CYCLIC ADP/RIBOSE-DEPENDENT CALCIUM RELEASE PATHWAY

细胞内钙离子的动员是一个重要的信号 在细胞中的机制，并由两个主要机制，肌醇 三磷酸(IP3)途径与钙诱导的钙释放(CICR) 进程。我们在海胆卵中发现了一种钙动员系统 它完全不依赖于IP3途径。此系统已激活 NAD+的代谢物称为环状ADP-核糖(CADPR)。此外 对于海胆卵，几种哺乳动物细胞类型已经被证明是 对cadpr的答复，表明该机制的一般性。 越来越多的证据表明，cADPR可能是一种内源性调节因子。 细胞内的CICR。最近的三个进展表明，cADPR的机制是 受到严格监管。首先，cADPR的合成途径已被证明是 受到cGMP依赖机制的刺激。第二，淋巴细胞蛋白 CD38已被证明是一种双功能酶，可以催化 CADPR和第三种可溶性蛋白的合成和水解性 已经证明，赋予cADPR敏感性所需的因素 对微粒体而言。 拟议的研究将研究cadpr的调节机制。 路径。(L)脑和海胆卵中的可溶性蛋白因子 使cADPR对卵子微粒体敏感的基因将被提纯并 特色化的。我们将调查可溶物质的可能性 因子作为cADPR受体的增敏剂对cADPR和/或 Ca~(2+)向Ca~(2+)释放机制。(2)cGMP依赖的调节 ADP-核糖环化酶和CD38的作用机制将被阐明。我们将使用 直接光亲和标记法和蛋白质测序鉴定 介导cGMP刺激血管内皮细胞生长的成分 CADPR的合成途径。(3)ADP-核糖基的酶机制 将对Cyclase和CD38进行研究。我们将研究它的形成 ADP-核糖化酶中间体及其催化部位的鉴定 用光亲和标记法测定酶的活性。(4)最后，我们会延长 从卵子微体到完整的卵子和哺乳动物大脑的结果 微粒体。

Mobilization of Ca+2 from intracellular stores is an important signaling mechanism in cells and is mediated by two major mechanisms, the inositol trisphosphate (IP3) pathway and the Ca+2-induced Ca+2 release (CICR) process. We have identified a Ca+2 mobilization system in sea urchin eggs which is totally independent of the IP3 pathway. This system is activated by a metabolite of NAD+ we called cyclic ADP-ribose (cADPR). In addition to sea urchin eggs, several mammalian cell types have been shown to be responsive to cADPR, indicating the generality of the mechanism. Increasing evidence suggests that cADPR may be an endogenous regulator of CICR in cells. Three recent advances indicate that the cADPR mechanism is tightly regulated. First, the synthetic pathway of cADPR has been shown to be stimulated by a cGMP-dependent mechanism. Second, a lymphocyte protein called CD38 has been shown to be a bifunctional enzyme that can catalyze both the synthesis and hydrolysis of cADPR and third, a soluble protein factor has been shown to be required for conferring the cADPR-sensitivity to microsomes. The proposed research will examine the regulation mechanisms of the cADPR pathway. (l) The soluble protein factors from brain and sea urchin eggs that confer the cADPR sensitivity to egg microsomes will be purified and characterized. We will investigate the possibilities that the soluble factor functions as a sensitizer of the cADPR-receptor to cADPR and/or the Ca+2 release mechanism to Ca+2. (2) The cGMP-dependent regulation mechanism of ADP-ribosyl cyclase and CD38 will be elucidated. We will use direct photoaffinity labeling and protein sequencing to identify the components involved in mediating the stimulatory effect of cGMP on the synthetic pathway of cADPR. (3) The enzymatic mechanisms of ADP-ribosyl cyclase and CD38 will be investigated. We will examine the formation of the ADP-ribosylated enzyme intermediates and identify the catalytic sites of the enzymes by photoaffinity labeling. (4) Finally, we will extend the results obtained from egg microsomes to intact eggs and to mammalian brain microsomes.