From the Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received for publication, December 23, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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Slp4-a
(synaptotagmin-like protein
4-a)/granuphilin-a is specifically localized on dense-core
vesicles in PC12 cells and negatively controls dense-core vesicle
exocytosis through specific interaction with Rab27A via the N-terminal
Slp homology domain (SHD) (Fukuda, M., Kanno, E., Saegusa, C., Ogata,
Y., and Kuroda, T. S. (2002) J. Biol. Chem. 277, 39673-39678). However, the mechanism of the inhibition by Slp4-a has
never been elucidated at the molecular level and is still a matter of
controversy. In this study, I discovered an unexpected biochemical
property of Slp4-a, that Slp4-a, but not other Rab27 effectors reported
thus far, is capable of interacting with both Rab27A(T23N), a dominant
negative form that mimics the GDP-bound form, and Rab27A(Q78L), a
dominant active form that mimics the GTP-bound form, whereas Slp4-a
specifically recognizes the GTP-bound form of Rab3A and Rab8A and does
not recognize their GDP-bound form. I show by deletion and mutation
analyses that the TGDWFY sequence in SHD2 is essential for Rab27A(T23N)
binding, whereas SHD1 is involved in Rab27A(Q78L) binding. I further
show by immunoprecipitation and cotransfection assays that Munc18-1, but not syntaxin IA, directly interacts with the C-terminal domain of
Slp4-a in a Rab27A-independent manner. Expression of Slp4-a mutants
that lack Rab27A(T23N) binding activity (i.e. specific binding to Rab27A(Q78L)) completely reverses the inhibitory effect of
the wild-type Slp4-a on high KCl-dependent neuropeptide Y
secretion in PC12 cells. The results strongly indicate that interaction of Slp4-a with the GDP-bound form of Rab27A, not with syntaxin IA or
Munc18-1, is the primary reason that Slp4-a expression inhibits dense
core vesicle exocytosis in PC12 cells.
Granuphilin-a was originally identified as a protein that is
abundantly expressed on insulin-containing vesicles in pancreatic Expression of Slp4-a (or Slp4-b/granuphilin-b, an alternatively spliced
isoform of Slp4-a lacking the C2B domain) has recently been shown to
inhibit regulated secretion in endocrine cells (2, 4, 5). The precise
mechanism of inhibition by Slp4-a, however, is still a matter of
controversy (2, 4) and has never been elucidated. In this study, I show
by means of neuropeptide Y (NPY) cotransfection assay combined with
deletion, mutation, and chimeric analyses that the SHD of Slp4-a is an
exception and binds both the GTP- and GDP-bound forms of Rab27A, and
that the Slp4-a·GDP-Rab27A complex has an inhibitory effect on high
KCl-dependent NPY secretion in PC12 cells. Based on these
results, I propose that binding of Slp4-a to GDP-Rab27A inhibits a
specific GTP/GDP exchange cycle required for dense-core vesicle exocytosis.
