articles
cAMP-GEFII is a direct target of cAMP
in regulated exocytosis
Nobuaki Ozaki*†‡, Tadao Shibasaki*‡, Yasushige Kashima*, Takashi Miki*, Kazuo Takahashi§, Hiroaki Ueno*,
Yasuhiro Sunaga*, Hideki Yano*, Yoshiharu Matsuura¶, Toshihiko Iwanaga#, Yoshimi Takai** and
Susumu Seino*††
*Department of Molecular Medicine, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan
§Core Research for Evolutional Science and Technology (CREST), Department of Molecular Immunology, Chiba University Graduate School of Medicine, Chiba
260- 8670, Japan
¶Department of Virology II, National Institute of Infectious Disease, Tokyo 162-8640, Japan
#Laboratory of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
**Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan
†On leave from the First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466-8550, Japan
‡These authors contributed equally to this work.
††e-mail: [email protected]
Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic
machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule,
Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMPinduced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA).
Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent,
PKA-independent exocytosis.
timulus–secretion coupling is an important event in many
cell types, including neuronal, endocrine, neuroendocrine
and exocrine cells. Exocytosis is the final vesicular-transport
step in the secretory pathway1–4. Although an increase in intracellular Ca2+ concentration is the primary signal in regulated exocytosis, other intracellular signals are also important; in particular,
cAMP is well known to regulate exocytosis in many secretory
cells. cAMP has been thought to induce long-term potentiation
(LTP)5–7 by increasing neurotransmitter release at mossy-fibre
synapses in the CA3 region in the hippocampus of the cerebrum8,
and at synapses between parallel fibres, the axons of cerebellar
granule cells, and Purkinje cells in the cerebellum9,10. cAMP
increases transmitter release at many synapses in peripheral ganglia and invertebrate preparations, including sympathetic11,12 and
parasympathetic13 ganglion neurons and the crayfish neuromuscular junction14. cAMP also regulates exocytosis in peripheral tissues, including the release of insulin from pancreatic β-cells15,16,
hormones from pituitary cells17,18, and amylase from parotid and
pancreatic acinar cells19–21. Although the effect of cAMP on regulated exocytosis is generally thought to be mediated mainly
through activation of PKA, which catalyses the phosphorylation
of regulatory proteins associated with exocytotic processes, it has
been proposed that cAMP also acts directly on the exocytotic
machinery in neuronal22 and non-neuronal23 cells in a PKA-independent manner. However, the direct target of cAMP in the exocytotic machinery is not known.
Here we show that the recently identified cAMP sensor cAMPGEFII24 binds both to Rim (now called Rim1), a putative Rab3
effector in the regulation of synaptic-vesicle fusion25, and to a new
isoform, Rim2, and that cAMP-GEFII, through its interaction
with Rim2, is responsible for cAMP-dependent, PKA-independent exocytosis.
S
Results
Identification of cAMP-GEFII and its interaction with Rim. ATPsensitive K+ channels (KATP channels) link cell metabolism to membrane potential and have an important function in stimulus–secretion coupling in neurons, neuroendocrine and endocrine cells26.
During the search by yeast two-hybrid screening of the mouse
insulin-secreting cell line MIN6 complementary DNA library for an
intracellular signalling molecule that is directly coupled to the
sulphonylurea receptor SUR1 (a subunit of the pancreatic β-cell KATP
channel27), we identified a protein called cAMP sensor (CAMPS).
CAMPS has two putative cAMP-binding domains, a Dishevelled,
Egl-10, Pleckstrin (DEP) domain, and a guanine nucleotide exchange
factor (GEF)-homology domain. cAMP-binding proteins that activate Rap1, a small G protein24,28, have recently been identified, and
CAMPS has incidentally been shown to be the mouse homologue of
cAMP-GEFII24. A previous study indicated the presence of only one
cAMP-binding domain in cAMP-GEFII, but sequence alignment of
cAMP-GEFII and various regulatory subunits of PKA29 have shown
the presence of two putative cAMP-binding domains (hereafter
referred to as cAMP-A and cAMP-B). A fusion protein consisting of
cAMP-A (amino-acid residues 43–153) and glutathione-S-transferase (GST) bound to [3H]cAMP with a Kd of 10.0 ± 2.3 µM
(n = 9), but binding of [3H]cAMP to GST–cAMP-B (amino-acid
residues 357–469) was not evident under the same conditions.
Because the identification of a target molecule of cAMP-GEFII
would indicate its physiological function, we searched for a molecule that interacts with cAMP-GEFII by a yeast two-hybrid screen
of the MIN6 cDNA library, using cAMP-GEFII as a bait.
Interestingly, cAMP-GEFII was found to interact with a new isoform (Rim2) of Rim1 (ref. 25), a putative effector of Rab3 that is
proposed to serve as a Rab3-dependent regulator of synaptic-vesicle fusion25. A full-length Rim2 cDNA encodes a 1,590-amino-acid
NATURE CELL BIOLOGY VOL 2 NOVEMBER 2000 http://cellbio.nature.com
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articles
Zinc-finger
PDZ
C2 A
C2 B
91.4%
78.7%
82.6%
80.7%
protein that has 61.6% identity with rat Rim1 (see Supplementary
Information). A zinc-finger domain, a PDZ domain and two C2
domains are highly conserved between Rim1 and Rim2 (Fig. 1a).
Because Rim1 is proposed to be a Rab3 effector25, we investigated
the possibility that Rim2 is also an effector of Rab3. Using yeast
two-hybrid assays, Rim2, like Rim1 (ref. 25), was found to interact
with constitutively active Rab3A (harbouring a Q81L mutation;
Fig. 1b). In addition, the immobilized GST–Rim2 bound only to
the GTPγS-bound form of Rab3A (Fig. 1c). These results indicate
that, like Rim1, Rim2 binds to the GTP-activated form of Rab3A.
We evaluated the specificity of the interaction between cAMPGEFII and Rim2 in in vitro and in vivo binding experiments. We
incubated lysate from COS-1 cells transfected with Flag-tagged,
full-length cAMP-GEFII with GST–Rim2. cAMP-GEFII was detected as a single band by immunoblotting with anti-Flag antibody
(Fig. 2a, left panel). Similarly, lysate from MIN6 cells, which express
endogenous cAMP-GEFII, was found to bind to GST–Rim2 by
immunoblotting with anti-cAMP-GEFII antibody (Fig. 2a, middle
panel). Homogenate from mouse brain, which expresses endogenous cAMP-GEFII, was also found to bind to GST–Rim1 (Fig. 2a,
right panel). We further confirmed that cAMP-GEFII and Rim2
interact in vivo by using COS-1 cells transfected with full-length
cAMP-GEFII and Rim2 (Fig. 2b). These results indicate that cAMPGEFII binds to both Rim1 and Rim2. We also investigated the
direct interaction of Rab3A, Rim, and cAMP-GEFII. The
Rab3A–Rim2–cAMP-GEFII complex was formed in vitro when
Rab3A was in the GTP-activated form (Fig. 2c). Because cAMPGEFII has been shown to have GEF activity towards Rap1 (ref. 24),
we investigated the possibility that cAMP-GEFII has also GEF
activity towards Rab3A. cAMP-GEFII did not stimulate the
Rim1
β-galactosidase activity
(Miller units)
b
500
Rab3A
Rab3A(Q81L)
400
300
c
200
100
Rim2
Rim1
GDP β S
+
+
GTP γ S
–
–
+
–
–
+
M r (K)
24
IB: anti-Rab3A
0
Rim2
Rim1 Rabphilin3
Figure 1 Comparison of the structures of Rim2 and Rim1, and the interaction between Rab3A and Rim2. a, Amino-acid identities of the zinc-finger,
PDZ and C2 domains of Rim2 and Rim1. b, Interaction of Rab3A with various
Rab3A-binding proteins, as determined by yeast two-hybrid assay. Interactions of
Rim2, Rim1 and rabphilin3 with wild-type Rab3A or constitutively active
Rab3A(Q81L) in various combinations were determined by transactivation of βgalactosidase activity in liquid culture. c, Interaction of Rab3A with Rim2 or Rim1
in vitro. GTPγS- and GDPβS-bound forms of Rab3A were incubated with
GST–Rim2 (residues 1–345) and GST–Rim1 (residues 1–204) immobilized on
glutathione beads, respectively. Rab3A was detected by immunoblotting (IB) with
anti-Rab3A antibody.
