Doc2 Enhances Ca
-dependent Exocytosis from PC12 Cells (*)
- Satoshi Orita(1),
- Takuya Sasaki(2),
- Ryutaro Komuro(2),
- Gaku Sakaguchi(1),
- Miki Maeda(1),
- Hisanaga Igarashi(1) and
- Yoshimi Takai(2)(3)(§)
- From the (1)Shionogi Institute for Medical Science, Settsu 566, the
- (2)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, and the
- (3)Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan
- §To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; ytakai{at}molbio.med.osaka-u.ac.jp.
Abstract
We previously isolated a new protein having two C2-like domains which interacted with Ca
and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated
on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca
-dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression
assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K
in an extracellular Ca
-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged
Doc2 stimulated the Ca
-dependent, high K
-induced release of co-expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with
the coding sequence of Doc2 in the antisense orientation, the high K
-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal
fragment containing two C2-like domains inhibited the high K
-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca
-dependent exocytosis of dense core vesicles from PC12 cells.
Footnotes
-
↵* The investigation at Osaka University Medical School was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, Sports, and Culture, Japan(1995), by grants-in-aid for abnormalities in hormone receptor mechanisms and for aging and health from the Ministry of Health and Welfare, Japan(1995), and by a grant from Uehara Memorial Foundation (1995). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- GH
-
growth hormone
- HA
-
hemagglutinin
- PSS
-
physiological salt solution.
-
↵2S. Orita, T. Sasaki, R. Komuro, G. Sakaguchi, M. Maeda, H. Igarashi, and Y. Takai, manuscript in preparation.
-
- Received November 3, 1995.
- Revision received January 11, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
