| Project/Area Number |
13670109
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | Tokyo Metropolitan Organization for Medical Research |
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Principal Investigator |
NAKATA Hiroyasu Tokyo Metropolitan Institute for Neuroscience Department of Molecular Cell Signaling, Director of Department, 東京都神経科学総合研究所, 副参事研究員 (00041830)
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| Co-Investigator(Kenkyū-buntansha) |
KAMIYA Toshio Tokyo Metropolitan Institute for Neuroscience, Staff scientist, 東京都神経科学総合研究所, 研究員 (80344068)
SAITOH Osamu Tokyo Metropolitan Institute for Neuroscience, Staff scientist, 東京都神経科学総合研究所, 研究員 (60241262)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | adenosine / purinergic / signal transduction mechanism / heterodimer / cross-talk / P2 receptor / P3 receptor / NECA / 神経伝達 |
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| Research Abstract |
Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors, respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of adenosine and ATP receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Therefore we have explored the possibility of hetero-oligomerization between G_<i/o> protein-coupled A_1 adenosine receptor (A_1R) and G_<q> protein-coupled P2Y_1 receptor (P2Y_1R) in transfected cultured cells by a new bioluminescence resonance energy transfer technology (BRET^2) in addition to indirect biochemical or pharmacological methods. The existence of A_1R/P2Y_1R hetero-oligomers in co-transfected HEK293T cells was first shown using co-immunoprecipitation methods. In the same co-transfected cells, ADPβS was able to
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reduce forskolin-evoked cAMP accumulation with pertussis toxin- and A_1R antagonist-sensitive manner, indicating that pharmacology of A_1R was significantly modified in the co-transfected cells, i.e. ADPβS binds A_1R and inhibits adenylyl cyclase activity via G_<i/O> proteins. Also, a high degree of A_1R and P2Y_1R co-localization was demonstrated in co-transfected cells by double immunofluorescence experiments with confocal laser microscopy. Then, the BRET^2 technique revealed constitutive heteromeric oligomerization between A_1R and P2Y_1R in living HEK293T cells. The BRET^2 signal also increased in a time-dependent manner upon addition of agonists for both receptors, which was inhibited by pretreatment with the P2Y_1R antagonist MRS2179, indicating that this process is promoted by the simultaneous activation of both receptors. These results suggest that the oligomeric association of A_1R with P2Y_1R generates A_1R with P2Y_1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purinergic signaling. Existence of this hybrid purinergic receptor may explain the controversial inhibition of synaptic transmission by adenine nucleotides. Less
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