CD38 Signaling in B Lymphocytes Is Controlled by Its Ectodomain but Occurs Independently of Enzymatically Generated ADP-Ribose or Cyclic ADP-Ribose This information is current as of September 27, 2021. Frances E. Lund, Hélène M. Muller-Steffner, Naixuan Yu, C. David Stout, Francis Schuber and Maureen C. Howard J Immunol 1999; 162:2693-2702; ; http://www.jimmunol.org/content/162/5/2693 Downloaded from References This article cites 39 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/162/5/2693.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 27, 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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD38 Signaling in B Lymphocytes Is Controlled by Its Ectodomain but Occurs Independently of Enzymatically Generated ADP-Ribose or Cyclic ADP-Ribose1 Frances E. Lund,2*He´le`ne M. Muller-Steffner,† Naixuan Yu,* C. David Stout,‡ Francis Schuber,† and Maureen C. Howard3* CD38 is a type II transmembrane glycoprotein that is expressed by many cell types including lymphocytes. Signaling through CD38 on B lymphocytes can mediate B cell activation, proliferation, and cytokine secretion. Additionally, coligation of CD38 and the B cell Ag receptor can greatly augment B cell Ag receptor responses. Interestingly, the extracellular domain of CD38 catalyzes the conversion of NAD1 into nicotinamide, ADP-ribose (ADPR), and cyclic ADPR (cADPR). cADPR can induce intracellular calcium release in an inositol trisphosphate-independent manner and has been hypothesized to regulate CD38-mediated signaling. Downloaded from We demonstrate that replacement of the cytoplasmic tail and the transmembrane domains of CD38 did not impair CD38 signaling, coreceptor activity, or enzyme activity. In contrast, independent point mutations in the extracellular domain of CD38 dramatically impaired signal transduction. However, no correlation could be found between CD38-mediated signaling and the capacity of CD38 to catalyze an enzyme reaction and produce cADPR, ADPR, and/or nicotinamide. Instead, we propose that CD38 signaling and coreceptor activity in vitro are regulated by conformational changes induced in the extracellular domain upon ligand/substrate binding, rather than on actual turnover or generation of products. The Journal of Immunology, 1999, 162: 2693–2702. http://www.jimmunol.org/ D38 is an evolutionarily conserved type II transmem- induce the release of intracellular Ca21 in mammalian cells such as brane glycoprotein that is expressed extensively on many pancreatic acinar and b-cells, neuronal cells, and heart and pitu- C cell types including lymphocytes (reviewed in Ref. 1). itary cells (reviewed in Ref. 12). CD38 is composed of a short cytoplasmic tail and an unremarkable Like the Aplysia cyclase enzyme, the extracellular domain of transmembrane domain, neither of which is homologous to any CD38 possesses ADP-ribosyl cyclase activity (13, 14). However, known protein (2–4). In contrast, the extracellular domain of unlike the Aplysia cyclase enzyme, CD38 is also able to mediate CD38 shares structural homology with a family of proteins that the catalysis of both NAD1 and cADPR into ADP-ribose (ADPR) includes the cytosolic ADP-ribosyl cyclase enzyme isolated from through its NAD1 and cADPR hydrolase activities (13). In fact, by guest on September 27, 2021 Aplysia californica (5). The ADP-ribosyl cyclase enzyme mediates CD38 is a more efficient NAD1 glycohydrolase enzyme than the catalysis of NAD1 into cyclic ADP-ribose (cADPR)4 (6, 7). In ADP-ribosyl cyclase enzyme, as .97% of the total product pro- sea urchin egg homogenates, cADPR has been shown to induce the duced by CD38 is ADPR (13). Nevertheless, it has been hypoth- mobilization of calcium from intracellular stores by an inositol esized that the cADPR produced via the cyclase activity of CD38 trisphosphate-independent, but caffeine- and ryanodine-sensitive, may be an important regulator of calcium signaling (15). pathway (8–10), suggesting that cADPR may induce calcium re- In the hemopoietic system, CD38 has been shown to regulate lease from the ryanodine receptor channel complex in the endo- lymphocyte activation and effector functions (1, 16). We and oth- plasmic reticulum (10, 11). Cyclic ADPR has also been shown to ers have shown that engagement of CD38 using anti-CD38 Abs can induce a proliferative response in B and T lymphocytes (17– 19), and that coligation of CD38 and the Ag receptor on B cells *DNAX Research Institute, Palo Alto, CA 94304; †Laboratoire de Chimie Bioor- (BCR) can augment BCR-mediated responses (20). Additionally, ganique, Unite´ Mixte