Molecular Cloning of Mouse Slac2-b, Slp5, and Munc18-1
cDNAs--
The chromosome locations and genome sequences of the
mouse slac2-b gene (GenBankTM accession number
AC079869; chromosome 9) and slp5 gene (XM_141661; chromosome
X) were obtained by data base searching (standard BLAST search) using a
human Slac2-b sequence and Slp5 cDNA sequence, respectively, as a
query (9, 10). cDNAs encoding the open reading frame of mouse
Slac2-b and Slp5 were estimated by sequence comparison with the human
Slac2-b and Slp5, respectively, and were then amplified from adult
mouse lung, testis, or spleen cDNA from mouse MTC panel I
(Clontech) by reverse transcriptase-PCR as
described previously (25). The following oligonucleotides with
appropriate restriction enzyme sites (underlined) or stop codons (in
boldface type) were used for amplification of the mouse Slac2-b and
Slp5 cDNAs: 5'-CGGATCCATGACGAAAGTTCCTCAGGG-3'
(Slac2-b Met primer, sense), 5'-CTCCTTTATCTTCCATCATA-3' (Slac2-b N1
primer, sense), 5'-AGGGTCTTCTCCAGAAAGGG-3' (Slac2-b N2 primer, sense), 5'-AGCCCTGAAAGTAAGGATGT-3' (Slac2-b N3 primer, sense),
5'-TCACCTATATGTAAGGGGTGGTT-3' (Slac2-b SHD-3' primer,
antisense), 5'-GTCAGACCAGGACGAATACT-3' (Slac2-b C1 primer, antisense),
5'-CTGTTCCACTTTCTTGGATG-3' (Slac2-b C2 primer, antisense),
5'-AGTCTCTCTATCTGGCAATT-3' (Slac2-b C3 primer, antisense),
5'-TCATAGTTCTGACTCTTTAT-3' (Slac2-b stop primer, antisense),
5'-GGATCCATGTCTAAGAACTCGGAGTT-3' (Slp5 Met primer, sense),
5'-CTAGCCATAGTCTCCCGTTTCAC-3' (Slp5
The purified PCR products were subcloned into the pGEM-T Easy vector
(Promega; Madison, WI), and both strands were completely sequenced as
described previously (25). Full-length mouse Slac2-b or Slp5 cDNA
was constructed on the pGEM-T Easy vector by using appropriate
restriction enzyme sites. I identified one alternative splicing site
(amino acid residues 387-408) in the mouse Slp5, and this splicing
event was tissue-specific (data not shown). The addition of the T7 tag
(or FLAG tag) to the N terminus of Slac2-b and Slp5 and construction of
expression vectors (pEF-T7-Slac2-b, pEF-T7-Slp5, and pEF-FLAG-Munc18-1)
were performed as described previously (25-28).
Construction of Deletion Mutants of the Slp4-a
SHD--
pEF-T7-GST (glutathione S-transferase)-Slp4-SHD1,
pEF-T7-Slp4- Site-directed Mutagenesis and Construction of a Chimera between
Slp3-a and Slp4-a--
Mutant Slp4-a plasmids carrying a
TGDWFY-to-AGAAAY substitution at amino acid positions 115-120 (named
Slp4-a(A4)) or a C102A/C105A substitution were obtained by two-step PCR
techniques using the following oligonucleotides with an artificial
AflII site (underlined) as described previously (30):
5'-CTTAAGCTCTATCTCCTTCGA-3' (AflII-5' primer, antisense) and
5'-CTTAAGAAAGCAGCTGGAGCTGCGGCTTATGACCAGAAA-3' (A4 primer,
sense) or 5'-CTTAAGCTCTATCTCCTTCGAGGCCACCTTGGCCCTCCA-3' (C102A/C105A primer, antisense) and
5'-CTTAAGAAAGCAACTGGAGA-3' (AflII-3'
primer, sense). A Slp4-a chimera plasmid containing a Slp3-a-SHD
at the N terminus (named Slp4-a(3-SHD)) was constructed by the same
techniques using the following oligonucleotides;
5'-CCCGCGGTGAATAAAAGAGA-3' (Slp4-SacII-5'
primer, antisense) and 5'-CCGCGGGAGATTGCAGGAGCTTTGCCCCAG-3' (Slp4(3-SHD) primer, sense). Dominant active forms and dominant negative forms of Rab3A (Q81L and T36N, respectively) and Rab8A (Q67L
and T22N, respectively) were similarly produced by PCR using the
following oligonucleotides:
5'-CGAATTCTTGCCCACGCTGCTGTTCC-3' (Rab3A-T36N-5'
primer, antisense),
5'-AAGAATTCGTTCCTCTTCCGCTACGC-3' (Rab-3A-T36N-3'
primer, sense), 5'-GCGGTACCGCTCTAGCCCTG-3' (Rab3A-Q81L primer, antisense),
5'-GGATCCATGGCGAAGACCTACGATTACCTGTTCAAGCTGCTGCTG ATCGGGGACTCGGGGGTAGGGAAGAACTGT-3' (Rab8A-T22N primer, sense), and
5'-CTGCAGATATGGGACACAGCCGGCCTGGAG-3'
(Rab8A-Q67L primer, sense). Other Slp4-a mutants (E14A,
I18A, V21A, and E32A) and Rab27A mutants (T23N and Q78L) were prepared
as described previously (5, 21).
