Doc2 enhances Ca2+-dependent exocytosis from PC12 cells.
We previously isolated a new protein having two C2-like domains which interacted with Ca2+ 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 Ca2+-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 Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged Doc2 stimulated the Ca2+-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 Ca2+-dependent exocytosis of dense core vesicles from PC12 cells.
Orita
S
S
Shionogi Institute for Medical Science, Japan.
Sasaki
T
T
Komuro
R
R
Sakaguchi
G
G
Maeda
M
M
Igarashi
H
H
Takai
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis; D013006:Growth Hormone / Q000096:biosynthesis*; D006388:Hemagglutinins / Q000096:biosynthesis; D007700:Kinetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism*; D016716:PC12 Cells; D010957:Plasmids; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016324:Sequence Tagged Sites; D014162:Transfection
D002135:Calcium-Binding Proteins; C490419:Doc2a protein, rat; D006388:Hemagglutinins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013006:Growth Hormone; D005982:Glutathione Transferase; D011188:Potassium; D002118:Calcium