A New Role for Platelet-Endothelial Cell Adhesion Molecule-1 (CD31): Inhibition of TCR-Mediated Signal Transduction This information is current as Debra K. Newton-Nash and Peter J. Newman of September 26, 2021. J Immunol 1999; 163:682-688; ; http://www.jimmunol.org/content/163/2/682 Downloaded from References This article cites 57 articles, 35 of which you can access for free at: http://www.jimmunol.org/content/163/2/682.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on September 26, 2021 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A New Role for Platelet-Endothelial Cell Adhesion Molecule-1 (CD31): Inhibition of TCR-Mediated Signal Transduction1 Debra K. Newton-Nash2 and Peter J. Newman Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress e PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3 mAbs, anti-PECAM-1 mAbs, Downloaded from or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction. The Journal of Immunology, 1999, 163: 682–688. http://www.jimmunol.org/ lymphocyte activation is a process that is controlled, in ciated with the CTLA-4 cytoplasmic domain dephosphorylates part, by regulation of the phosphorylation state of tyrosine tyrosine residues in the ITAMs of the TCR z-chain (5). Similarly, T residues in the cytoplasmic domains of TCR-associated KIRs contain an ITIM within their cytoplasmic domain, which, signal transduction units. In the initial activation phase, tyrosine upon tyrosine phosphorylation, supports the binding of the non- residues in immunoreceptor tyrosine-based activation motifs transmembrane PTP, SHP-1 (6, 7). The substrates for KIR-asso- 3 (ITAM) of the signal transduction units of the TCR complex be- ciated SHP-1 have not yet been determined. come phosphorylated by members of the src family of protein Platelet-endothelial cell adhesion molecule-1 (PECAM-1), or tyrosine kinases (PTKs), supporting recruitment of z-associated CD31, is a 130-kDa member of the Ig gene superfamily that is protein of 70 kDa (ZAP-70), which itself becomes activated by expressed on the surface of endothelial cells and circulating plate- by guest on September 26, 2021 tyrosine phosphorylation and enables transmission of downstream lets, monocytes, neutrophils, and certain T cell subsets. PECAM-1 signals for T cell activation (1). The level of protein tyrosine phos- was originally assigned to the family of Ig-like cellular adhesion phorylation is balanced by the activity of protein tyrosine phos- molecules (CAMs) based on sequence similarity between its six phatases (PTPs), which negatively regulate the initial phase of T extracellular Ig domains and those of other Ig domain-containing cell activation. The mechanism by which the initial phase of T cell CAMs (8). An adhesive function for PECAM-1 is supported by the activation is modulated involves active transmission of negative observations that amino-terminal domains 1 and 2 mediate ho- signals through membrane-associated molecules, such as CTLA-4 mophilic interactions (9, 10) that facilitate adhesion between and killer cell inhibitory receptors (KIRs) (2, 3). CTLA-4 contains PECAM-1-expressing cells (11). Recent evaluation of the structure an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like se- and biochemical properties of its cytoplasmic domain, however, quence that supports the binding of Src homology 2 domain-bear- ing PTP (SHP)-2 (4). Upon docking and activation, SHP-2 asso- suggest that PECAM-1 may also belong to the family of Ig-like, ITIM-containing inhibitory receptors (12). The PECAM-1 cyto- plasmic domain contains numerous potential sites for phosphory- Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI lation of serine, threonine, and tyrosine residues (8). Of the five 53233 tyrosine residues in the human PECAM-1 cytoplasmic domain, Received for publication March 4, 1999. Accepted for publication April 29, 1999. two are surrounded by sequences that conform to an ITIM, for The costs of publication of this article were defrayed in part by the payment of page which the consensus sequence is (I/V/L)xpYxx(L/V) (6). The charges. This article must therefore be hereby marked advertisement in accordance PECAM-1 cytoplasmic domain contains two ITIMs, one around with 18 U.S.C. Section 1734 solely to indicate this fact. tyrosine 663 and the other around tyrosine 686 (reviewed in Ref. 1 This work was supported by National Institutes of Health Grant P01 HL44612 and the Blood Center Research Foundation. 12). The long distance between ITIMs in the PECAM-1 cytoplas- 2 Address correspondence and reprint requests to Dr. Debra K. Newton-Nash, Blood mic domain (17 residues) contrasts with the relatively short dis- Research Institute, P.O. Box 2178, Milwaukee, WI 53201-2178. E-mail address: tance (6–8 residues) that separates ITAM sequences, and is con- [email protected] sistent with the ability of the PECAM-1 cytoplasmic domain to 3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation support interactions with the relatively widely spaced tandem Src motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; AP, alkaline phospha- tase; BCR, B cell receptor for Ag; CB, calcium buffer; GAM, goat anti-mouse IgG; homology 2 (SH2) domains of PTPs, as opposed to the relatively KIR, killer inhibitory receptor; PECAM-1, platelet-endothelial cell adhesion mole- closely spaced tandem SH2 domains of PTKs (reviewed in Ref. cule-1; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SHP, Src homology 2 domain-bearing PTP; SH2, Src homology 2; PerCP, peridinin chlorophyl 12). The tyrosine residues at positions 663 and 686 of the PE- protein. CAM-1 cytoplasmic domain become phosphorylated in response Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 683 to numerous stimuli, including platelet aggregation (13), cross- CA), murine monoclonal anti-SHP-2 (Santa Cruz Biotechnology, Santa linking of PECAM-1-specific mAbs (14, 15), mechanical stimu- Cruz, CA), rabbit polyclonal anti-human PECAM-1 (SEW32-34; prepared lation of endothelial cells (16), engagement of the FceRI on ba- in our laboratory), and HRP-conjugated GAM (Jackson ImmunoResearch, West Grove, PA) or alkaline phosphatase (AP)-conjugated goat anti-rabbit sophils (17), aggregation of the Ag receptor on T cells (18), and IgG (Zymed Laboratories) Abs were used for Western blot analysis. treatment with the PTP inhibitor pervanadate (19). When phos- phorylated, these tyrosine residues support the binding of SHP-2 Induction and assessment of PECAM-1 tyrosine phosphorylation (13, 18, 20–23) and possibly SHP-1 (18, 22, 23). Thus, both struc- and SHP-2 binding tural and biochemical properties of the PECAM-1 cytoplasmic do- Jurkat cells in log phase growth were pelleted by centrifugation at 300 3 main suggest that it may belong to the Ig-like, ITIM-containing g for 10 min at room temperature and resuspended at a final concentration 7 family of inhibitory receptors. of 10 cells/ml in cold RPMI (complete)/10% FBS. Cell suspensions (1 ml) were incubated with no Ab, with the human CD3e-specific Ab, UCTH1 (1 The pattern of PECAM-1 expression on T lymphocytes also mg/ml), with a human PECAM-1-specific Ab, or with both anti-CD3e and suggests a potential inhibitory function for this molecule. Specif- anti-PECAM-1 for 30 min on ice. To remove unbound Ab, cell suspensions ically, the majority of CD41 (24–27) and half of CD81 T lym- were washed twice with 5 ml of cold 0.145 M sodium chloride containing phocytes (24, 25) lose PECAM-1 expression as they make the 10 mM HEPES, 2.8 mM KCl, 2 mM MgCl2,10mMD-glucose, and 0.1% BSA (calcium buffer (CB)), and 10 mM CaCl2. Cells were resuspended in transition from naive to memory cells. This pattern of expression the same buffer at a final concentration of 2 3 107 cells/ml.