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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
1R01HD032040-01
Application #
2204936
Study Section
Biochemistry Study Section (BIO)
Project Start
1994-07-15
Project End
1998-04-30
Budget Start
1994-07-15
Budget End
1995-04-30
Support Year
1
Fiscal Year
1994
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
Lee, H C (1998) Calcium signaling by cyclic ADP-ribose and NAADP. A decade of exploration. Cell Biochem Biophys 28:1-17
Graeff, R M; Franco, L; De Flora, A et al. (1998) Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate. J Biol Chem 273:118-25
Graeff, R M; Walseth, T F; Lee, H C (1997) Radioimmunoassay for measuring endogenous levels of cyclic ADP-ribose in tissues. Methods Enzymol 280:230-41
Munshi, C; Lee, H C (1997) High-level expression of recombinant Aplysia ADP-ribosyl cyclase in offhia pastoris by fermentation. Protein Expr Purif 11:104-10
Munshi, C B; Fryxell, K B; Lee, H C et al. (1997) Large-scale production of human CD38 in yeast by fermentation. Methods Enzymol 280:318-30
De Flora, A; Guida, L; Franco, L et al. (1997) CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose. J Biol Chem 272:12945-51
Walseth, T F; Aarhus, R; Gurnack, M E et al. (1997) Preparation of cyclic ADP-ribose antagonists and caged cyclic ADP-ribose. Methods Enzymol 280:294-305
Lee, H C; Graeff, R M; Walseth, T F (1997) ADP-ribosyl cyclase and CD38. Multi-functional enzymes in Ca+2 signaling. Adv Exp Med Biol 419:411-9
Walseth, T F; Wong, L; Graeff, R M et al. (1997) Bioassay for determining endogenous levels of cyclic ADP-ribose. Methods Enzymol 280:287-94
Lee, H C; Aarhus, R; Gee, K R et al. (1997) Caged nicotinic acid adenine dinucleotide phosphate. Synthesis and use. J Biol Chem 272:4172-8

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