論文 - 緑川 良介
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Temporal and quantitative analysis of the functional expression of Ca(2+)-permeable AMPA receptors during LTP.
Wakazono Y, Midorikawa R, Takamiya K
Neuroscience research 2023年7月
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Morise J., Yamamoto S., Midorikawa R., Takamiya K., Nonaka M., Takematsu H., Oka S.
International Journal of Molecular Sciences 21 ( 14 ) 1 - 11 2020年7月
記述言語：日本語 掲載種別：研究論文（学術雑誌） 出版者・発行元：International Journal of Molecular Sciences
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1–4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits’ ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.
DOI： 10.3390/ijms21145101
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Monitoring the glycosylation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors using specific antibodies reveals a novel regulatory mechanism of N-glycosylation occupancy by molecular chaperones in mice.
Midorikawa R, Takakura D, Morise J, Wakazono Y, Kawasaki N, Oka S, Takamiya K
Journal of neurochemistry 2020年1月
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Kandel M., Yamamoto S., Midorikawa R., Morise J., Wakazono Y., Oka S., Takamiya K.
Journal of Neurochemistry 147 ( 6 ) 730 - 747 2018年12月
担当区分：筆頭著者, 責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Neurochemistry
The AMPA-type glutamate receptor (AMPA-R) plays a primary role in principal excitatory synaptic transmission and many neuronal functions including synaptic plasticity that underlie learning and memory. N-glycosylation is one of the major post-translational modifications of membrane proteins, but its specific roles in neurons remain largely unknown. AMPA-R subunits are N-glycosylated at their extracellular domains during their biosynthesis in the lumen of the endoplasmic reticulum and Golgi system. Six N-glycosylation sites are presumed to exist in the extracellular domain of GluA1, which is a member of the AMPA-R subunits. We observed that the intracellular trafficking and cell surface expression were strongly suppressed in the GluA1 mutants lacking N-glycans at N63/N363 in HEK293T cells. Multimer analysis using Blue Native-PAGE displayed the impaired tetramer formation in the glycosylation mutants (N63S and N363S), indicating that the mis-transport was caused by impaired tetramer formation. N63S and N363S mutants were primarily degraded via the lysosomal pathway. Flag-tagged N363S GluA1, but not N63S GluA1, expressed in primary cortical neuron cultures prepared from GluA1 knockout mice was observed to localize at the cell surface. Co-expression of GluA2 partially rescued tetramer formation and the cell surface expression of N363S GluA1 but not N63S GluA1, in HEK293T cells. Electrophysiological analysis also demonstrated functional heteromers of N363S GluA1 with GluA2. These data suggest that site-specific N-glycans on GluA1 subunit regulates tetramer formation, intracellular trafficking, and cell surface expression of AMPA-R. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. (Figure presented.).
DOI： 10.1111/jnc.14565
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Autophagy-dependent rhodopsin degradation prevents retinal degeneration in Drosophila. 査読あり
Midorikawa R, Yamamoto-Hino M, Awano W, Hinohara Y, Suzuki E, Ueda R, Goto S.
Journal of Neuroscience 2010年8月
記述言語：英語 掲載種別：研究論文（学術雑誌）
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KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity. 査読あり
Midorikawa R, Takei Y, Hirokawa N
Cell 125 ( 2 ) 371 - 83 2006年4月
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Kato K., Kishi T., Kamachi T., Akisada M., Oka T., Midorikawa R., Takio K., Dohmae N., Bird P., Sun J., Scott F., Miyake Y., Yamamoto K., Machida A., Tanaka T., Matsumoto K., Shibata M., Shiosaka S.
Journal of Biological Chemistry 276 ( 18 ) 14562 - 14571 2001年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 ± 0.22 × 106 m-1 s -1, showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively.
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Characterization of recombinant and brain neuropsin, a plasticity- related serine protease 査読あり
Shimizu C., Yoshida S., Shibata M., Kato K., Momota Y., Matsumoto K., Shiosaka T., Midorikawa R., Kamachi T., Kawabe A., Shiosaka S.
Journal of Biological Chemistry 273 ( 18 ) 11189 - 11196 1998年5月
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Journal of Biological Chemistry
Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys32-Ile33bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.