Abstract

The molecular mechanisms involved in the regulation of calcium entry into immune system cells are poorly understood. My preliminary data describe the molecular characterization of a novel calcium entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific vertebrate ADP-Ribose Hydrolase (ADPRH-1), and the functional demonstration that a previously identified gene encodes a protein product which is an ADPR gated calcium channel. Based on these data, I propose to explore ADPR metabolism and the mechanisms which regulate ADPR-mediated calcium entry within the immune system through three specific aims:
Specific Aim 1 : Characterization of free ADPR metabolism in the immune system.
This aim will characterize the cell biology, structure/function relationships, and physiologic roles of ADPRH-1 in the regulation of ADPR metabolism. These analyses will include the production of an ADPRH- 1 deficient cell line and its complementation using various forms of ADPRH-1.
Specific Aim 2 : Immune system mechanisms for the regulation of free ADPR levels.
This aim will focus on analyses of ADPR metabolism in relation to immune cell manipulations plausibly linked to the production of free ADPR. It will utilize both classic methods for measurement of total cellular free ADPR and a novel electrophysiologic assay for cytosolic free ADPR.
Specific Aim 3 : Characterization of the cell biology and structure/function relationships of the ADPR gated cation channel: a possible key effector of free ADPR. These analyses will include screening of cell lines and primary cell types for channel expression and ADPR-mediated currents, and a structure/function analysis of the role of an ADPRH- 1 homology region of the ADPR gated channel in the gating process. Together, the experiments in these aims will provide us with substantial new information regarding the metabolism of free ADPR and the role of ADPR dependent calcium entry in immune system function.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064091-03
Application #
6628959
Study Section
Immunobiology Study Section (IMB)
Program Officer
Marino, Pamela
Project Start
2001-02-01
Project End
2006-01-31
Budget Start
2003-02-01
Budget End
2004-01-31
Support Year
3
Fiscal Year
2003
Total Cost
$262,280
Indirect Cost

  Institution

Name
University of Washington
Department
Pediatrics
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195

  Publications

Buelow, Ben; Scharenberg, Andrew M (2008) Characterization of parameters required for effective use of tamoxifen-regulated recombination. PLoS One 3:e3264
Song, Yumei; Buelow, Ben; Perraud, Anne-Laure et al. (2008) Development and validation of a cell-based high-throughput screening assay for TRPM2 channel modulators. J Biomol Screen 13:54-61
Scharenberg, Andrew M; Humphries, Lisa A; Rawlings, David J (2007) Calcium signalling and cell-fate choice in B cells. Nat Rev Immunol 7:778-89
Song, Yumei; Dayalu, Rashmi; Matthews, Sharon A et al. (2006) TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes. Eur J Cell Biol 85:1253-64
Grubisha, Olivera; Rafty, Louise A; Takanishi, Christina L et al. (2006) Metabolite of SIR2 reaction modulates TRPM2 ion channel. J Biol Chem 281:14057-65
Scharenberg, Andrew M (2005) TRPM2 and TRPM7: channel/enzyme fusions to generate novel intracellular sensors. Pflugers Arch 451:220-7
Perraud, Anne-Laure; Shen, Betty; Dunn, Christopher A et al. (2003) NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase. J Biol Chem 278:1794-801
Launay, Pierre; Fleig, Andrea; Perraud, Anne Laure et al. (2002) TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell 109:397-407