MIN6
m1
Ri
T–
GS
GS
T
T–
Ri
Ri
T
T–
GS
GS
Flag–
cAMP-GEFII
Flag–Rim2
+
+
Myc–cAMP-GEFII
Myc–luciferase
+
–
–
+
–
–
–
d
–
+
+
+
His6–Rim2
+
+
+
+
GDPβS–Rab3A
–
–
+
–
GTPγS–Rab3A
–
–
IB: anti-Rab3A
IB: anti-His
–
+
M r (K)
24
60,000
40,000
20,000
Rab3A
60,000
40,000
20,000
175
0
0
0
Figure 2 Interaction of cAMP-GEFII, Rim and Rab3A, and the GEF activity of
cAMP-GEFII. a, Lysate from COS-1 cells transfected with Flag-tagged cAMP-GEFII
(left panel), from MIN6 cells (middle panel), or from mouse-brain homogenate (right
panel) was evaluated for binding to GST–Rim2, GST–Rim1 or GST alone. cAMPGEFII was detected by immunoblotting with anti-Flag antibody (left panel) or
immunoglobulin G-purified antibody against the carboxy terminus (residues
1,001–1,011, QMSHRLEPRRP) of mouse cAMP-GEFII (middle and right panels). b,
Interaction of cAMP-GEFII and Rim2 in vivo. The interaction of cAMP-GEFII and Rim2
was evaluated by immunoprecipitation (IP) with anti-Flag antibody, followed by
immunoblotting of COS-1 cells transfected with Myc-tagged cAMP-GEFII and Flagtagged Rim2. As a negative control, COS-1 cells transfected with Myc-tagged
luciferase and Flag-tagged Rim2 were used. cAMP-GEFII (Mr 110K) and luciferase
806
60
Rap1B
[3H]GDP bound (d.p.m.)
c
[3H]GDP bound (d.p.m.)
IB: anti-Myc
GST
GST–cAMP-GEFII
+
M r (K)
110
IB: anti-cAMP-GEFII
cAMP-GEFII
cAMP-GEFII
IP: anti-Flag
b
Brain
m2
m2
COS-1
GS
a
T
Identity
GS
a
Rim2
5
10
15
cAMP-GEFII (pmol)
0
5
10
15
cAMP-GEFII (pmol)
(Mr 60K) were analysed by immunoblotting (IB) with specific antibody against cAMPGEFII and with anti-Myc antibody, respectively. c, Direct interaction of cAMP-GEFII,
Rim2 and Rab3A. The His6 –Rim2/GST–cAMP-GEFII complex was obtained by first
incubating His6–Rim2 (40 pmol) with GST–cAMP-GEFII (10 pmol), and then by incubation with glutathione beads for 2 h at 4 °C. After extensive washing, complexes
immobilized on beads were incubated with GDPβS– or GTPγS–Rab3A (50 pmol
each) for 2 h at 4 °C. Bound proteins were subjected to SDS–PAGE and then to
immunoblotting to detect Rab3A and His6–Rim2 using anti-Rab3A and anti-His antibodies, respectively. d, GEF activity of cAMP-GEFII towards Rap1B (left panel) and
Rab3A (right panel). The GEF activity of cAMP-GEFII was measured as described in
Methods. Data represent GEF activity in the absence (open circles) or presence
(filled circles) of 1 mM cAMP.
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*
*
150
Rim1
6.4
Rim2
7.2
5.4
100
50
0
– forskolin
+ forskolin
b
Growth-hormone secretion
(% of control)
Growth-hormone secretion
(% of control)
200
Size (kb)
4.0
3.5
cAMP-GEFII
– forskolin
+ forskolin
a
PC12
At-T20
Ovary
Pituitary
Adrenal
Pancreatic islets
MIN6
RINm5F
Stomach
Jejunum
Colon
Testis
Kidney
Skeletal muscle
a
Heart
Lung
Liver
Cerebrum
Cerebellum
articles
β-gal cAMP
-GEFII
150
100
50
β-gal cAMP
-GEFII
0
β-gal
WT
H-89
b
Cx
T810A G114E
G422D
cAMP-GEFII
c
– 8-Br-cAMP
+ 8-Br-cAMP
c
*
C
100
e
50
0
Figure 3 Comparison of the distributions of cAMP-GEFII, Rim1 and Rim2
mRNA. a, Northern-blot analyses of cAMP-GEFII, Rim1 and Rim2 in various rat tissues and endocrine- and neuroendocrine-derived cell lines. Lanes were loaded with
10 µg (5 µg for pancreatic islets) of total RNA from the indicated tissues and cell
lines. Hybridizations and washing were carried out under standard conditions. The
faint Rim2 signals detected in cerebrum and cerebellum are the result of crosshybridization with the Rim1 cDNA probe used. b–d, In situ hybridization of cAMPGEFII (b), Rim1 (c) and Rim2 (d) in mouse brain. Cb, cerebellum; Cp, caudoputamen; Cx, cortex; Hi, hippocampus; Ob, olfactory bulb; Po, pons; Th, thalamus. e,
In situ hybridization of cAMP-GEFII, Rim1 and Rim2 in mouse pituitary. Scale bars
represent 1 mm.
GDP/GTP exchange activity involving Rab3A under the same conditions in which it showed the GDP/GTP exchange activity towards
Rap1B (Fig. 2d).
Comparison of the expression patterns of cAMP-GEFII, Rim1,
and Rim2. Northern blotting (Fig. 3a) revealed that Rim2 messenger RNA is expressed predominantly in endocrine tissues and
endocrine- and neuroendocrine-derived cell lines, including pituitary, pancreatic islets, MIN6 cells and rat pheochromocytomaderived PC12 cells. Rim2 mRNA in brain was detected by polymerase chain reaction with reverse transcription (data not shown).
