Unintended exposure to radiation via a nuclear accident, or explosion of a """"""""dirty bomb,"""""""" can have devastating consequences. We developed a novel analog of the phospholipid growth factor lysophosphatidic acid (LPA), octadecenyl thiophosphate (OTP), which when applied up to 24h post-irradiation in mice irradiated with lethal doses of gamma-irradiation shows strong radiomitigating action by rescuing cells from apoptosis and saving life. Experiments indicate that the molecular target of OTP is the LPA2 G protein-coupled receptor. The objective of this application is to elucidate the radiomitigating mechanism of action unique to the LPA2 receptor. The central hypothesis of the proposed work is that macromolecular protein complexes formed at the C-terminal tail of the LPA2 receptor are required for the radioprotective effect of OTP. This is a significant departure from the concept that the LPA2 receptor coupled G protein-mediated signaling events are sufficient to elicit radiomitigation. LPA2 is distinctly different from the other LPA receptor subtypes in its C- terminal tail, where it forms protein-protein interactions with PDZ domain-binding proteins and with LIM domain proteins. We hypothesize that these LPA2-protein interactions regulate multiple prosurvival signaling pathways (ERK1/2 and AKT) and also arrest the actions Siva-1, a proapoptotic protein that links DNA damage to the apoptotic cascade. This hypothesis goes beyond the LPA2 GPCR because the sequence motifs responsible for these protein-protein interactions are present in many other GPCR.
The specific aims are: 1. Test the hypothesis that the C-terminal PDZ motif is required for the radioprotective effect. 2. Characterize the motif in the C terminal 55 amino acids of the LPA2 receptor required for the interactions with the LIM domain proteins TRIP6 and Siva-1 by determining whether physical interaction with them is required for the antiapoptotic effect. 3. Dissect the role of LPA2-TRIP6 - c-Src - ERK/AKT signaling axis in the antiapoptotic effect. 4. Elucidate the role of the LPA2 - Siva-1- BCL-XL signaling axis in the antiapoptotic effect. An in-depth understanding of OTP's mechanism of action is of paramount importance for scientific, regulatory, and therapeutic reasons. Not negligible is the fact that OTP might also provide potential therapeutic applications in the attenuation of the side effects of radiation- and chemotherapy, as well as diseases associated with increased program cell death. Understanding the signaling interactions between pro-survival GPCR and the apoptotic machinery will pave the way to the identification of new therapeutic targets as well as GPCR ligands. Unintended exposure to radiation via a nuclear accident, or explosion of a """"""""dirty bomb,"""""""" can have devastating consequences to the public. We developed a novel analog of the phospholipid growth factor lysophosphatidic acid (LPA), octadecenyl thiophosphate (OTP). When applied up to 24 h post-irradiation in mice irradiated with lethal doses of gamma-irradiation, OTP shows significant radiomitigating action by rescuing cells from apoptosis and saving life. OTP has many of the properties of an ideal radioprotectant/radiomitigator: a) it is stable at room temperature for years; b) it can be formulated for a wide variety of patient types; c) it is relatively easy to synthesize and manufacture, and inexpensive; and d) it is effective when given up to 12 h after radiation injury. Experiments conducted in vitro and with knockout (KO) mice indicate that the molecular target of OTP is the LPA2 G protein-coupled receptor (GPCR). The objective of this grant application is to elucidate the an- tiapoptotic mechanism of action unique to the LPA2 receptor subtype ? ? ? ?
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