Molecular and Functional Characterization of CD300b, a New Activating Immunoglobulin Receptor Able to Transduce Signals through Two Different Pathways This information is current as of September 26, 2021. Águeda Martínez-Barriocanal and Joan Sayós J Immunol 2006; 177:2819-2830; ; doi: 10.4049/jimmunol.177.5.2819 http://www.jimmunol.org/content/177/5/2819 Downloaded from References This article cites 47 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/177/5/2819.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Molecular and Functional Characterization of CD300b, a New Activating Immunoglobulin Receptor Able to Transduce Signals through Two Different Pathways1 A´ gueda Martı´nez-Barriocanal and Joan Sayo´s2 In this study, we describe the characterization of human CD300b, a novel member of the CMRF-35/immune receptor expressed by myeloid cell (IREM) multigene family of immune receptors. Immune receptor expressed by myeloid cell-3 cDNA was cloned from a PHA-activated PBMC library and RT-PCR revealed the gene to be expressed preferentially in cells of myeloid origin. The CD300b cDNA open reading frame encodes a 201-aa type I protein composed of a single extracellular Ig V-type domain followed by a transmembrane region containing a positively charged residue (lysine) which is a common feature among receptors that associate with activating adaptor proteins. Indeed, CD300b was able to associate with DNAX-activating protein of 12 kDa (DAP- Downloaded from 12) and deliver different activating signals through this ITAM-based adaptor. Unusually for an activating receptor, the 29-aa cytoplasmic tail of CD300b contains a tyrosine-based motif that, upon c-Fyn phosphorylation, became a docking site for the intracellular signaling mediator growth factor receptor-bound protein 2. Moreover, in the absence of DAP-12, CD300b was able to activate NFAT/AP-1-dependent transcriptional activity in RBL-2H3 cells. This activity could be abolished only by mutating both the cytoplasmic tyrosine and the transmembrane lysine. Our data suggest the existence of an unidentified molecule capable of interacting with CD300b through a charged residue of the transmembrane region and allowing receptor signaling independent http://www.jimmunol.org/ of DAP-12. Therefore, CD300b defines a nonclassical Ig receptor able to trigger signals by coupling distinct mediators and thus initiating different signaling pathways. The Journal of Immunology, 2006, 177: 2819–2830. mmune function is regulated by a balance between positive gion that allows the receptor to associate with transmembrane and negative signals delivered by activating and inhibitory adaptor proteins. To date, two types of transmembrane adaptor I receptors on the surface of leukocytes. These receptors are proteins have been described: the ITAM-bearing adaptors, includ- responsible for the correct transduction of information from the ing DNAX-activating protein of 12 kDa (DAP-12)/killer cell-ac- exterior to the interior of the cell and elicit a response that can tivating receptor-associated protein (KARAP), Fc␧RI␥, and CD3␨; range from reactivity to quiescence depending on the stimulus (1). and the PI3K-associated adaptor DAP-10. Engagement of activat- by guest on September 26, 2021 Inhibitory receptors display long cytoplasmic tails presenting a ing receptors promotes tyrosine phosphorylation of transmem- variable number of ITIMs. Upon ligand binding, ITIMs are ty- brane adaptors and subsequent recruitment of either protein ty- rosine phosphorylated by Src kinases and become docking sites for rosine kinases containing SH2 domains such as Syk or Zap70, or 3 Src homology 2 (SH2) -containing phosphatases, such as SHIP PI3K that would contribute to activation of AKT and phospho- and SH2 domain-containing phosphatase 1 (SHP-1). Dephosphor- lipase C-␥ pathways. Activating signaling events will lead to se- ylation of key signaling molecules will lead to inhibition of cel- cretion of cell mediators, migration, proliferation, and/or differen- lular activation (2, 3). In contrast, classical activating receptors tiation, among other processes (4–8). display both a short cytoplasmic tail having no known signaling We recently identified immune receptor expressed by myeloid motifs, and a positively charged residue in the transmembrane re- cell (IREM)-1 and IREM-2, members of the CMRF-35/IREM family (CD300) of Ig receptors (9, 10). The genes of this family Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, are clustered in a region on