In contrast, Rim1 mRNA was found by northern blotting to be
expressed in cerebrum, cerebellum, and pituitary. In addition to the
principal transcripts (6.4 kilobases (kb) for Rim1; 7.2 and 5.4 kb for
Rim2), there were several minor transcripts present for both Rim1
and Rim2, probably as a result of alternative splicing (data not
shown). cAMP-GEFII mRNA was generally co-expressed with
Rim1 or Rim2 mRNA in the tissues and cell lines in which regulated exocytosis is known to occur. In situ hybridization of mouse
brain (Fig. 3b–d) revealed that Rim1 mRNA is expressed in cerebral
cortex, hippocampus (especially CA3 and dentate gyrus), olfactory
bulb, and cerebellar cortex (Fig. 3c), whereas Rim2 mRNA is
expressed only in cerebellar cortex (Fig. 3d). The distribution of
cAMP-GEFII mRNA largely overlapped that of Rim1 mRNA in
brain (Fig. 3b, c). Rim2 and cAMP-GEFII mRNAs were coexpressed in anterior pituitary (Fig. 3e). Immunoblot analysis of
A
None
Control Antisense
– 8-Br-cAMP
+ 8-Br-cAMP
d
**
300
Insulin secretion
(% of control)
Insulin secretion
(% of control)
150
d
*
NS
200
200
100
0
None
Control Antisense
Figure 4 Effect of cAMP-GEFII on Ca2+-dependent secretion. a, Effects of
forskolin and the PKA inhibitor H-89 on high-K+ (60 mM, 15 min)-induced secretion
of growth hormone from cAMP-GEFII-transfected PC12 cells. H-89 (10 µM) was
added to the incubation buffer 10 min before treatment with forskolin (50 µM).
Secretion of growth hormone was determined by the amount released into the
medium relative to the total cellular content41. High-K+-induced secretion of growth
hormone in the absence of forskolin in β-galactosidase (β-gal)-transfected PC12
cells was used as the respective control (100%) for the effect of forskolin (left) and
for the effect of forskolin plus H-89 (right). Values are percentages of growth-hormone secretion relative to the respective control. b, Effects of cAMP-GEFII mutants
on forskolin-induced secretion of growth hormone from PC12 cells. PC12 cells
transfected with β-gal, wild-type (WT) cAMP-GEFII, or the indicated cAMP-GEFII
mutants were treated with high K+ in the presence or absence of forskolin (50 µM).
High-K+-induced secretion of growth hormone in the absence of forskolin in β-galtransfected PC12 cells was used as control (100%). Values are percentages of
growth-hormone secretion relative to control. c, Insulin-secretory response to 8-BrcAMP in MIN6 cells. MIN6 cells were treated with control ODNs or antisense ODNs
against cAMP-GEFII. Values are percentages of insulin secretion in the absence of
8-Br-cAMP (100%). Inset, effect of antisense ODNs on levels of cAMP-GEFII. Cell
lysate incubated with GST–Rim2 immobilized on glutathione beads was subjected
to immunoblotting with anti-cAMP-GEFII antibody. C, control ODNs; A, antisense
ODNs. d, Insulin-secretory response to 8-Br-cAMP in mouse pancreatic islets, determined as in c. None, MIN6 cells or pancreatic islets without treatment; control,
MIN6 cells (c) or pancreatic islets (d) treated with control ODNs; antisense, MIN6
cells (c) or pancreatic islets (d) treated with antisense ODNs. Data in a–d were
obtained from 3–5 independent experiments (n = 6–21); values are means ±
s.e.m. * denotes P < 0.001; ** denotes P < 0.002; NS, not significant.
subcellular fractions of MIN6 cells showed that the majority of
cAMP-GEFII is present in cytosolic fractions (see Supplementary
Information).
cAMP-GEFII mediates cAMP-dependent, PKA-independent exocytosis. The interaction of cAMP-GEFII and Rim indicates that
cAMP-GEFII is involved in regulated exocytosis. To determine its
functional significance, we examined the effect of cAMP on Ca2+dependent secretion in PC12 cells transfected with growth hormone and cAMP-GEFII. Because PC12 cells endogenously express
Rim2 but not cAMP-GEFII, we reasoned that exogenously introduced cAMP-GEFII might form a complex with endogenous Rim2.
NATURE CELL BIOLOGY VOL 2 NOVEMBER 2000 http://cellbio.nature.com
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807
articles
+
+
+
∆A
)
–
+
2(
+
cAMP-GEFII
im
Rim2(∆A)
PC12
b
IB: anti-HA
HA–Rim2
T
3
+
T–
R
2
+
GS
1
–
GS
a
Rim2
cAMP-GEFII
anti-HA
HA–Rim2(∆A)
anti-Myc
Myc–cAMP-GEFII
– 8-Br-cAMP
+ 8-Br-cAMP
Growth-hormone secretion
(% of control)
*
150
IP: anti-Myc
HA–Rim2
IB: anti-HA
HA–Rim2(∆A)
MIN6
GS
T
GS
T–
Ri
m2
(
∆A
)
c
100
50
0
cAMP-GEFII
Rim2(∆A)
––
+ +
+ +
––
+ +
+ +
cAMP-GEFII
C-peptide secretion
(% of control)
8-Br-cAMP(–)
8-Br-cAMP(+)
150
*
100
50
0
Luciferase Rim2(∆A)
Figure 5 Effects of Rim2(∆A) on the interaction of cAMP-GEFII and fulllength Rim2 and on cAMP-GEFII-mediated exocytosis. a, Effect of Rim2(∆A)
on the interaction of cAMP-GEFII and full-length Rim2 in COS-1 cells. Upper three
panels, immunoblot (IB) analysis. Lysates from COS-1 cells transfected with the
expression vectors for HA-tagged full-length Rim2, HA-tagged Rim2(∆A) and Myctagged cAMP-GEFII were subjected to SDS–PAGE. Immunoblotting was carried out
using anti-HA or anti-Myc antibodies. There was no difference in expression of HAtagged full-length Rim2 (lanes 2 and 3), HA-tagged Rim2(∆A) (lanes 1 and 3), and
Myc-tagged cAMP-GEFII (lanes 1, 2 and 3) in transfected COS-1 cells. Lower two
panels, immunoprecipitation of Rim2 and Rim2(∆A). Lysates from transfected COS1 cells were incubated with anti-Myc antibody, and then incubated with protein
G–sepharose. Washed complexes were subjected to SDS–PAGE. Full-length Rim2
and Rim2(∆A) were detected by immunoblotting with anti-HA antibody. Rim2(∆A)
binds to cAMP-GEFII, as does full-length Rim2. b, Interaction of Rim2(∆A) with
cAMP-GEFII expressed in PC12 cells (upper panel) and the effect of Rim2(∆A) on 8Br-cAMP-induced secretion of growth hormone (lower panel). Upper panel, lysate
from PC12 cells transfected with full-length cAMP-GEFII was incubated with
GST–Rim2(∆A) immobilized on glutathione beads. cAMP-GEFII was detected by
immunoblotting with a specific antibody against cAMP-GEFII. Lower panel, PC12
cells transfected with growth hormone, cAMP-GEFII and Rim2(∆A) were treated with
high K+ (60 mM, 15 min) in the presence or absence of 1 mM 8-Br-cAMP. High-K+induced secretion of growth hormone in the absence of 8-Br-cAMP in Rim2(∆A)transfected PC12 cells was used as control (100%). Values are percentages of
growth-hormone secretion relative to control. c, Interaction of Rim2(∆A) with cAMPGEFII expressed in MIN6 cells (upper panel) and the effect of Rim2(∆A) on 8-BrcAMP-induced secretion of C-peptide (lower panel). Upper panel, lysate from MIN6
cells was analysed as in b. Lower panel, human C-peptide secretory response to 8Br-cAMP in MIN6 cells transfected with human proinsulin together with luciferase or
Rim2(∆A). There was no difference in C-peptide secretion in the absence of 8-BrcAMP between luciferase-transfected and Rim2(∆A)-transfected MIN6 cells. Values
are percentages of C-peptide secretion in the absence of 8-Br-cAMP (100%). Data
in b and c were obtained from 3–5 independent experiments (n = 9–22); values
are means ± s.e.m. *denotes P < 0.001.
