Interaction of neuronal nitric-oxide synthase with alpha1-syntrophin in rat brain.
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 alpha1-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 alpha1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and alpha1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and alpha1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and alpha1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with alpha1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons.
Hashida-Okumura
A
A
Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamada-Oka, Suita, Osaka 565-0871, Japan. hashida@protein.osaka-u.ac.jp
Okumura
N
N
Iwamatsu
A
A
Buijs
R M
RM
Romijn
H J
HJ
Nagai
K
K
D000818:Animals; D001921:Brain / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002478:Cells, Cultured; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; C091825:syntrophin alpha1; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat