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.

and to interact with Ca 2ϩ and phospholipid (8,10,11). Synaptotagmin was originally found to be specifically located on synaptic vesicles (8,12) and shown by genetic and biochemical analyses to regulate neurotransmitter release as a Ca 2ϩ sensor (10,13) (for reviews, see Refs. 14 and 15). Rabphilin-3A was isolated as a downstream target molecule of Rab3A, which specifically interacts with GTP-Rab3A (9,16). Rabphilin-3A is specifically expressed in neuron, where it is highly concentrated on synaptic vesicles (17,18). Synaptotagmin has a transmembrane segment and is anchored on synaptic vesicles through this segment (8,19), but rabphilin-3A has no transmembrane segment and anchors on synaptic vesicles through an anchoring protein (20). These properties of rabphilin-3A, together with the observation that Rab3A is involved in Ca 2ϩdependent exocytosis (for a review, see Ref. 21), strongly suggest that rabphilin-3A may serve as both a downstream target molecule of Rab3A and a Ca 2ϩ sensor for neurotransmitter release. Consistently, Chung et al. (22) took advantage of the GH 1 co-expression assay system, which was originally developed by Wick et al. (23), and showed evidence that rabphilin-3A is indeed involved in Ca 2ϩ -dependent exocytosis from bovine adrenal chromaffin cells. In this assay system, expressed GH is stored in dense core vesicles of chromaffin cells and released in response to various agonists in an extracellular Ca 2ϩ -dependent manner (23,24).
We recently isolated a third protein having two C2-like domains, C2A (127 amino acids, 90 -216 amino acids) and C2B (132 amino acids, 240 -371 amino acids), and named Doc2 (Double C2) (25). Doc2 is a protein with a calculated M r of 44,071 and 400 amino acids. Doc2 has neither a transmembrane segment nor a Rab3A-binding domain but is abundantly expressed in brain where it is highly concentrated on the synaptic vesicle fraction. In this study, we have taken advantage of the GH co-expression assay system of PC12 cells and examined whether Doc2 is also involved in Ca 2ϩ -dependent exocytosis.

EXPERIMENTAL PROCEDURES
Materials and Chemicals-PC12 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 5% horse serum at 37°C in 10% CO 2 as described (26). A rabbit antiserum was generated against a fusion protein of glutathione Stransferase and the N-terminal fragment (1-85 amino acids) of mouse Doc2 2 and affinity-purified by using the same fusion protein and glutathione S-transferase as described (27). Human GH was expressed using pXGH5 in which GH expression is driven by the mouse metallothionein-I promoter (28).
Northern and Western Blot Analyses-Northern blot analysis was performed using 4 g of poly(A) ϩ RNA isolated from rat cerebrum, rat liver, and PC12 cells. The blot was hybridized with a 32 P-labeled 1.2kilobase DNA fragment of the mouse Doc2 cDNA. Several tissues of adult female rat and PC12 cells were homogenized as described (29). Twenty g of the membrane fractions of rat cerebrum, rat liver, and PC12 cells were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis by use of the anti-Doc2 polyclonal antibody.
Construction of Expression Vectors-A DNA fragment encoding the HA (YPYDVPDYA) epitope with the methionine codon was inserted into the XbaI site of pEF-BOS (30) to express the HA-tagged fusion proteins (pEF-HA). To generate sense (1-400 amino acids, pEF-HA-Doc2), antisense (1-400 amino acids, pEF-HA-AS), and deletion mu-* 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 be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Transfection of Various Doc2 cDNA Constructs and Measurement of Release of Expressed GH-PC12 cells were plated at a density of 5 ϫ 10 5 cells/35-mm dish and were incubated for 18 -24 h. The cells were then co-transfected with 2 g of pXGH5 and 2 g of the indicated expression plasmid using lipofectAMINE reagent according to the manufacturer's instruction (Life Technologies, Inc.). GH release experiments were performed 48 h after transfection. PC12 cells were washed with a PSS (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 , 20 mM HEPES, pH 7.4, and 11 mM glucose) and incubated for 10 min with a high K ϩ solution (PSS containing 60 mM KCl and 85 mM NaCl) or a low K ϩ solution (PSS containing 4.7 mM KCl and 140 mM NaCl). The amounts of the GH released into the medium and retained in the cells were measured using a radioimmunoassay kit (Nichols Institute, San Juan Capistrano, CA).
Immunocytochemistry-Immunocytochemistry was performed on PC12 cells that had been fixed with 4% paraformaldehyde and permeabilized with methanol (Ϫ20°C). Human GH was detected with a rabbit anti-human GH antibody (Dako Co., Carpintenia, CA) and a donkey anti-rabbit antibody conjugated to fluorescein (Chemicon International, Inc., Temecula, CA). The HA epitope was detected with a mouse anti-HA antibody (Berkeley Antibody Co., Richmond, CA) and a donkey anti-mouse antibody conjugated to rhodamine (Chemicon). Cells were analyzed with a Zeiss Axioplan microscope (Carl Zeiss, Oberkochen, Germany).

