(Received for publication, November 3, 1995; and in revised form, January 11, 1996)
From the
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.
The C2 domain was first found in protein kinase C activated by
Ca and phospholipid (1, 2) (for a
review, see (3) ). The C2-like domain was subsequently found in
many other important intracellular signaling elements, including
phospholipase C
(4) , Ras GTPase-activating
protein(5) , phospholipase A
(6) , and Unc
13(7) . All of these proteins have one C2-like domain. In
contrast to these proteins, synaptotagmin and rabphilin-3A were shown
to have two C2-like domains, C2A and C2B (8, 9) and to
interact with Ca
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
sensor (10, 13) (for reviews, see (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
-dependent exocytosis (for a review, see (21) ), strongly suggest that rabphilin-3A may serve as both a
downstream target molecule of Rab3A and a Ca
sensor
for neurotransmitter release. Consistently, Chung et al. (22) took advantage of the GH (
)co-expression assay
system, which was originally developed by Wick et al. (23) , and showed evidence that rabphilin-3A is indeed involved
in Ca
-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
-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 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
-dependent exocytosis.
Figure 1: Expression of Doc2 in PC12 cells. A, Northern blot analysis of Doc2 mRNA in PC12 cells. Lane 1, rat cerebrum; lane 2, PC12 cells; lane 3, rat liver. The arrowhead indicates the position of Doc2. B, immunoblot analysis of Doc2 in PC12 cells by use of the anti-Doc2 polyclonal antibody. Lane 1, recombinant human Doc2 (9-400 amino acids) expressed as a fusion protein with the N-terminal HA epitope using the insect/baculovirus system (10 ng of protein); lane 2, the membrane fraction of rat cerebrum (25 µg of protein); lane 3, the membrane fraction of PC12 cells (25 µg of protein); lane 4, the membrane fraction of rat liver (25 µg of protein).
Figure 2: Co-expression of HA-tagged Doc2 with GH in PC12 cells. PC12 cells were co-transfected with pXGH5 and pEF-HA-Doc2. A, phase image; B, immunofluorescence staining for GH; C, immunofluorescence staining for HA-tagged Doc2.
Figure 3:
Ca-dependent, high
K
-induced release of expressed GH from PC12 cells. A, time course for release of expressed GH in the presence of
extracellular Ca
. The cells were incubated for the
indicated periods of time.
, with the high K
solution;
, with the low K
solution. B, effect of extracellular Ca
on the high
K
-induced release of GH. Bar 1, with the high
K
solution in the presence of 2.5 mM CaCl
; bar 2, with the high K
solution in the presence of 1 mM EGTA instead of 2.5
mM CaCl
; bar 3, with the low K
solution in the presence of 2.5 mM CaCl
; bar 4, with the low K
solution in the
presence of 1 mM EGTA instead of 2.5 mM CaCl
. Data are expressed as the average percentage
released of the total GH stores. The values are representative of three
independent experiments.
Figure 4:
Effect of overexpression of Doc2 on
release of expressed GH from PC12 cells. A, effect of
pEF-HA-Doc2 and pEF-HA-AS on the high K-induced
release of expressed GH. B, dose-response effect of
pEF-HA-Doc2 and pEF-HA-AS.
, with pEF-HA-Doc2;
, with
pEF-HA-AS. C, effect of pEF-HA-Doc2 and pEF-HA-AS on the low
K
-induced release of expressed GH. Bar 1,
with pEF-HA; bar 2, with pEF-HA-Doc2; bar 3, with
pEF-HA-AS. Data are expressed as the average percentage released of the
total GH stores. The values are representative of three independent
experiments.
Figure 5:
Effect of Doc2 mutants on release of
expressed GH from PC12 cells. A, effect of pEF-HA-Doc2N and
pEF-HA-Doc2C on the high K-induced release of
expressed GH. B, effect of pEF-HA-Doc2N and pEF-HA-Doc2C on
the low K
-induced release of expressed GH. Bar
1, with pEF-HA; bar 2, with pEF-HA-Doc2N; bar 3,
with pEF-HA-Doc2C. Data are expressed as the average percentage
released of the total GH stores. The values are representative of three
independent experiments.
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 and phospholipid, strongly suggest that Doc2
as well as synaptotagmin and rabphilin-3A plays a role in
Ca
-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-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
-dependent release. These results suggest that Doc2
is involved in Ca
-dependent exocytosis. However, we
cannot exclude from these results a possibility that Doc2 affects the
endocytosis of GH followed by its exocytosis. To clarify this issue, we
measured internalization of surface-bound
I-labeled GH in
PC12 cells. GH was not internalized significantly in low K
and high K
solutions containing 1 nM GH
(data not shown). The concentration of GH maximally secreted from PC12
cells in response to high K
was about 0.5 nM (data not shown). This result indicates that overexpression of
Doc2 and its various fragments affects Ca
-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 (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
-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
-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
-dependent
exocytosis, suggest that these three proteins having two C2-like
domains are involved in and play different roles in
Ca
-dependent exocytosis. Doc2 may interact with
specific components involved in Ca
-dependent
exocytosis, which are different from those which synaptotagmin and
rabphilin-3A interact with, and introduction of the N- or 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-dependent exocytosis requires several
Ca
sensors (37) (for a review, see (38) ). One of the most probable Ca
sensors
is synaptotagmin(10, 13, 14, 15) .
However, there are several lines of evidence that Ca
sensors other than synaptotagmin are also involved in
Ca
-dependent exocytosis(13, 39) .
Doc2 and rabphilin-3A, which have two C2-like domains, may be possible
candidates for these Ca
sensors. Further studies are
necessary to clarify the role of Doc2 as a Ca
sensor.