Immunoprecipitation and Immunoblotting--
PC12 cells
(confluent 10-cm dish) were homogenized in a buffer containing 500 µl
of 50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 0.5 mM GTP NPY Release Assay--
NPY-T7-GST secretion assay in PC12 cells
was essentially performed as described previously (5, 31). Briefly,
PC12 cells (6-cm dish) were cotransfected with pShooter-NPY-T7-GST and
pEF-T7-Slp, pEF-T7-Slac2, or pEF-BOS, a vector control by using
LipofectAMINE 2000 reagent (Invitrogen) according to the
manufacturer's notes (31). Since the expression levels of recombinant
T7-tagged proteins varied among the Slp (or Slac2) family, the amounts
of pEF-T7-Slp (or Slac2) plasmids used for transfection were varied so
that similar amounts of T7-tagged proteins would be expressed in total cell lysates of PC12 cells. Three days after transfection, cells were
stimulated with high KCl buffer (56 mM KCl, 95 mM NaCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 15 mM HEPES-KOH, pH 7.4) for 10 min at 37 °C. Released
NPY-T7-GST was recovered by incubation with glutathione-Sepharose beads
and analyzed by immunoblotting with horseradish peroxidase
(HRP)-conjugated anti-T7 tag antibody (Novagen, Madison, WI). The
intensity of the immunoreactive bands on x-ray film was quantified by
Lane Analyzer (version 3.0) (ATTO Corp., Tokyo, Japan) and normalized
by total expressed NPY-T7-GST.
Miscellaneous Procedures--
T7-tagged Slp4-a mutants,
HA-tagged Rab27A, and/or various FLAG-tagged proteins (Rab3A, Rab8A,
Rab27A, syntaxin IA, SNAP-25, VAMP-2, or Munc18-1) were coexpressed in
COS-7 cells (32), and associations between these proteins were
evaluated by immunoprecipitation as described previously (18, 25). The
amounts of pEF-FLAG vectors used for transfection were varied so that
similar amounts of FLAG-tagged proteins would be expressed in total
cell lysates of COS-7 cells. For instance, because Rab27A(T23N) protein
seemed to be unstable in COS-7 cells, a 5 times greater amount of
pEF-FLAG-Rab27A(T23N) plasmids was used for transfection than
pEF-FLAG-Rab27A(Q78L) or the wild-type pEF-FLAG-Rab27A to achieve the
same protein expression levels (see Fig. 2A, top
panel). Direct interaction of GST-Slp4-a with FLAG-Munc18-1
was also assessed as described previously (10). The blots shown
in this paper are representative of at least two or three
independent experiments.
The Slp Homology Domain of Slp4-a Interacts with Both GDP- and
GTP-bound Forms of Rab27A--
The SHD basically consists of two
Next, I investigated the GTP/GDP dependence of the Rab binding activity
of the Slp4-a deletion mutants. To my surprise, the entire SHD of
Slp4-a bound both the dominant active form (Q78L; mimics GTP-bound
state) and the dominant negative form of Rab27A (T23N; mimics GDP-bound
form), despite the fact that the SHD1 alone (or the
The interaction between SHD and Rab27A(T23N) was found to be a unique
event in Slp4-a (asterisk in Fig. 2A), because,
except for Slp4-a, all of the Rab27-binding proteins reported thus far specifically recognize Rab27A(Q78L) and do not recognize Rab27A(T23N) (Fig. 2A, compare lanes 1 and
2). Based on the results of deletion analysis of the Slp4-a
SHD, I hypothesized that the SHD1 alone can function as a
GTP-Rab27A-binding site, the same as the Slac2-a SHD1 (i.e.
