Cells possess various mechanisms for transducing information from the external environment to intracellular responses. Binding of ligands to surface receptors can lead to production of second messengers inside the cell and the first such messenger identified is cAMP. Receptor activation can also elevate Ca2+ due to mobilization of internal Ca2+ stores. The discovery of inositol trisphosphate (IP3) as a messenger for this process has centralized Ca2+ mobilization in signaling. Our research establishes that, in addition to IP3, the internal Ca2+ stores can be mobilized by two new messengers via totally independent pathways. Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) were discovered and their structures determined in my lab. CADPR is a new cyclic nucleotide derived from NAD, but unlike cAMP, its main signaling function is through direct modulation of Ca2+-induced CA2+ release (CICR), a major mechanism of Ca2+ mobilization beside the IP3-pathway. A variety of cells from plant to mammalian species are shown to be responsive to cADPR, indicating the generality of the signaling pathway. Similar to cADPR, NAADP, a metabolite of NADP, can also mobilize Ca2+ stores, but the release mechanism and the stores it acts on are distinct from cADPR. These two Ca2+ agonists are, nevertheless, intimately related, since the same metabolic enzymes can, under appropriate conditions, synthesize either one, suggesting a unified mechanism may regulate both pathways. Elucidation of the signaling pathways mediated by cADPR and NAADP is likely to have an important impact on our understanding of signal transduction mechanisms.
Aim I is to develop and use a RIA for measuring cellular content of cADPR.
Aim 2 is to purify and characterize a cGMP-dependent ADP-ribosyl cyclase.
Aim 3 is to characterize the cGMP-dependent activation of the cyclase. These three Aims focus on identifying the stimuli and the activation mechanism of the cADPR-pathway. Results will provide the necessary evidence for establishing cADPR as a second messenger.
Aim 4 is to determine the role of cADPR in the propagation of Ca2+ waves. It is generally believed that the CICR mechanism is crucial in propagating Ca2+ waves. Our finding that cADPR can modulate the Ca2+ sensitivity of CICR makes it relevant to investigate its role in the process.
Aim 5 is to characterize the Ca2+ signaling mechanism mediated by NAADP. We will probe the structure-function relationship of NAADP, explore the regulatory mechanisms of its synthesis and investigate its possible role in mediating Ca2+ oscillations in cells.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD017484-16
Application #
2673476
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-04-01
Project End
2001-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
16
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Physiology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Graeff, Richard; Lee, Hon Cheung (2002) A novel cycling assay for nicotinic acid-adenine dinucleotide phosphate with nanomolar sensitivity. Biochem J 367:163-8
Graeff, Richard; Lee, Hon Cheung (2002) A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity. Biochem J 361:379-84
Lee, H C (2001) Physiological functions of cyclic ADP-ribose and NAADP as calcium messengers. Annu Rev Pharmacol Toxicol 41:317-45
Graeff, R; Munshi, C; Aarhus, R et al. (2001) A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities. J Biol Chem 276:12169-73
Lee, H C; Aarhus, R (2000) Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose. J Cell Sci 113 Pt 24:4413-20
Khoo, K M; Han, M K; Park, J B et al. (2000) Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in hepatocyte nucleus. J Biol Chem 275:24807-17
Lee, H C (2000) NAADP: An emerging calcium signaling molecule. J Membr Biol 173:8-Jan
Munshi, C; Aarhus, R; Graeff, R et al. (2000) Identification of the enzymatic active site of CD38 by site-directed mutagenesis. J Biol Chem 275:21566-71
Lee, H C (1999) A unified mechanism of enzymatic synthesis of two calcium messengers: cyclic ADP-ribose and NAADP. Biol Chem 380:785-93
Wong, L; Aarhus, R; Lee, H C et al. (1999) Cyclic 3-deaza-adenosine diphosphoribose: a potent and stable analog of cyclic ADP-ribose. Biochim Biophys Acta 1472:555-64

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