Expression of Doc2 in PC12 Cells-In
Northern blot analysis, a 2.2-kilobase transcript was detected in PC12 cells and rat cerebrum but not in rat liver (Fig. 1A). In Western blot analysis, one immunoreactive band with an M r of about 47,000 was detected in PC12 cells and rat cerebrum but not in rat liver (Fig. 1B). These results indicate that Doc2 is indeed expressed in PC12 cells as well as in rat cerebrum.
Co-expression of Doc2 with Human GH in PC12 Cells-The plasmid encoding human Doc2 (pEF-HA-Doc2) was co-transfected with pXGH5 (encoding human GH) in PC12 cells. An epitope tag (HA) was attached to the N terminus of the Doc2 protein to detect it in the cells. When transfected Doc2 and GH were analyzed by immunocytochemistry using the respective antibodies, both proteins were detected in the same cell (Fig. 2). About 90% of the GH-expressing cells expressed detectable amounts of the HA-tagged Doc2 protein as determined by fluorescence microscopy.
Ca 2ϩ -dependent Release of Co-expressed GH from PC12 Cells-When human GH is transfected into PC12 cells, GH is stored in dense core vesicles and released upon stimulation by high K ϩ in an extracellular Ca 2ϩ -dependent manner (23,24). Consistent with these earlier observations, high K ϩ stimulated release of co-expressed GH in a time-dependent manner in the presence of extracellular Ca 2ϩ but not in the absence of extracellular Ca 2ϩ (Fig. 3).
Enhancement of Release of Co-expressed GH by Overexpression of Doc2-When 2 g of pEF-HA-Doc2 was transfected into PC12 cells, the high K ϩ -induced release of co-expressed GH was further enhanced about 40% (Fig. 4A). This effect was dependent on the amount of the transfected plasmid DNA (Fig.  4B). In contrast, when a plasmid with the coding sequence of Doc2 inserted in the antisense orientation (pEF-HA-AS) was transfected into PC12 cells, the high K ϩ -induced release of co-expressed GH was inversely inhibited about 37% (Fig. 4A). This effect was also dependent on the amount of the transfected plasmid DNA (Fig. 4B). Overexpression of Doc2 or the reduced expression of endogenous Doc2 by transfection with an antisense Doc2 construct did not affect the basal GH release (Fig. 4C).
Inhibition of Release of Co-expressed GH by Overexpression of Doc2 Deletion Mutants-PC12 cells were co-transfected with pXGH5 and several deletion mutants of Doc2. The mutant lacking the C2-like domains but containing the N-terminal portion of Doc2 (1-89 amino acids, pEF-HA-Doc2N) inhibited the high K ϩ -induced release of co-expressed GH about 45% (Fig. 5A). The mutant containing the C2-like domains (90 -400 amino acids, pEF-HA-Doc2C) also inhibited the release about 45% (Fig. 5A). Overexpression of the mutants did not affect the basal GH release (Fig. 5B). DISCUSSION In our preceding paper, we have shown that Doc2 is abundantly expressed in brain where it is enriched in the synaptic vesicle fraction (25). Consistently, we have shown here that Doc2 is also expressed in PC12 cells. These results, together with the property of Doc2 that it has two C2-like domains responsible for interaction with Ca 2ϩ and phospholipid, strongly suggest that Doc2 as well as synaptotagmin and rabphilin-3A plays a role in Ca 2ϩ -dependent exocytosis.
To obtain evidence supporting this role of Doc2, we have taken advantage of the GH co-expression assay system of PC12 cells. By use of this assay system, we have shown here that overexpression of Doc2 enhances Ca 2ϩ -dependent release of co-expressed GH from PC12 cells and that reduction of endogenous Doc2 by transfection with an antisense Doc2 construct inversely reduces the release. We have moreover shown here that several Doc2 deletion mutants inhibit the Ca 2ϩ -dependent release. These results suggest that Doc2 is involved in Ca 2ϩdependent exocytosis. However, we cannot exclude from these results a possibility that Doc2 affects the endocytosis of GH Doc2 and Ca 2ϩ -dependent Exocytosis 7258 followed by its exocytosis. To clarify this issue, we measured internalization of surface-bound 125 I-labeled GH in PC12 cells. GH was not internalized significantly in low K ϩ and high K ϩ solutions containing 1 nM GH (data not shown). The concentration of GH maximally secreted from PC12 cells in response to high K ϩ was about 0.5 nM (data not shown). This result indicates that overexpression of Doc2 and its various fragments affects Ca 2ϩ -dependent release of co-expressed GH but not the endocytosis.
The C2-like domains of synaptotagmin interact with several important molecules involved in endocytosis and exocytosis, such as AP2 (clathrin adaptor complex-2) (32), IP 4 (inositol 1,3,4,5-tetrakisphosphate) (33), syntaxin (34), and ␤-SNAP (␤soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) (35), and microinjection of the fragments containing these domains into PC12 cells inhibits the Ca 2ϩ -dependent exocytosis (36). The N-terminal fragment of rabphilin-3A interacts with Rab3A (11), and overexpression of this fragment in chromaffin cells inhibits the Ca 2ϩ -dependent exocytosis (22). These earlier results, together with the present results that overexpression of the N-terminal fragment or the fragment containing the C2-like domains of Doc2 in PC12 cells also inhibits the Ca 2ϩ -dependent exocytosis, suggest that these three proteins having two C2-like domains are involved in and play different roles in Ca 2ϩ -dependent exocytosis. Doc2 may interact with specific components involved in Ca 2ϩ -dependent exocytosis, which are different from those which synaptotagmin and rabphilin-3A interact with, and introduction of the Nor C-terminal fragment of Doc2 into PC12 cells may disrupt these interactions. It is important to isolate these Doc2-interacting molecules in the future.
Electrophysiological studies demonstrated that Ca 2ϩ -dependent exocytosis requires several Ca 2ϩ sensors (37) (for a review, see Ref. 38). One of the most probable Ca 2ϩ sensors is synaptotagmin (10,(13)(14)(15). However, there are several lines of evidence that Ca 2ϩ sensors other than synaptotagmin are also involved in Ca 2ϩ -dependent exocytosis (13,39). Doc2 and rabphilin-3A, which have two C2-like domains, may be possible candidates for these Ca 2ϩ sensors. Further studies are necessary to clarify the role of Doc2 as a Ca 2ϩ sensor. Doc2 and Ca 2ϩ -dependent Exocytosis 7259