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

视黄酸（RA）诱导HL-60细胞产生的外源型NAD酶活性是由CD 38引起的，CD 38的氨基酸序列与拟南芥ADP-核糖基环化酶同源。CD 38不仅催化NAD^+的水解，而且催化环ADP-核糖（cADPR）的形成和水解，后者是细胞内Ca ^2+库释放Ca^2+的新型介质或调节剂。在本研究中，我们开发了一种放射免疫分析法（RIA）测定cADPR，并获得了以下结果。1.用抗CD 38单克隆抗体（IgG 1-subcless）刺激RA分化的HL-60细胞，诱导细胞蛋白质（包括c-cbl原癌基因产物p120）快速酪氨酸磷酸化<c-cbl>。抗CD 38单克隆抗体显著增强了对甲酰-Met-Leu-Phe的超氧化物形成反应。Fc γ-II受体似乎参与通过抗CD 38 mAb诱导的酪氨酸磷酸化介导的信号转导途径。2.CD38在大鼠脑内除淋巴细胞外还大量存在。在原代培养的大鼠胶质细胞和神经元中，在星形胶质细胞表面观察到最丰富的CD 38。激光共聚焦显微镜分析显示，免疫反应性CD 38和酶活性定位于星形胶质细胞的细胞表面的点状形状。细胞表面的CD 38聚集和内化后，添加的酶底物培养的星形胶质细胞。3.在CD 38基因的第一内含子中发现了一个由两个直接重复的TGACCT样六聚体基序和一个5-核苷酸间隔区组成的RA反应元件（RARE）。该RARE与RA受体和维甲酸X受体组成的异源二聚体相互作用。因此，RA诱导的人CD 38基因的表达被证明是通过位于第一内含子中的RARE介导的。4.海胆卵受精后，细胞内cADPR表达增加。

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.