CD38 and ADP-ribosyl Cyclase Catalyze the Synthesis of a Dimeric ADP-ribose That Potentiates the Calcium-mobilizing Activity of Cyclic ADP-ribose (original) (raw)
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Cell Biochemistry and Biophysics, 1998
CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD § to nicotinamide and cyclic ADPribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca 2+mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca 2+ response to KCl-induced depolarization, a process that can be qualified as a Ca2+-induced Ca 2+ release (CICR) mechanism. On the other hand, in the CD38 § human Namalwa B lymphoid cells, NAD § (and thiol compounds *Author to whom all correspondence and reprint requests should be addressed.
FEBS Letters, 1998
Cyclic ADP-ribose (cADPR) is a natural metabolite of L L-NAD + with a potent Ca 2+ -mobilizing activity in different cell types, including T-lymphocytes. We investigated (i) whether stimulation of T-lymphocytes with different agonists affects the intracellular concentration of cADPR, and (ii) whether the lymphocyte antigen CD38, through its ectocellular ADP-ribosyl cyclase and cADPR-hydrolase enzymatic activities, can account for the regulation of the intracellular levels of cADPR and the Ca 2+ -mobilizing effects of this nucleotide in Jurkat and HPB.ALL T-lymphocytes. The anti-CD3 antibody OKT3, the sphingolipid sphingosine and lysophosphatidic acid induced an increase in intracellular cADPR with concomitant increases in the intracellular Ca 2+ concentration ([Ca 2+ ] i ). In contrast, activation of an ectocellular ADP-ribosyl cyclase by preincubation of cells with L L-NAD + led to a dose-dependent increase in cADPR, but no changes in [Ca 2+ ] i were observed. However, extensive washing of the cells following preincubation with NAD + demonstrated that the increases in cADPR were not intracellular but due to cell surface-associated nucleotide. Accordingly, measurements of ADP-ribosyl cyclase activity in intact T-cells showed ectocellular synthesis of cADPR, but no evidence was obtained for a shift of this activity into the cells which could account for intracellular accumulation of cADPR. Taken together, the results indicate no direct involvement of the ADP-ribosyl cyclase activity of CD38 on the regulation of the cADPR-mediated intracellular Ca 2+ -signalling in T-lymphocytes.
Essential cysteine residues for cyclic ADP-ribose synthesis and hydrolysis by CD38
Journal of Biological Chemistry
We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as a second messenger for glucoseinduced insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373) and that human leukocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Takasawa, S., Tohgo, A, Noguchi, N., Koguma, T., Nata, K, Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J.
Journal of Biological Chemistry
Ca2+ mobilization in the endoplasmic reticulum in the process of insulin secretion (Takasawa, s., Nata, K., Yonekura, &, and Okamoto, H. (1993) Science 259,370473). In the present study, we isolated a cDNA for CD38, which has been reported to be a human leukocyte antigen, from a human insulinoma and expressed the cDNAin COS-7 cells. CD38 expression was observed in the plasma membrane and the microsome fractions of the COS-7 cells. When we incubated the plasma membrane fraction with NAD+ and analyzed the reaction products by high pressure liquid chromatography, the formation of cADPR was observed in addition to the ADP-ribose (ADPR) formation. When the plasma membrane fraction was incubated with cADPR, cADPR was converted to ADPR stoichiometrically. These results suggest that CD38 has both cADPR-forming and -hydrolyzing activities. Moreover, we found that ATP (2-10 m~) , generated in the glucose metabolism in p-cells, inhibited the cADPR-hydrolyzing activity, resulting in the increased formation of cADPR These findings indicate a role for CD38 in the synthesis and hydrolysis of cADPR in the process of insulin secretion in pancreatic p-cells.
Journal of Biological Chemistry, 2004
Native human HL-60 cells do not express CD38, a multifunctional ectoenzyme, which generates cyclic ADP-ribose (cADPR), a potent calcium mobilizer. However, when HL-60 cells are induced to differentiate to granulocytes by treatment with retinoic acid (RA), they express CD38 and accumulate cADPR. Both processes play a causal role in RA-induced differentiation. Other granulocyte differentiation-inducers, including dimethyl sulfoxide (Me 2 SO), fail to induce CD38 expression. We investigated whether treatment of HL-60 cells with Me 2 SO involves any changes in the cADPR/intracellular calcium ([Ca 2؉ ] i) signaling system and, specifically, whether Me 2 SO affects those nucleoside transporters (NT) (both equilibrative (ENT) and concentrative (CNT)) that mediate influx of extracellular cADPR. Semiquantitative polymerase chain reaction analysis of transcripts, binding of [ 3 H]nitrobenzylthioinosine (NBMPR) to intact cells, and influx experiments of extracellular cADPR (with selective inhibitors of NT as NBMPR or in specific conditions) were performed in native and Me 2 SO-differentiated HL-60 cells. The native cells showed uptake of cADPR across ENT2, whereas influx of cADPR into the Me 2 SOdifferentiated cells occurred mostly by concentrative processes mediated by CNT3 and by an NBMPR-inhibitable concentrative NT designated cs-csg. Me 2 SO-differentiated, but not native HL-60 cells, accumulated cADPR and showed increased [Ca 2؉ ] i levels when grown in a transwell co-culture setting over CD38-transfected 3T3 fibroblasts where nanomolar cADPR concentrations are present in the medium. NBMPR inhibited both responses of Me 2 SO-induced cells. Thus, concentrative influx of extracellular cADPR across CNT3 and cs-csg NT could substitute in the absence of CD38 in eliciting cADPR-dependent [Ca 2؉ ] i increases in granulocyte-differentiated HL-60 cells, as well as in other CD38 ؊ cells. ADP-ribosyl cyclases play a major role in regulating intracellular calcium levels ([Ca 2ϩ ] i) 1 by converting NAD ϩ to nicotina
Journal of Biological Chemistry, 1997
CD38 catalyzes not only the formation of cyclic ADPribose (cADPR) from NAD ؉ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD ؉ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999
CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38(+) cells with either NAD+ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that t...
The FASEB Journal
CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38 ؉ cells with either NAD ؉ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR-generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD ؉ , or GSH, or N-acetylcysteine. This effect of CD38-internalizing ligands on intracellular calcium levels was found to involve a two-step mechanism: 1) influx of cytosolic NAD ؉ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8-N 3 -NAD ؉ , and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD ؉ to cADPR, followed by outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigarginsensitive stores. This unknown intracellular trafficking of NAD ؉ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38 ؉ cells.-Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., Passalacqua, M., De Flora, A. Ligand-induced internalization of CD38 results in intracellular Ca 2؉ mobilization: role of NAD ؉ transport across cell membranes. FASEB J. 13, 273-283 (1999)