chromosome 17 (17q25.1) spanning Barcelona, Spain ϳ250 kb, and code for both activating and inhibitory receptors. Received for publication February 21, 2006. Accepted for publication June 8, 2006. IREM-1 and IRp60 have been shown to recruit SHP-1/2 phospha- The costs of publication of this article were defrayed in part by the payment of page tases and deliver inhibitory signals (9, 11). Oppositely, IREM-2 charges. This article must therefore be hereby marked advertisement in accordance has been found to associate with the DAP-12 adaptor and therefore with 18 U.S.C. Section 1734 solely to indicate this fact. deliver activating signals (10). The first identified member, 1 This work was supported by grants from Plan Nacional de IϩD (SAF2004-00972). A.M.-B. was supported by a fellowship from the Ministerio de Ciencia y Tecnologı´a, CMRF-35, has a short cytoplasmic tail and a transmembrane re- and J.S. was supported by a contract Ramon y Cajal from Ministerio de Ciencia y gion containing a negatively charged residue, structural features Tecnologı´a. that make its classification uncertain (12). 2 Address correspondence and reprint requests to Dr. Joan Sayo´s, Molecular Immu- nopathology Unit, Departament de Cie`ncies Experimentals i de la Salut, Universitat In the present study, we report the identification and functional Pompeu Fabra, Doctor Aiguader 80, 08003 Barcelona, Spain. E-mail address: characterization of a novel Ig-like receptor belonging to the [email protected] CMRF-35/IREM family. CD300b, expressed mainly on myeloid 3 Abbreviations used in this paper: SH2, Src homology 2; Grb2, growth factor re- cells, is a nonclassical activating receptor able to deliver signals ceptor-bound protein 2; HA, hemagglutinin; WT, wild type; mIREM, murine IREM; Tx-100, Triton X-100; TREM, triggering receptor expressed on myeloid cell; SH3, not only by associating with the transmembrane adaptor protein Src homology 3; SHP-1, SH2 domain-containing phosphatase 1; PVDF, polyvinyli- DAP-12, but also by recruiting growth factor receptor-bound pro- dene difluoride; DAP-12, DNAX-activating protein of 12 kDa; KARAP, killer cell- activating receptor-associated protein; IREM, immune receptor expressed by myeloid tein 2 (Grb-2) through a tyrosine-based motif present in the recep- cell. tor’s cytoplasmic tail. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 2820 CLONING OF CD300b Materials and Methods lowed by a FLAG epitope into the HindIII site of pCDNA3. Sequences ␨ Cells and Abs encoding DAP-12 and CD3 transmembrane adaptor proteins without their signal peptides were amplified by PCR and cloned into the BamHI/EcoRI Human T (Jurkat, Molt-4, T-All 103/102), B (Ramos, Daudi, RPMI 8866), and EcoRI/XhoI sites, respectively, of pCDNA3-FLAG. DAP-12 was am- NK (YT, NKL), and myelomonocytic cell lines (U937, THP-1, HL60, plified from pJFE14-SR␣/DAP-12 using oligos 9–10 and CD3␨ was am- MonoMac6), and KU812, P815, and Raw264.7 cell lines were maintained plified from pSR␣/CD3␨ using oligos 11–12 (10). A pBabePuro-hemag- in RPMI 1640/L-glutamine medium supplemented with 10% heat-inacti- glutinin (HA) expression vector was generated by cloning the Ig␬ signal vated FBS, 25 mM HEPES, 2 mM glutamine, 100 IU/ml penicillin, and peptide followed by an HA epitope into the BamHI/EcoRI sites of pBa- 100 ␮g/ml streptomycin. T-All 103/102 and NKL culture medium were bePuro. CD300b wild-type (WT), CD300b Y188F, CD300b K158L, and additionally supplemented with 100 U/ml human IL-2 (BD Pharmingen). CD300b K158L Y188F were cloned into the EcoRI/SalI sites of pBabe- COS-7 and RBL-2H3 cells were grown in DMEM containing 10% heat- Puro-HA vector by PCR amplification from the pDisplay constructs using inactivated FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 IU/ml oligos 13-2. For the three-hybrid system assay, sequence encoding the penicillin, and 100 ␮g/ml streptomycin. Human PBMC were obtained from CD300b cytoplasmic tail, obtained using oligos 14–2, was cloned into the heparinized venous blood of healthy donors by Ficoll-Hypaque gradient EcoRI/SalI sites of pBridge/c-Fyn420Y-F, 531Y-F, 176R-Q MCSI cassette de- centrifugation. Human NK cells were isolated and cultured as described scribed previously (15). The pBridge/CD300b Cyto/c-Fyn vector was di- (13). gested with BglII endonuclease and religated to obtain a pBridge/ Anti-HA.11 mAb was obtained from Covance. Mouse anti-phospho- CD300bCyto/c-Fyn catalytic mutant construct. tyrosine mixture coupled to HRP was obtained from Zymed Laboratories.