Neuronal calcium sensor protein visinin-like protein-3 interacts with microsomal cytochrome b5 in a Ca2+-dependent manner.
Visinin-like protein-3, which is one of the neuronal calcium sensors, has been shown to be mainly expressed in cerebellar Purkinje cells, but cellular function of this protein has not yet been elucidated. We examined the tissue distribution of murine visinin-like protein-3 transcripts using real-time reverse transcription-PCR. Visinin-like protein-3 mRNA was found to be expressed in peripheral tissues. Particularly, the expression of the transcript in the thymus was significantly higher than in other peripheral tissues. In addition, B6RVTC1 thymoma cells robustly expressed visinin-like protein-3 mRNA. To identify a target protein of visinin-like protein-3, we performed a pull-down experiment using glutathione S-transferase-tagged visinin-like protein-3 and two-dimensional electrophoresis. We demonstrated that microsomal cytochrome b(5) was a Ca(2+)-dependent binding partner of visinin-like protein-3. In a co-immunoprecipitation experiment, it was observed that hippocalcin, as well as visinin-like protein-3, could interact with cytochrome b(5). Furthermore, we confirmed that the sequence Val(114)-Tyr(127) at the C-terminal tail of cytochrome b(5) is the minimal structural requirement for binding to visinin-like protein-3. In addition, the loop His(19)-His(25) at the N terminus of visinin-like protein-3 is essential for binding to cytochrome b(5). Microsomal cytochrome b(5) was also shown to be a potential activator of cytochrome P450. The present findings raise the possibility that visinin-like protein-3 may link Ca(2+) signaling to the machinery of microsomal monooxygenase complex composed of cytochrome b(5), cytochrome P450, and some reductases. This report provides the first evidence of an interaction between visinin-like protein-3 and microsomal cytochrome b(5).
Oikawa
Kensuke
K
Department of Pathology, School of Nursing, Asahikawa Medical College, Midorigaoka Higashi 2-1, Asahikawa, Hokkaido 078-8510, Japan.
Kimura
Shoji
S
Aoki
Naoko
N
Atsuta
Yoshiaki
Y
Takiyama
Yumi
Y
Nagato
Toshihiro
T
Yanai
Mitsuru
M
Kobayashi
Hiroya
H
Sato
Keisuke
K
Sasajima
Tadahiro
T
Tateno
Masatoshi
M
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D051596:Neurocalcin; D009474:Neurons / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D014633:Valine / Q000737:chemistry
D002135:Calcium-Binding Proteins; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D012333:RNA, Messenger; D044169:Receptors, Calcium-Sensing; D011993:Recombinant Fusion Proteins; C485222:VILIP-3 protein, mouse; C496688:VSNL1 protein, human; D014443:Tyrosine; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase; D014633:Valine; D002118:Calcium