808
Ca2+-dependent (60 mM K+) secretion of growth hormone in the
absence of forskolin was not different in β-galactosidase (β-gal)transfected (control) and cAMP-GEFII-transfected PC12 cells, but
secretion in the presence of forskolin (50 µM) was significantly
higher in cAMP-GEFII-transfected cells than in those transfected
with β-gal (Fig. 4a, left). Ca2+-dependent growth-hormone secretion induced by 8-bromoadenosine 3′, 5′ cyclic monophosphate
(8-Br-cAMP, 1 mM) was also enhanced by cAMP-GEFII (cAMPGEFII-transfected, 139.2 ± 4.8%; control, 100.0 ± 6.8%, n = 9,
P < 0.005). These results indicate that cAMP-GEFII mediates Ca2+dependent, cAMP-induced exocytosis. We then examined the effect
of the PKA inhibitor H-89 (10 µM) on forskolin-induced secretion
of growth hormone. Of the forskolin-induced secretion in cAMPGEFII-transfected PC12 cells, 42% was not blocked by H-89
(Fig. 4a, right). As forskolin-induced secretion in β-gal-transfected
PC12 cells was completely abolished by H-89, the secretion that was
not blocked by H-89 is probably mediated by cAMP-GEFII. To
determine further the functional role of cAMP-GEFII in cAMPdependent exocytosis, we prepared two mutants. Forskolininduced secretion of growth hormone was not altered in the
mutant cAMP-GEFII(T810A), in which the potential PKA phosphorylation site is disrupted (Fig. 4b), indicating that cAMP promotes Ca2+-dependent secretion through cAMP-GEFII without
involving its phosphorylation by PKA. Forskolin-induced secretion
of growth hormone in the mutant cAMP-GEFII(G114E, G422D),
in which both cAMP-binding sites are disrupted, was reduced to
the level of secretion observed in β-gal-transfected PC12 cells (Fig.
4b), indicating that binding of cAMP to cAMP-GEFII is required
for cAMP-GEFII-mediated secretion. Together, these results indicate that cAMP-GEFII is responsible for cAMP-dependent, PKAindependent exocytosis. To clarify the physiological relevance of
cAMP-GEFII, we investigated the function of endogenous cAMPGEFII in secretion. We used antisense oligodeoxynucleotides
(ODNs) against cAMP-GEFII in mouse insulin-secreting MIN6
cells and mouse pancreatic islets for this purpose because cAMP is
known to potentiate glucose-induced exocytosis of insulin granules
from pancreatic β-cells15,16. Levels of cAMP-GEFII were significantly reduced in MIN6 cells treated with antisense ODNs, relative to
controls (Fig. 4c, inset), and 8-Br-cAMP-induced insulin secretion
from MIN6 cells in the presence of high glucose (16.7 mM) was
markedly inhibited by treatment with the antisense ODNs (Fig. 4c).
Moreover, 8-Br-cAMP-induced insulin secretion from normal pancreatic islets was reduced to 53% of that in controls by the antisense
ODNs treatment (Fig. 4d). These results indicate that cAMP-GEFII
is a direct target of cAMP in regulated exocytosis in native secretory cells.
The effects of cAMP-GEFII on exocytosis are mediated by Rim2.
To determine whether the effects of cAMP-GEFII on exocytosis are
mediated by Rim2, we prepared the deletion mutant Rim2(∆A),
which lacks the zinc-finger and C2 domains but retains the cAMPGEFII-binding region. Overexpression of Rim2(∆A) inhibited
binding of full-length Rim2 to cAMP-GEFII in transfected COS-1
cells (Fig. 5a). In addition, Rim2(∆A) was shown to bind to exogenous and endogenous cAMP-GEFII in PC12 cells and MIN6 cells,
respectively (Fig. 5b, c). These results indicate that Rim2(∆A) has a
dominant negative effect on the interaction between cAMP-GEFII
and Rim2 in these cells. We then transfected PC12 cells with growth
hormone, cAMP-GEFII and Rim2(∆A), and examined the effect of
Rim2(∆A) on cAMP-GEFII-mediated secretion of growth hormone. We reasoned that if the interaction of cAMP-GEFII and
Rim2 is necessary for cAMP-GEFII-mediated exocytosis, overexpression of Rim2(∆A) should lead to inhibition of cAMP-GEFIImediated exocytosis. As expected, overexpression of Rim2(∆A) in
PC12 cells transfected with cAMP-GEFII inhibited 8-Br-cAMPinduced, Ca2+-dependent secretion of growth hormone (Fig. 5b).
To confirm further that the effect of cAMP-GEFII is mediated by
Rim2, we examined the effect of Rim2(∆A) on 8-Br-cAMP-induced
exocytosis from MIN6 cells expressing endogenous cAMP-GEFII
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articles
a
b
M r (K)
175
HA–Rim2
– 8-Br-cAMP
+ 8-Br-cAMP
C-peptide secretion
(% of control)
100
50
+ +
+ +
ti-M
100
50
0
0
+ +
– –
an
150
150
C-peptide secretion
(% of control)
Growth-hormone
secretion (% of control)
0
cAMP-GEFII
Rim2(∆B)
33
– 8-Br-cAMP
+ 8-Br-cAMP
– 8-Br-cAMP
+ 8-Br-cAMP
150
50
M r (K)
175
HA–syntenin
105
100
IP
:
an
Ly
sa
te
ti-M
yc
HA–Rim2(∆B)
IP
:
Ly
sa
te
HA–Rim2
Luciferase Rim2(∆B)
Figure 6 Effects of Rim2(∆B) and syntenin on cAMP-GEFII-mediated exocytosis. a, Interaction of cAMP-GEFII and Rim2(∆B) (upper panel) and the effects of
Rim2(∆B) on 8-Br-cAMP-induced secretion of growth hormone (lower-left panel) and
C-peptide (lower-right panel). Upper panel, lysate from COS-1 cells transfected with
Myc-tagged cAMP-GEFII together with HA-tagged full-length Rim2 or HA-tagged
Rim2(∆B) was subjected to SDS–PAGE, and then to immunoblotting (IB) with anti-HA
antibody (left lane). Lysate was also subjected to immunoprecipitation with anti-Myc
antibody, and then to immunoblotting with anti-HA antibody (right lane). Rim2(∆B)
did not bind to cAMP-GEFII. Lower-left panel, 8-Br-cAMP-induced secretion of growth
hormone from PC12 cells transfected with growth hormone, cAMP-GEFII and
Rim2(∆B). Rim2(∆B) did not affect 8-Br-cAMP-induced secretion of growth hormone
in PC12 cells. PC12 cells were treated with high K+ (60 mM, 15 min) in the presence or absence of 1 mM 8-Br-cAMP. High-K+-induced secretion of growth hormone
in the absence of 8-Br-cAMP in cAMP-GEFII-transfected PC12 cells was used as con-
Luciferase Syntenin
trol (100%). Values are percentages of growth-hormone secretion relative to control. Experimental conditions were as described in Fig. 5b. Lower-right panel,
human C-peptide secretory response to 8-Br-cAMP in MIN6 cells transfected with
human proinsulin together with luciferase or Rim2(∆B). Rim2(∆B) did not affect 8-BrcAMP-induced secretion of C-peptide in MIN6 cells. Experimental conditions were as
described in Fig. 5c. b, Interaction of cAMP-GEFII and syntenin (upper panel) and
the effect of syntenin on 8-Br-cAMP-induced secretion of C-peptide (lower panel).
