Interaction of Neuronal Nitric-oxide Synthase with α1-Syntrophin in Rat Brain*
- Akiko Hashida-Okumura‡¶,
- Nobuaki Okumura‡,
- Akihiro Iwamatsu‖,
- Ruud M. Buijs**,
- Herms J. Romijn‖ and
- Katsuya Nagai‡
- From the ‡Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamada-Oka, Suita, Osaka 565-0871, Japan, ‖Central Laboratories for Key Technology, Kirin Brewery Company, Limited, 1-13-5 Fukuura, Kanazawa-Ku, Yokohama, Kanagawa 236-0004, Japan, and the **Netherlands Institute for Research, Meibergdreef 33, 1105 AZ, Amsterdam Zuidoost, Netherlands
Abstract
Neuronal nitric-oxide synthase (nNOS) has a PSD-95/Dlg/ZO-1 (PDZ) domain that can interact with multiple proteins. nNOS has been known to interact with PSD-95 and a related protein, PSD-93, in brain and with α1-syntrophin in skeletal muscle in mammals. In this study, we have purified an nNOS-interacting protein from bovine brain using an affinity column made of Sepharose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as α1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and α1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and α1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and α1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with α1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons.
Footnotes
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↵* This work was supported by Grant-in-aid for Encouragement of Young Scientists 09770039 from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed. Tel.: 81-6-6879-8632; Fax: 81-6-6879-8633; E-mail:hashida{at}protein.osaka-u.ac.jp.
- Abbreviations:
- nNOS
-
neuronal nitric-oxide synthase
- SCN
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suprachiasmatic nucleus
- PVN
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paraventricular nucleus
- GST
-
glutathione S-transferase
- TBS
-
Tris-buffered saline
- PAGE
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polyacrylamide gel electrophoresis
- HPLC
-
high performance liquid chromatography
- PDZ
-
PSD-95/Dlg/ZO-1
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- Received October 13, 1998.
- Revision received January 5, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
