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

视黄酸（RA）诱导HL-60细胞的外链NADase活性是由于CD38，其氨基酸序列与葡聚糖adp -核糖基环化酶同源。CD38不仅可以催化NAD^+的水解，还可以催化环adp核糖（cADPR）的形成和水解，cADPR是细胞内Ca^<2+>储存释放Ca^<2+>的新型介质或调节剂。在本研究中，我们开发了一种用于cADPR测量的放射免疫分析法（RIA），并获得了以下发现。1.用抗cd38单克隆抗体（igg1 -亚隐性）刺激ra分化的HL-60细胞诱导细胞蛋白快速酪氨酸磷酸化，包括c-cbl原癌基因产物p120^<c-cbl>。抗cd38单抗显著增强了甲酰基met -亮氨酸反应的超氧化物形成。Fcgamma-II受体似乎参与了通过抗cd38单抗诱导的酪氨酸磷酸化介导的信号转导途径。2.除淋巴细胞外，CD38在大鼠脑中也大量存在。在大鼠神经胶质细胞和神经元原代培养中，星形胶质细胞表面的CD38含量最高。共聚焦激光显微镜分析显示，免疫反应性CD38和酶活性定位在星形胶质细胞表面呈点状。在培养的星形胶质细胞中加入酶底物后，细胞表面CD38聚集并内化。这种内化伴随着细胞内cadpr的增加。在CD38基因的第一个内含子中发现了一个RA应答元件（RARE），该元件由两个定向的tgacct样六聚体基序和一个5核苷酸间隔组成。该罕见菌与RA受体和类视黄醇X受体组成的异二聚体相互作用。因此，ra诱导的人CD38基因的表达是通过位于第一个内含子的RARE介导的。4.在海胆卵受精后，观察到细胞中cADPR的增加。

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.