Upper panel, similarly to Rim2(∆B) described in a, syntenin did not bind to cAMPGEFII. Lower panel, human C-peptide secretory response to 8-Br-cAMP in MIN6 cells
transfected with human proinsulin together with luciferase or syntenin. Syntenin did
not affect 8-Br-cAMP-induced secretion of C-peptide in MIN6 cells. Experimental
conditions were as described in Fig. 5c. Data in a and b were obtained from 2–3
independent experiments (n = 6–10); values are means ± s.e.m.
human insulin crossreact with endogenous mouse insulin, whereas
antibodies against human C-peptide do not crossreact with
endogenous mouse C-peptide, we monitored secretion by measuring the release of human C-peptide from MIN6 cells transfected
with human proinsulin and Rim2(∆A). Overexpression of
Rim2(∆A) in MIN6 cells significantly suppressed 8-Br-cAMPinduced secretion of C-peptide in the presence of 16.7 mM glucose
(Fig. 5c). To confirm the specificity of the dominant negative effect
of Rim2(∆A), we used a deletion mutant, Rim2(∆B), lacking the
cAMP-GEFII-binding region and another PDZ-domain-containing protein, syntenin30, for the binding and secretion experiments.
Neither Rim2(∆B) nor syntenin bound to cAMP-GEFII (Fig. 6a, b).
Overexpression of Rim2(∆B) in PC12 cells and MIN6 cells did not
inhibit 8-Br-cAMP-induced secretion of growth hormone or of Cpeptide, respectively (Fig. 6a). In addition, overexpression of syntenin in MIN6 cells did not inhibit 8-Br-cAMP-induced secretion
of C-peptide (Fig. 6b). These results indicate that the effects of
cAMP-GEFII on cAMP-induced, Ca2+-dependent exocytosis are
specifically mediated by Rim2.
cAMP
PKA-dependent
mechanism
(phosphorylation)
IB: anti-HA
yc
IB: anti-HA
PKA-independent
mechanism
(Rim–cAMP-GEFII)
Exocytosis
Discussion
Figure 7 Model of cAMP-dependent exocytosis. cAMP regulates exocytosis by
PKA-dependent and PKA-independent mechanisms, the former being mediated by
PKA phosphorylation of regulatory proteins that are associated with secretory or
synaptic vesicles and the latter by the interaction of cAMP-GEFII and Rim.
and Rim2. Proinsulin is converted into insulin and C-peptide during the secretory process in pancreatic β-cells. As antibodies against
During our search for an intracellular signalling molecule that couples to the sulphonylurea receptor SUR1, we found that the cAMPbinding protein cAMP-GEFII24 interacts with SUR1. Whether
cAMP-GEFII modulates the function of SUR1 or the activity of
KATP channels is not known at present. cAMP-A in cAMP-GEFII
binds to cAMP with much lower affinity, compared with the PKA
regulatory subunit type-I (PKARIα), but cAMP-B does not bind to
cAMP under the same conditions. cAMP-B contains a Glu/Lys substitution at position 423, a residue that is important for cAMP
binding29. PKARIα harbouring an equivalent mutation (E200K)
NATURE CELL BIOLOGY VOL 2 NOVEMBER 2000 http://cellbio.nature.com
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articles
dissociates from cAMP more rapidly than does the wild type31; similarly, cAMP-B may also rapidly dissociate from cAMP, so that
binding of cAMP to cAMP-B remains possible.
Because the identification of a target of cAMP-GEFII would
indicate its physiological function, we attempted to find a molecule
that interacts with cAMP-GEFII. Interestingly, cAMP-GEFII was
found to bind both to Rim1 and to a new isoform, Rim2. As Rim1
has been proposed to be a Rab3-dependent regulator of fusion of
synaptic vesicles to the plasma membrane25, the observed interaction of cAMP-GEFII with Rim1 or Rim2 indicates that cAMPGEFII may participate in regulated exocytosis. Although cAMP is
well known to regulate secretion of neurotransmitters, hormones
and enzymes7,15,16,20,32,33, the direct target of cAMP in the exocytotic
machinery was not previously known. We have shown that exogenously introduced cAMP-GEFII into PC12 cells co-transfected
with growth hormone mediates forskolin-induced and 8-BrcAMP-induced growth-hormone secretion. In addition, cAMPGEFII also mediates forskolin-induced secretion of growth hormone in PC12 cells; this secretion is not blocked by the PKA
inhibitor H-89. Furthermore, treatment with antisense ODNs
against cAMP-GEFII in MIN6 cells and pancreatic islets, which
express endogenous cAMP-GEFII, markedly inhibited 8-Br-cAMPinduced insulin secretion. Finally, overexpression of the mutant
Rim2(∆A), which lacks the zinc-finger and C2 domains but binds to
cAMP-GEFII, significantly inhibited cAMP-GEFII-mediated exocytosis, whereas the mutant Rim2(∆B), which does not bind to
cAMP-GEFII, did not. Together, these findings indicate that cAMPGEFII is a direct target of cAMP in regulated exocytosis and that
cAMP-GEFII, through its interaction with Rim, mediates cAMPdependent, PKA-independent exocytosis.
As well as its role in PKA phosphorylation of proteins that are
associated with secretory processes7,16,32,33, cAMP has been proposed
to act directly on the exocytotic machinery 20,22,35. For example, in
insulin-secreting pancreatic β-cells, cAMP has been proposed to
have a dual stimulatory effect on exocytosis — a rapid, PKA-independent phase and a late, PKA-dependent phase23. In fact, we found
that 8-Br-cAMP-induced insulin secretion from pancreatic islets
treated with antisense ODNs against cAMP-GEFII was reduced to
~53% of that in controls (Fig. 4d), indicating the physiological significance of cAMP-GEFII in cAMP-dependent, PKA-independent
insulin secretion in normal pancreatic β-cells. In acinar cells, cAMP
may directly stimulate amylase release21. In the CA3 region of the
brain hippocampus, cAMP induces LTP by enhancing Ca2+dependent exocytosis of synaptic vesicles7,33,35. Although cAMP has
been thought to induce LTP by activation of PKA in mossy-fibre
synapses of the CA3 region, a recent study has indicated that cAMP
has multiple actions, of which only the direct activation of the exocytotic machinery requires Rab3A22, a vesicle protein that is essential for mossy-fibre LTP36. Rab3A acts through the structurally
related proteins rabphilin3 (ref. 37) and Rim1 (ref. 25). However,
although Rab3A and its effectors, rabphilin3 and Rim1, are ubiquitously expressed in most synapses in the brain25,38, cAMP-enhanced
glutamate release occurs in synaptosomes of the CA3 region but
not the CA1 region22, a finding that is consistent with the idea that
cAMP-GEFII and Rim1 are predominantly co-expressed in the
CA3 region. Interestingly, SUR1 mRNA has recently been reported
to be expressed at moderate to high levels in the CA2 and CA3
regions of the hippocampus and in the cerebellar cortex39, regions
in which cAMP-GEFII mRNA is also expressed. In pancreatic βcells, as well as inhibiting the KATP channel by binding to SUR1 at
the plasma membrane, sulphonylureas have been proposed to
stimulate exocytosis of insulin granules by acting directly on
sulphonylurea receptors, which constitute a functional part of the
regulatory exocytotic protein40. Together, these findings indicate
that SUR1, through its interaction with cAMP-GEFII or with a
complex of Rim–cAMP-GEFII, may mediate a signal that leads to
exocytosis in some neurons in the brain as well as in some
endocrine cells.
