Essential roles for Dok2 and RasGAP in CD200 receptor-mediated regulation of human myeloid cells.
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.
Mihrshahi
Robin
R
Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.
Barclay
A Neil
AN
Brown
Marion H
MH
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D064236:Cartilage Oligomeric Matrix Protein; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000276:immunology / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology; D016209:Interleukin-8 / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D064235:Matrilin Proteins; D051379:Mice; D022423:Myeloid Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D064446:Orexin Receptors; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000276:immunology / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology; D020729:p120 GTPase Activating Protein / Q000235:genetics / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000954:Antigens, Surface; C477337:CD200R1 protein, human; C508606:CXCL8 protein, human; D064236:Cartilage Oligomeric Matrix Protein; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C115089:DOK2 protein, human; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C508598:IL4 protein, human; D016209:Interleukin-8; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins; D064446:Orexin Receptors; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; C577553:TSP5 protein, human; D020729:p120 GTPase Activating Protein; D020703:ras GTPase-Activating Proteins; D015847:Interleukin-4; C042739:antigens, CD200