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Essential Roles for Dok2 and RasGAP in CD200 Receptor-Mediated Regulation of Human Myeloid Cells¹

Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

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 1 µM at 37°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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by an Oxford University Clarendon Fund award to R.M. and by the U.K. Medical Research Council (G0601169 and G0400808).

² Address correspondence and reprint requests to Dr. Marion H. Brown, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, U.K. E-mail address: marion.brown{at}path.ox.ac.uk

³ Abbreviations used in this paper: CD200R, CD200 receptor; Dok, downstream of tyrosine kinase; PTB, phosphotyrosine binding; RasGAP, RAS p21 protein activator 1; RU, response unit; SH2, SRC homology 2; SHP, SRC homology 2 domain-containing phosphatase; siRNA, small interfering RNA; SPR, surface plasmon resonance.