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. 2009 Oct 15;183(8):4879-86.
doi: 10.4049/jimmunol.0901531. Epub 2009 Sep 28.

Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells

Robin Mihrshahi  1 , A Neil Barclay, Marion H Brown
Affiliations

Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells

Robin Mihrshahi et al. J Immunol. .

Abstract

The CD200 receptor (CD200R) acts as a negative regulator of myeloid cells by interacting with its widely expressed ligand CD200. Using mutants expressed in U937 cells, we show that inhibition is mediated by the PTB domain binding motif (NPLY) in the receptor's cytoplasmic region. The adaptor protein downstream of tyrosine kinase 2 (Dok2) bound directly to the phosphorylated NPLY motif with a 10-fold higher affinity (K(D) of approximately 1 microM at 37 degrees C) than the closely related Dok1. Both of these proteins have been suggested to play a role in CD200R signaling in murine cells. Dok2 was phosphorylated in response to CD200R engagement and recruited RAS p21 protein activator 1 (RasGAP). Knockdown of Dok2 and RasGAP by RNA interference revealed that these proteins are required for CD200R signaling, while knockdown of Dok1 and the inositol 5-phosphatase SHIP did not affect CD200R-mediated inhibition. We conclude that CD200R inhibits the activation of human myeloid cells through direct recruitment of Dok2 and subsequent activation of RasGAP, which distinguishes this receptor from the majority of inhibitory receptors that utilize ITIMs and recruit phosphatases.

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Figures

Fig. 1
Fig. 1
CD200R mutants are expressed on transduced U937 cells. U937 cells were lentivirally transduced to express wild-type (Wt) or signaling deficient (cytoplasmic domain truncated [Tr] or with each of its three tyrosines mutated to phenylalanines [Y1F, Y2F, Y3F]) human CD200R. Expression of wild-type and mutant CD200R is shown after magnetic (MACS) beads separation using OX108 mAb. Cells were stained with OX108 followed by phycoerythrin-conjugated anti-mouse IgG F(ab’)2.
Fig. 2
Fig. 2
CD200R engagement inhibits activation of U937 cells. (A) Wild-type (black) and truncated (grey) CD200R were engaged on U937 cells using different reagents as indicated. Plate-bound OX108 or isotype control were immobilized at 40 μg/ml, soluble OX108 was used at 10 μg/ml and crosslinking anti-mouse IgG at 20 μg/ml. Cells were then stimulated overnight with 20 ng/ml LPS and culture supernatants assayed for IL-8 by ELISA. (B) Wild-type or truncated CD200R was engaged on U937 using 100 μg/ml plate-bound OX108 mAb and cells were stimulated overnight using different reagents as indicated. Neisseria = ethanol-killed Neisseria meningitides diluted in culture medium. * = p < 0.05 according to two-tailed student’s t-test, ** = p < 0.005, N.S. = non-significant. Results are expressed as means of duplicate or triplicate wells ± standard deviation and are representative of two or more independent experiments.
Fig. 3
Fig. 3
The innhibitory effect of CD200R is mainly mediated by its third intracellular tyrosine. CD200R (wild-type and mutants) was engaged on U937 cells using 100 μg/ml plate-bound OX108 mAb and stimulated and assayed as in Fig. 2. * = p < 0.05, *** = p < 0.0005, N.S. = non-significant. Results are expressed as means of triplicate wells ± standard deviation and are representative of six experiments. No significant effects of tyrosine mutations were observed in the absence of OX108 mAb or in the presence of an isotype control mAb (data not shown).
Fig. 4
Fig. 4
Dok2 binds the cytoplasmic tail of CD200R with higher affinity than Dok1. Singly (pY3) or doubly (pY1,3) phosphorylated peptides corresponding to parts of the cytoplasmic tail of human CD200R were immobilized on a BIAcore chip. (A) Binding of various concentrations (μM) of recombinant PTB domain of human Dok2 passed over flow cells containing immobilized unphosphorylated (pY0) control or pY1,3 phosphopeptide. (B) and (C), Equilibrium binding values at each concentration for Dok2 (B) and Dok1 (C) binding to pY3 and pY1,3 phosphopeptides. The hyperbolas represent best fits used for affinity calculations. Results are representative of two independent experiments.
Fig. 5
Fig. 5
Phosphorylation of Dok2 and recruitment of RasGAP following engagement of CD200R. U937 cells expressing wild-type or truncated CD200R were incubated for 5 min at 37°C in the presence of CD200-COMP. Cells were then lysed and Dok2 (A) or SHIP (B) was immunoprecipitated from lysates. Immunoprecipitates were blotted with biotinylated anti-phosphotyrosine mAb. Membranes were then stripped and re-blotted with specific antibodies against the indicated proteins. Results are representative of at least three independent experiments. (C) IL-4 activated human macrophages were treated with CD200R mAb (OX108) or isotype control (OX21) for 7 min, followed by Dok2 immunoprecipitation and Western blotting with the indicated antibodies. Results are representative of two independent experiments conducted with macrophages from three different donors.
Fig. 6
Fig. 6
Knockdown of Dok2 and RasGAP abrogates CD200R signaling in U937 cells. U937 cells expressing wild-type (black) or truncated (grey) CD200R were transfected twice with siRNA against Dok2 (A), Dok1 (B), SHIP or RasGAP (C). 24–48 h after the second transfection, cells were incubated overnight in the presence of 2.5 μg/ml soluble OX108 mAb and 50 ng/ml LPS and the supernatants assayed for IL-8. The remaining cells were lysed and lysates were western blotted to assess the degree and specificity of protein knockdown. A scrambled (siScr) Dok2 siRNA sequence or a non-targeting control siRNA sequence (siNeg) was used as negative control. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, N.S. = non-significant. Results are expressed as means of triplicate wells normalized to the amount of IL-8 produced by cells expressing the truncated CD200R ± standard deviation. Results are representative of at least three independent experiments.
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References

    1. Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH, Barclay AN. Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity. 2000;13:233–242. - PubMed
    1. Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, Song Y, Jenmalm M, Gorman D, McClanahan T, Liu MR, Brown MH, Sedgwick JD, Phillips JH, Barclay AN. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J. Immunol. 2003;171:3034–3046. - PubMed
    1. Hatherley D, Barclay AN. The CD200 and CD200 receptor cell surface proteins interact through their N-terminal immunoglobulin-like domains. Eur. J. Immunol. 2004;34:1688–1694. - PubMed
    1. Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, Blom B, Homola ME, Streit WJ, Brown MH, Barclay AN, Sedgwick JD. Down-regulation of the macrophage lineage through interaction with OX2 (CD200) Science. 2000;290:1768–1771. - PubMed
    1. Cherwinski HM, Murphy CA, Joyce BL, Bigler ME, Song YS, Zurawski SM, Moshrefi MM, Gorman DM, Miller KL, Zhang S, Sedgwick JD, Phillips JH. The CD200 receptor is a novel and potent regulator of murine and human mast cell function. J. Immunol. 2005;174:1348–1356. - PubMed

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