810
We have shown that cAMP regulates exocytosis by PKA-independent, as well as PKA-dependent, mechanisms and that cAMPGEFII, a cAMP sensor, through its interaction with Rim, a putative
Rab3 effector, is responsible for the PKA-independent mechanism
(Fig. 7). We have found that a Rab3A–Rim2–cAMP-GEFII complex
can be formed in vitro when Rab3A is in the GTP-activated form,
but it is not known whether such a three-molecule complex can be
formed in vivo. The Rab3–Rim1 complex has been proposed to
serve as a clamp for Ca2+-dependent exocytosis25, and GTP hydrolysis or protein phosphorylation would inactivate this clamp25. As
cAMP-GEFII does not exhibit GDP/GTP exchange activity towards
Rab3A, it is unlikely that the GEF activity of cAMP-GEFII modulates the Rab3A–Rim complex. Whether and how cAMP and Ca2+
might modulate the Rim–cAMP-GEFII complex or the
Rab3A–Rim–cAMP-GEFII complex upon stimulation in vitro and
in vivo requires further investigation.
Methods
Yeast two-hybrid screening and isolation of full-length cDNAs.
A plasmid cDNA library in the vector pVP16 was made from the mouse insulin-secreting cell line
MIN6. Yeast two-hybrid bait vectors were constructed in pBTM116 using a DNA fragment encoding
partial rat SUR1 (amino-acid residues 598–1003; GenBank accession no. L40624) and a full-length
mouse cAMP-GEFII cDNA. Yeast two-hybrid screening of the plasmid MIN6 cDNA library was carried out as described41. Prey clones encoding a partial cAMP-GEFII (residues 187–730) and a partial
Rim2 (residues 53–863) were isolated. Their full-length cDNAs were obtained from the MIN6 cDNA
library42.
GST pull-down assay and co-immunoprecipitation.
Full-length cAMP-GEFII, Rim1 (residues 530–806), Rim2 (residues 538–863), and Rim2(∆A) (residues
198–830) were expressed as GST-fusion proteins and purified according to the manufacturer’s instructions (Amersham). Full-length Rim2, expressed in Sf9 cells as a 6 × histidine (His6)-fusion protein, was
purified according to the manufacturer’s instructions (Qiagen). Lipid-modified Rab3A was purified
from the membrane fraction of Sf9 cells overexpressing Rab3A. The full-length cAMP-GEFII cDNA
was subcloned into pFLAG-CMV-2 (Sigma) or pCMV-Tag3 (Stratagene). Full-length Rim2 cDNA was
subcloned into pFLAG-CMV-2 or the modified pCMV containing haemagglutinin (HA) epitope
(pCMV-HA). Rim2(∆A), Rim2(∆B) (residues 1–200 and 831–1,590) and full-length syntenin cDNAs
were also subcloned into pCMV-HA. For the GST pull-down assay, cell lysates were incubated with
GST-fusion proteins (Rim1, Rim2, or Rim2(∆A)) immobilized on glutathione beads. The
His6–Rim2/GST–cAMP-GEFII complex immobilized on glutathione beads was incubated with
GDPβS- or GTPγS-Rab3A; immunoblotting was then carried out using anti-His (Qiagen) and antiRab3A (Transduction Laboratories) antibodies. For co-immunoprecipitation, cell lysates were incubated with anti-FLAG M2 (Sigma), anti-Myc (Santa Cruz Biotechnology) or anti-HA (Roche) antibodies,
and then incubated with protein G–sepharose. Proteins were analysed by immunoblotting.
cAMP-binding assay.
cAMP-A (residues 43–153), cAMP-B (residues 357–469) and full-length rat PKARIα were expressed as
GST-fusion proteins and purified. The cAMP-binding assay was carried out as described 31, with slight
modifications. Briefly, GST-fusion protein (1 µg) was incubated in binding buffer (200 µl) containing
various concentrations of [3H]cAMP, 50 mM potassium phosphate pH 6.8, 150 mM NaCl, 1 mM
EDTA, 5 mM 2-mercaptoethanol and 0.5 mg ml–1 BSA with or without 40 mM unlabelled cAMP for 2
h on ice.
Assay for binding of Rab3A to Rim.
Full-length cDNAs for wild-type mouse Rab3A and constitutively active bovine Rab3A(Q81L) were
subcloned into the yeast bait vector pBTM116. The zinc-finger regions of bovine rabphilin3 (residues
1–283), rat Rim1 (residues 1–204) and mouse Rim2 (residues 1–345) were subcloned into the prey
vector pVP16. β-galactosidase activity was assayed in liquid culture according to the manufacturer’s
instructions (Clontech). Data were obtained from three independent clones from each transformation
and normalized by cell numbers determined by absorbance at 600 nm. Rat Rim1 (residues 1–204) and
mouse Rim2 (residues 1–345) were expressed as GST fusions and purified. The GTPγS- or GDPβSbound form of lipid-modified Rab3A was incubated for 90 min at 4 °C with GST–Rim1 or GST–Rim2
(30 pmol each) immobilized on glutathione beads in reaction buffer. Rab3A was detected by
immunoblotting with anti-Rab3A antibody.
Measurement of GEF activity of cAMP-GEFII towards small G proteins.
The GEF activity of cAMP-GEFII was estimated by measuring the dissociation of [3H]GDP from
[3H]GDP–Rab3A or [3H]GDP–Rap1B. Full-length cAMP-GEFII expressed as a His6-fusion protein in
Escherichia coli was purified with Ni–NTA sepharose (Qiagen). Lipid-modified Rap1B was purified in a
similar way to Rab3A. The [3H]GDP-bound form of Rab3A and Rap1B (2 pmol each) were separately
incubated with His6–cAMP-GEFII (8 pmol) and 60 µM GTPγS in the presence or absence of 1 mM
cAMP for 20 min at 30 °C. After incubation, dissociation of [3H]GDP from Rab3A or Rap1B was
assayed by measuring the radioactivity of [3H]GDP bound to each small G protein, using the nitro
cellulose-filtration method.
Northern blotting and in situ hybridization.
Northern blotting was carried out under standard stringent hybridization conditions. The probes used
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were mouse cAMP-GEFII (bases 606–2,237), rat Rim1 (1,035–1,491) and mouse Rim2 (586–1,490)
cDNAs. In situ hybridization was carried out as described43. The antisense oligonucleotide probes
(45mers) used for mouse cAMP-GEFII and Rim2 were the regions corresponding to bases 2,746–2,790
and 1,376–1,420, respectively. For the antisense oligonucleotide probe for Rim1, Rim1 cDNA was partially cloned from mouse brain; the probe used was 5′-TTGCGCTCACTCTTCTGGCCTCCCTTGCCATTCTGCTCTGAAAGC.
