Assay of Cyclic ADP-ribose and Analysis of Its Target Molecules

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Ecto-form NADase activity induced by retinoic acid (RA) in HL-60 cells is due to CD38, which has an amino acid sequence homologous to Aplysia ADP-ribosyl cyclase. CD38 catalyzes not only the hydrolysis of NAD^+, but also the formation and hydrolysis of cyclic ADP-ribose (cADPR), that is a novel mediator or modulator of Ca^<2+> release from intracellular Ca^<2+> stores. In the present study, we developed a radioimmunoassay (RIA) for cADPR measurement and obtained the following findings. 1.Stimulation of RA-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (IgG1-subcless) induced rapid tyrosine phosphorylation of cellular proteins including the c-cbl proto-oncogene product, p120^<c-cbl>. Superoxide formation in response to formyl-Met-Leu-Phe was markedly enhanced by the anti-CD38 mAbs. Fcgamma-II receptors appeared to be involved in the signal transduction pathway mediated through the anti-CD38 mAb-induced tyrosine phosphorylation. 2.CD38 was abundantly present in rat brain in addition to lymphocytes. In primary culture of rat glial cells and neurons, CD38 was most abundantly observed in astrocyte cell surface. Confocal laser microscopic analysis revealed that immunoreactive CD38 and the enzyme activity were localized on the cell surface of astrocytes in a dot-like shape. The cell-surface CD38 was clustered and internalized upon the addition of the enzyme substrates to the cultured astrocytes. This internalization accompanied with the increase of intracellular cADPR.3.An RA response element (RARE) consisting of two directrepeated TGACCT-like hexamer motifs with a 5-nucleotide spacer was found to be located in the first intron of CD38 gene. This RARE interacted with heterodimer composed of RA receptor and retinoid X receptor. Thus, the RA-induced expression of human CD38 gene was demonstrated to be mediated through the RARE located in the first intron. 4.An increase in cellular cADPR was observed upon the fertilization of see urchin eggs.