de Recherche 7514 Centre National de la Recherche anti-CD38 stimulation has been shown to be a potent inducer of Scientifique-Universite´ Louis Pasteur, Faculte´ de Pharmacie, Illkirch, France; and cytokine production in T and B cells (20–22). Finally, CD38 ‡Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037 clearly plays an important role in the immune system in vivo, as Received for publication September 1, 1998. Accepted for publication November animals deficient in CD38 have marked deficiencies in their ability 23, 1998. to mount humoral immune responses (23). The costs of publication of this article were defrayed in part by the payment of page To examine the molecular requirements for anti-CD38-mediated charges. This article must therefore be hereby marked advertisement in accordance activation and costimulation in murine B cells, we recently estab- with 18 U.S.C. Section 1734 solely to indicate this fact. lished an in vitro CD38 signaling model system using the A20 1 DNAX Research Institute is fully funded by Schering Plough Corp. F.S. and H.M.-S. were supported by the Centre National de la Recherche Scientifique Pro- murine B cell lymphoma (20). A20 cells are CD38 negative but gramme Physique et Chimie du Vivant (Grant 1997–4053). C.D.S. was supported by express a functional BCR complex, which, when cross-linked, will National Science Foundation Grant MCB9513421. initiate the synthesis of IL-2 (24–26). When CD38 was stably 2 Address correspondence and reprint requests to Dr. Frances E. Lund, The Trudeau expressed in A20 cells, the resultant CD381 clones inducibly pro- Institute,P.O.Box59,SaranacLake,NY12983.E-mailaddress:flund@trudeauinstitute. org duced IL-2 after CD38 was cross-linked with a polyclonal Ab (polyanti-CD38) (20). Furthermore, coligation of CD38 and the 3 Current address: Anergen, Inc., 301 Penobscot Dr., Redwood City, CA 94063. BCR induced a potent synergistic IL-2 response in these cells, 4 Abbreviations used in this paper: cADPR, cyclic adenosine diphosphate ribose; ADPR, adenosine diphosphate ribose; HA, hyaluronate; BCR, B cell Ag receptor; suggesting that CD38 might function as a BCR coreceptor. Inter- ARAP, Ag receptor-associated protein. estingly, CD38-mediated signaling and coreceptor activity in this Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 2694 CD38 SIGNALING OCCURS INDEPENDENTLY OF cADPR GENERATION system was found to be completely dependent upon expression of clones. For example, 10 independently selected WILD-CD38 clones were a functional BCR complex (20). Surprisingly, a large truncation expanded in log phase for 1 wk. Equal numbers of these 10 clones were of the cytoplasmic tail of CD38 did not noticeably depress then mixed together, and 30 identical aliquots of the mixed WILD-CD38 cells were frozen (280°C) in 10% DMSO/90% FBS. CD38-mediated signal transduction, suggesting that the cytoplas- mic tail of CD38 might be unnecessary for anti-CD38-mediated FACS staining of mixed mutant clones signaling (20). An aliquot of all the mutant mixed clones was thawed, grown in tissue In this report we have attempted to reconcile the signaling ca- culture, and then analyzed for CD38 expression on a FACS Calibur (Bec- pacity of murine CD38 with its enzymatic properties. We have ton Dickinson, Mountain View, CA) after staining. Cells were first blocked performed site-directed mutagenesis to produce mutated forms of with 1 mg of purified Fc block (24G2, PharMingen, San Diego, CA) and CD38 and have examined the consequences of these mutations on then stained on ice with either protein A-purified rabbit anti-mouse CD38 or purified normal rabbit IgGs. The cells were washed, stained with rat CD38-mediated signal transduction and enzyme activity in our anti-rabbit FITC (Zymed, South San Francisco, CA), and analyzed. A20 B lymphoma model system. The results are presented and discussed in the context of the predicted structure for CD38. Preparation of cross-linked Ab conjugates Cross-linked Abs were prepared as previously described (20). Briefly, 2 mg Materials and Methods of the purified rabbit anti-CD38 Ab and 8 mg of purified normal rabbit Cell lines, culture conditions, and Abs IgGs were mixed per 1.0 ml of recombinant protein A beads (Zymed). To prepare the substimulatory anti-CD38 cross-linked beads, 200 mg of anti- The A20/2J cell line was obtained from American Type Culture Collection CD38 Ab plus 10 mg of normal rabbit IgGs were incubated per 1.0 ml of (Manassas, VA), and the cytokine-dependent cell line HT-2 was a gift from protein A beads. To prepare the substimulatory anti-Ig beads, 4 mgof Dr.