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Receptors and Transporters

開催日 2014/9/11
時間 11:00 - 12:00
会場 Poster / Exhibition(Event Hall B)

An investigation of post-translational cleavage of an orphan metabotropic receptor, Prrt3

  • P1-033
  • 山本 泉 / Izumi Yamamoto:1 山本 友美 / Tomomi Yamamoto:1 今野 幸太郎 / Kohtaro Konno:2 渡辺 雅彦 / Masahiko Watanabe:2 久保 義弘 / Yoshihiro Kubo:1,3 
  • 1:生理研・神経機能素子 / Div Biophys and Neurobiol, NIPS, Okazaki, Japan 2:北大院・医・解剖・解剖発生 / Dept Anatomy, Hokkaido Univ Grad Schl Med, Sapporo, Japan 3:総研大院生命科学生理 / Physiol Sci SOKENDAI, Hayama, Japan 

Proline-rich transmembrane protein 3 (Prrt3) is an orphan G-protein coupled receptor (GPCR). The physiological functions of Prrt3 still remain unknown, yet our behavioral studies using Prrt3 heterozygous knock out (KO) mice suggested that Prrt3 is involved in the long-term retention of memory. Using our Prrt3 specific antibodies, we observed it is expressed in various regions in mouse brain including hippocampus, thalamus and cerebellar cortex. We also explored subcellular localization of Prrt3 by biochemical fractionation, and observed Prrt3 protein is distributed in synaptosome but not in post synaptic densities, implying its localization at extrasynaptic or presynaptic regions.
In our western blot analysis using antibodies to the extracellular N-terminus or the cytoplasmic C-terminus, we noticed the presence of multiple sizes of Prrt3 protein. The anti – C-terminal antibody, but not anti – N-terminal antibody, detected specific bands of cleaved Prrt3 at 80 and 70 kDa, which are smaller than the non-cleaved bands of 120 and 140 kDa. In this study, we investigated the molecular mechanisms of the generation of these cleaved forms of Prrt3. To examine the effects of various extracellular proteases on Prrt3, we isolated non-cleaved form of Prrt3 from mouse brain by immune-precipitation using N-terminal antibody, and incubated it with proteases in vitro. After 12 hours of incubation with thrombin or plasmin, Prrt3 was digested completely. Interestingly, we observed cleaved form of Prrt3 at 80 kDa faintly when Prrt3 was incubated with tissue plasminogen activator (tPA). We further examined the effect of tPA on Prrt3 using tPA homozygous KO mouse brain. Prrt3 specific 80 kDa band was detected also in tPA homozygous KO mice lane, demonstrating tPA is not the main protease which is responsible for the cleavage of Prrt3 N-terminal domain. Neurotrypsin and neuropsin homozygous KO mice were also examined, yet we still observed Prrt3 specific bands at 80 kDa. The results show the involvement of other extracellular proteases or processing enzymes in the formation of cleaved form of Prrt3.

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