Chidambaram N, Chang C F
Faculty of Medicine, The National University of Singapore, Singapore, 119260.
Arch Biochem Biophys. 1999 Mar 15;363(2):267-72. doi: 10.1006/abbi.1999.1103.
CD38 is a 46-kDa type II transmembrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose (cADPR) from NAD+. cADPR is a second messenger known to regulate intracellular Ca2+-induced Ca2+-release (CICR). A recent study has revealed that CD38 in Namalwa B cells undergoes internalization upon exposure to external NAD+. In this study, recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells and the possibility of the protein to undergo internalization upon exposure to a substrate analog NADP+ was examined. It was found that such treatment of CHO cells resulted in a decrease of ADP-ribosyl cyclase activity, as well as immunofluorescence of CD38 on the cell surface. The same treatment of CHO cells also resulted in intracellular clustering of CD38 molecules as revealed by confocal microscopic analysis. The internalized CD38 was purified using a streptavidin/biotin-based method and was found to exhibit both ADP-ribosyl cyclase and cADPR hydrolase activities. On immunoblot, the internalized CD38 appeared as a monomer of 46 kDa under reducing condition of SDS-PAGE. Our data demonstrate that NADP+ can efficiently induce internalization of CD38, a process that may be important in the production of cADPR intracellularly to regulate CICR.
CD38是一种46 kDa的II型跨膜糖蛋白，可催化从NAD +合成环ADP核糖（cADPR）。cADPR是一种已知可调节细胞内钙诱导的钙释放（CICR）的第二信使。最近的一项研究表明，Namalwa B细胞中的CD38在暴露于外部NAD +后会发生内化。在本研究中，重组大鼠CD38在中国仓鼠卵巢（CHO）细胞中表达，并研究了该蛋白在暴露于底物类似物NADP +后发生内化的可能性。发现对CHO细胞进行这种处理会导致ADP核糖基环化酶活性降低，以及细胞表面CD38的免疫荧光降低。共聚焦显微镜分析显示，对CHO细胞进行相同处理也会导致CD38分子在细胞内聚集。使用基于链霉亲和素/生物素的方法纯化内化的CD38，发现其同时具有ADP核糖基环化酶和cADPR水解酶活性。在免疫印迹上，在SDS-PAGE的还原条件下，内化的CD38表现为46 kDa的单体。我们的数据表明，NADP +可以有效地诱导CD38的内化，这一过程可能在细胞内产生cADPR以调节CICR中起重要作用。