Measurements of growth-hormone and insulin secretion.
Secretion of growth hormone from transfected PC12 cells was measured as described41. Expression
vectors pSRα for wild-type cAMP-GEFII, cAMP-GEFII(T810A), cAMP-GEFII(G114E, G422D),
Rim2(∆A) and Rim2(∆B) were prepared; β-galactosidase was used as a control. PC12 cells were transfected with growth-hormone-expression vector pXGH5 (Nichols Institute, San Juan, Capistrano,
California) plus each vector described above, using LipofectAMINE (Life Technologies). PC12 cells
were incubated with a low-K+ (4.7 mM) or high-K+ (60 mM) solution41, in the presence or absence of
forskolin (50 µM) or 8-Br-cAMP (1 mM). Forskolin or 8-Br-cAMP was added 10 min before incubation with low- or high-K+ solution. In some experiments, H-89 (10 µM) was added 10 min before
forskolin treatment. Insulin secretion was measured in MIN6 cells and isolated mouse pancreatic islets
as described42. Both were treated for 96 h with 4 µM of antisense phosphorothioate-substituted ODNs
(16mers) against mouse cAMP-GEFII (the region corresponding to bases 104–119) or control ODNs
(5′-ACCTACGTGACTACGT; Biognostik, Göttingen, Germany). The insulin-secretory response to 8Br-cAMP (1 mM) in the presence of high glucose concentration (16.7 mM) for 60 min was assessed.
Islets were isolated by the collagenase-digestion method.
Measurement of C-peptide secretion.
MIN6 cells were transfected with human proinsulin expression vector pCMV-hproinsulin, plus
pCMV-Tag3–luciferase, pCMV-HA–Rim2(∆A), pCMV-HA–Rim2(∆B) or pCMV-HA–syntenin.
Luciferase was used as a control. Three days after transfection, the C-peptide-secretory response to 8Br-cAMP (1 mM) in the presence of glucose (16.7 mM) for 60 min was assessed, using a human Cpeptide radioimmunoassay kit (Linco Research, St Charles, Missouri).
RECEIVED 6 DECEMBER 1999; REVISED 5 MAY 2000; ACCEPTED 30 JUNE 2000;
PUBLISHED 10 OCTOBER 2000.
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ACKNOWLEDGEMENTS
We thank T. Sasaki for helpful advice and K. Yamaguchi for technical assistance. We also thank M.
Takahashi for critical reading of the manuscript. This work was supported by a Grant-in-Aid for
Creative Basic Research (10NP0201) from the Ministry of Education, Science, Sports and Culture; by
Scientific Research Grants from the Ministry of Health and Welfare, Japan; by the Uehara Memorial
Foundation; by a grant from Novo Nordisk Pharma Ltd.; by a grant for studies on the pathophysiology
and complications of diabetes from Tsumura Pharma Ltd.; and by the Yamanouchi Foundation for
Research on Metabolic Disorders. T.S. is supported by Research Fellowships of the Japan Society for
the Promotion of Science for Young Scientists.
Correspondence and requests for materials should be addressed to S.S. The nucleotide sequences of
mouse CAMPS (cAMP-GEFII) and mouse Rim2 have been deposited at GenBank under accession
numbers AB021132 and AB021131, respectively. Supplementary Information is available on Nature
Cell Biology’s website (http://cellbio.nature.com) or as paper copy from the London editorial office of
Nature Cell Biology.
NATURE CELL BIOLOGY VOL 2 NOVEMBER 2000 http://cellbio.nature.com
© 2000 Macmillan Magazines Ltd
811
supplementary information
Splice variant A
Rim2
Rim1
92
96
MSAPLGPRGRPAPTPAASQPPPQPEMPDLSHLTEEERKIILAVMDRQKKEEEKEQSVLKIKEEHKAQPTQ-WFPFSGITELVNNVLQ-------PQQKQP
MSSAVGPRGPRPPTV----PPPMQELPDLSHLTEEERNIIMAVMDRQKEEEEKEEAMLKCVVRDMAKPAACKTPRNAESQPHQPPLNIFRCVCVPRKPSS
Zinc finger
Rim2
Rim1
NEKEPQT--KLHQQFEMYKEQVKKMGEESQQ-QQEQKGDAPTCGICHKTKFADGCGHNCSYCQTKFCARCGGRVSLRSNK----VMWVCNLCRKQQEILT
EEGGPERDWRLHQQFESYKEQVRKIGEEARRYQGEHKDDAPTCGICHKTKFADGCGHLCSYCRTKFCARCGGRVSLRSNNEDKVVMWVCNLCRKQQEILT
185
196
Rim2
Rim1
KSGAWFYNSGSNTLQQPDQKVPRGLRNE----EAPQEKKAKLHEQPQFQGAPGDLSVPAVEKGRAHG-LTRQ---DTIKNGSGVKHQIAS----DMPSDR
KSGAWFFGSGP---QQPSQDGTLSDTATGAGSEVPREKKARLQERSRSQTPLSTAAVSSQDTATPGAPLHRNKGAEPSQQALGPEQKQASRSRSEPPRER
273
293
Rim2
Rim1
KRSPSVSRDQNRRYEQSEEREDYSQYVPSDGTMPRSPSDYADRRSQREPQFYEEPGHLNYRDSNRRGHRHSKEYIVDDEDVESRDEYERQRREEEYQARY
KKAPGLS-EQNGKGGQKSERKRVPKSVVQPGE---GIADERERKERRETRRLEKGRSQDYSD---RPEKRDNGRVAEDQ---------KQRKEEEYQTRY
373
377
Rim2
Rim1
RSDPNLARYPVKPQPYEEQMRIHAEVSRARHERRHSDVSLANAELEDSRISLLRMDRPSRQRSVSERRAAMENQRSYSMERTREAQGQSSYPQRTSNHSP
RSDPNLARYPVKAPPEEQQMRMHARVSRARHERRHSDVALPHTEAAAA----------APAEATAGKRAPATAR----VSPPESPRARAAAAQPPTEHGP
473
463
Rim2
Rim1
PTPRRSPIPLDRPDMRRADSLRKQHHLDPSSAV--RKTKREKMETMLRNDSLSSDQSESVRPPPPRPHKSKKGGKMRQVSLSSSEEELASTPEYTSCDDV
