REGULATION OF CYCLIC ADP RIBOSE SIGNALING SYSTEM

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The overall objectives of the proposed research are to gain insight into the mechanism by which cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and to elucidate the mechanisms by which cADPR levels are regulated in mammalian cells. cADPR is a naturally occurring metabolite of beta-nicotinamide adenine dinucleotide )beta-NAD) that has been found to mobilize calcium from intracellular stores in a variety of cell types. The specific aims are: (1) to identify and characterize cADPR binding proteins in mammalian systems, (2) to examine the regulation of endogenous levels of cADPR in mammalian systems, and (3) to characterize the mechanisms involved in the regulation of cADPR metabolic enzymes. cADPR binding proteins will be studied by conventional binding as well as by photoaffinity labeling techniques. 3-deaza-cADPR, a potent, non- hydrolyzable analog of cADPR, has recently been developed, and will be an important probe in the examination of proteins to which cADPR interacts. The regulation of endogenous levels of cADPR will be examined in several mammalian cell lines using a radioimmunoassay for cADPR developed in this laboratory. Particular attention will be focused on agents that are known to alter intracellular calcium concentrations, either through calcium influx or by mobilization of interal stores of calcium. The mechanism(s) by which cADPR levels are regulated will be examined by characterizing the enzymes responsible for the synthesis (ADP-ribosyl cyclase) and degradation (cADPR hydrolase) from systems shown to have alterations in cADPR levels. The regulation of the intracellular concentration of calcium is a critical process in all cells. Calcium plays an important role in a number of processes, including neurotransmitter release, cytoskeleton changes, muscle contraction, gene expression, etc. Accumulating evidence suggests that cADPR may be the endogenous regulator of calcium induced- calcium release (CICR) through ryanodine receptor type calcium release channels. cADPR appears to increase the sensitivity of CICR to calcium ions in a manner very similar to caffeine. Caffeine has many physiological actions and is one of the most socially abused drugs in the world. The proposed studies should provide useful information on the role cADPR plays in the regulation of calcium homeostasis and may also provide valuable insight into some of caffeine's biological actions.