Mechanism of cyclic ADP-ribose hydrolysis in mammalian cells.

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1.We isolated a cDNA for CD38, which has been reported to be a human leukocyte atigen, from a human insulinoma and expressed the cDNA in COS-7 cells. When we incubated the plasma membrane with fraction NAD^+ or cyclic ADP-ribose (cADPR) and analyzed the reaction products by HPLC,the formation and hydrolysis of cADPR (ADP-ribosyl cyclase and cADPR hydrolase activities) were detected. Moreover, we found that ATP (2-10 mM), generated in the glucose metabolism in beta-cells, inhibited the cADPR-hydrolyzing activity, resulting in the accumulation of cADPR.2.We isolated rat CD38 cDNA from islets of Langerhans and determined its primary structure. Rat CD38 is composed of 303 amino acids and shares 89% homology with human CD38. The membrane fraction of the COS-7 cells into which rat CD38 expression vector had been introduced exhibited both ADP-ribosyl cyclase and cADPR hydrolase activities. Rat CD38 mRNA is expressed in various tissues including pancreatic islets but not in RINm5F cells.3.We p … More roduced transgenic mice overexpressing human CD38 in pancreatic beta cells. The enzymatic activity of CD38 in transgenic islets was greatly increased, and ATP efficiently inhibited the cADPR hydrolase activity. Glucose- and ketoisocaproate (which, like glucose, generates ATP during the metabolism) -induced but not tolbutamide- nor KCI-induced insulin secretion from transgenic islets were 1.7-2.3-fold higher than that of control. In glucose-tolerance tests, the transgenic serum insulin level was higher than that of control. Thus, the cADPR-CD38 signaling system has a regulatory role in insulin secretion by glucose in beta-cells, suggesting that not only Ca^<2+> from extracellular sources (Ca^<2+> influx through voltage-dependent Ca^<2+> channels evoked by glucose-induced cell membrane depolarization) but also Ca^<2+> release from intracellular stores (cADPR-induced Ca^<2+> release from the endoplasmic reticulum) play important roles in insulin secretion.4.We introduced site-directed mutations to CD38 and found that C119K- and/or C202E-CD38 exhibited only ADP-ribosyl cyclase activity. Furthermore, Aplysia ADP-ribosyl cyclase into which we introduced the mutations K95C and E176C,which correspond to residues 119 and 201 of human CD38, exhibited not only ADP-ribosyl cyclase activity but also cADPR hydrolase.5.We isolated human CD38 gene and determined its structure. The CD38 is consists of 8 exons that extending -100 kbp on the human genome, and is mapped to human chromosome 4p 15 by fluorescent in situ hybridization. Less