PPPRPAPGPAEPPEPRVPEPLRKQGRLDPGSAVLLRKAKREKAESMLRNDSLSSDQSESVRPSPPKPHRPKRGGKRRQMSVSSSEEEGVSTPEYTSCEDV
571
563
Rim2
Rim1
ELESESVSEKGDSQKGKRKTSEQGVLSDSNTRSERQKKRMYYGGHSLEEDLEWSEPQIKDSGVDTCSSTTLNEEHSHSDKHPVTWQPSKDGDRLIGRILL
ELESESVSEKGDLD--------------------------YY----------WLDPATWHS-----------RETSPISSHPVTWQPSKEGDRLIGRVIL
671
616
Rim2
Rim1
NKRLKDGSVPRDSGAMLGLKVVGGKMTESGRLCAFITKVKKGSLADTVGHLRPGDEVLEWNGRLLQGATFEEVYNIILESKPEPQVELVVSRPIGDIPRI
NKR---TTMPKESGALLGLKVVGGKMTDLGRLGAFITKVKKGSLADVVGHLRAGDEVLEWNGKPLPGATNEEVYNIILESKSEPQVEIIVSRPIGDIPRI
771
713
Rim2
Rim1
PDSTHAQLESSSSSFESQKMDRPSISVTSPMSPGMLRDVPQFLSGQLSIKLWFDKVGHQLIVTILGAKDLPSREDGRPRNPYVKIYFLPDRSDKNKRRTK
PESSHPPLESSSSSFESQKMERPSISVISPTSPGALKDAPQVLPGQLSVKLWYDKVGHQLIVNVLQATDLPPRVDGRPRNPYVKMYFLPDRSDKSKRRTK
871
813
Rim2
Rim1
TVKKTLEPKWNQTFIYSPVHRREFRERMLEITLWDQARVREEESEFLGEILIELETALLDDEPHWYKLQTHDVSSLPLPRPSPYLPRRQLHGESPTRRLQ
TVKKLLEPKWNQTFVYSHVHRRDFRERMLEITVWDQPRVQDEESEFLGEILIELETALLDDEPHWYKLQTHDESSLPLPQPSPFMPRRHIHGESSSKKLQ
Rim2
Rim1
RSKRISDSEVSDYDCEDGVGVVS--DYRHNGRDLQSSTLSVPEQVMSSNHCSPSGSPHRVDVIGRTRSWSPSAPPPQRNVEQGH--RGTRATGHYNTISR 1,067
RSQRISDSDISDYEVDDGIGVVPPVGYRASARESKATTLTVPEQQRTTHHRSRSVSPHRGDDQGRPRSRLPNVPL-QRSLDEIHPTRRSRSPTRHHDASR 1,012
Rim2
Rim1
M---DRHRVMDDHYSSDRDRDCEAADRQPYHRS-----RSTEQRPLLERTTTRSRSSERPDTNLM---RSMPSLMTGRSA-------PPSPALSRSHPRT 1,149
SPADHRSRHVESQYSSEPDSELLMLPRAKRGRSAESLHMTSELQPSLDRARSASTNCLRPDTSLHSPERERHSRKSERCSIQKQSRKGTASDADRTHRQG 1,112
Rim2
Rim1
GSVQTSPSSTPGTGRRGRQLPQLPPK-GTLERSAMDIEERNRQMKL--NKYKQVAGSDPR--LEQDYHSKYRSGWDPHRGADTVSTKSSDSDVSDVSAVS
SPTQSPPADTSFGSRRGRQLPQVPVRSGSIEQASLVVEERTRQMKVKVHRFKQTTGSGSSQELDHEQYSKYNIHKDQYRSCDNASAKSSDSDVSDVSAIS
Rim2
Rim1
RTSSASRFSSTSYMSVQSERPRGNRKISVFTSKMQNRQMGVSGKNLTKSTSISGDMCSLEKNDGSQSDTAVGALGTSGKKRRSSIGAKMVAIVGLSRKSR 1,344
RASSTSRLSSTSFMSEQSERPRG--RISSFTPKMQGRRMGTSGRAIIKSTSVSGEIYTLERNDGSQSDTAVGTVGAGGKKRRSSLSAKVVAIV--SRRSR 1,308
Rim2
Rim1
SASQLSQTEGGGKKLRSTVQRSTETGLAVEMRNWMTRQASRESTDGSMNSYSSEGNLIFPGVRLASDSQFSDFLDGLGPAQLVGRQTLATPAMGDIQVGM 1,444
STSQLSQTESGHKKLKSTIQRSTETGMAAEMRK-MVRQPSRESTDGSINSYSSEGNLIFPGVRVGPDSQFSDFLDGLGPAQLVGRQTLATPAMGDIQIGM 1,407
Rim2
Rim1
MDKKGQLEVEIIRARGLVVKPGSKTLPAPYVKVYLLDNGVCIAKKKTKVARKTLEPLYQQLLSFEESPQGRVLQIIVWGDYGRMDHKSFMGVAQILLDEL 1,544
EDKKGQLEVEVIRARSLTQKPGSKSTPAPYVKVYLLENGACIAKKKTRIARKTLDPLYQQSLVFDESPQGKVLQVIVWGDYGRMDHKCFMGVAQILLEEL 1,507
Rim2
Rim1
ELSNMVIGWFKLFPPSSLVDPTSAPLTRRASQSSLESSTGPSYSRS
DLSSMVIGWYKLFPPSSLVDPTLAPLTRRASQSSLESSSGPPCIRS
PDZ
C2A
971
913
Splice variant B
1,244
1,212
C2B
1,590
1,553
Figure S1 Alignment of the amino-acid sequences of mouse Rim2 and rat Rim1. Splice variants detected in Rim2 are shown, as are the positions of the
zinc-finger, PDZ and C2 domains. The principal form of Rim2 in MIN6 cells contains sequence A but lacks sequence B. Amino-acid numbers are shown on the right.
Sucrose concentration (M)
Fraction
cAMP-GEFII
0.16
0.24
0.31
0.39
1
2
3
4
0.55
0.63
0.78
0.94
1.10
5
6
7
8
9
*
*
1.33
1.58
10
11
1.64
12
Synaptotagmin III
Synaptophysin
Figure S2. Subcellular localization of cAMP-GEFII in MIN6 cells. Immunoblot analysis of sucrose-gradient fractions of MIN6 cells. Sucrose gradient
fractionation was carried out using the method of Wendland and Scheller (Mol. Endocrinol. 8, 1070; 1994). MIN6 cells from 5 confluent 10-cm plates were
collected by 2 ml buffer (200 mM sucrose, 50 mM NaCl, 2 mM EGTA, 10 mM HEPES pH 7.2 and 1 mM phenylmethylsulfonyl fluoride) and homogenized with a
Potter homogenizer. The homogenate was centrifuged at 1,770g for 6 min at 4 ˚C. The resulting postnuclear supernatant (1.2 ml) was applied to the tops of
gradients of 1.8-ml sucrose steps (0.4, 0.6, 0.8, 1.0, 1.4 or 1.8 M sucrose in 10 mM HEPES pH 7.2 and 2 mM EGTA), and centrifuged at 55,000g for 2 h at
4 ˚C in a P40ST rotor (Hitachi Koki, Japan). Fractions (1 ml) were collected from the top to the bottom, precipitated with 15% trichloroacetic acid, and
subjected to immunoblotting using anti-cAMP-GEFII (upper panel), anti-synaptotagmin III (middle panel) and anti-synaptophysin (lower panel) antibodies. Fraction
numbers and the density of each fraction are indicated. cAMP-GEFII is present in fractions 1 and 2 (cytosolic fractions) and cannot be detected in either large
dense-core vesicles (synaptotagmin III as a marker) or synaptic-like vesicles (synaptophysin as a marker; Diabetes 46, 2002, 1997). Non-specific bands
indicated by * are observed in fractions 1, 8 and 9.
NATURE CELL BIOLOGY VOL 2 NOVEMBER 2000 http://cellbio.nature.com
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