6. For the specific amount of experiments conducted per mutant and per residue, see S2 Table. F) Heatmap of the means of the data shown in Fig B-F. A double gradient from blue to red was used for the percentage of inhibition.