RBD27, Rab-binding domain specific
for Rab27 isoforms) (33), that the SHD2 is involved in an
auxiliary GDP-Rab27A binding, because the SHD2 alone does not recognize
Rab27A at all, and that the zinc finger motifs are not necessary for
Rab27A recognition. To verify this hypothesis, I performed Ala-based
site-directed mutagenesis (Fig.
3A). When one of the conserved
amino acids among the SHD1 of the Slp and Slac2 families (Glu-14,
Ile-18, Val-21, or Asp-32 of Slp4-a) (5, 33) was replaced by Ala, the
mutant Slp4-a SHD1 (T7-GST-Slp4-SHD1) completely abolished Rab27A
binding activity (Fig. 3B, lanes
2-5), indicating that the Slp4-a SHD1 is critical for
GTP-Rab27A binding, the same as the Slac2-a SHD1 (33). When the TGDWFY
sequence in the SHD2 was mutated to AGAAAY, the mutant Slp4-a(A4)
specifically bound Rab27A(Q78L) but did not bind Rab27A(T23N) (Fig.
3C, lanes 1 and 2). By
contrast, mutation of the zinc finger motifs (C102A/C105A) of Slp4-a
had no effect on interaction with either Rab27A(T23N) or Rab27A(Q78L)
(Fig. 3C, lanes 3 and 4)
but has been shown to reduce the Rab3A binding activity (4). These
results indicate that the SHD1 and the SHD2 of Slp4-a are involved in
recognition of GTP-Rab27A and GDP-Rab27A, respectively.
Slp4-a, but Not Other Slps and Slac2s, Inhibits Dense-core Vesicle
Exocytosis in PC12 Cells--
Since appropriate GTP/GDP exchange (or
GTPase activity) of Rab proteins is generally believed to be essential
for expression of their function and Rab effectors are also generally
believed to bind the GTP-bound, activated form of Rab proteins, I
hypothesized that binding of Slp4-a to the GDP-bound form of Rab27A is
responsible for the inhibition of dense-core vesicle exocytosis in
endocrine cells (2, 4, 5). Consistent with this hypothesis, expression of other Slps and Slac2s that specifically interact with the GTP-bound form of Rab27A did not inhibit high KCl-dependent NPY
secretion in PC12 cells (Fig.
4A, shaded
bars); only the Slp4-a expression significantly inhibited
high KCl-dependent NPY secretion (Fig. 4A,
cross-hatched bar). Expression of Slp3-a and Slp5,
Ca2+-dependent-type Slp (5, 6, 9, 16),
significantly promoted high KCl-dependent NPY secretion,
and promotion by Slp3-a was always greater than by Slp5 expression.
Expression of Slp1 and Slp2-a slightly stimulated high
KCl-dependent NPY secretion, but their effect was not
statistically significant under my experimental conditions. Expression
of Slac2-a, Slac2-b, and Slac2-c, on the other hand, had no effect at
all. Since, with the exception of Slac2-b, the expression levels of the
recombinant proteins did not differ much (Fig. 4A,
insets), the effects observed in Fig. 4A were
unlikely to be attributable to differences in protein expression
levels. Expression of Slac2-b protein was difficult to detect in total
cell lysates (Fig. 4A, inset, lane
8), and as a result the effect of Slac2-b expression on NPY
secretion may have been underestimated. Since similar expression levels of NPY were observed both in Slp4-a (or Slp3-a)-transfected cells and
in nontransfected cells and the Slp4-a (or Slp3-a) expression had no
significant effect on low KCl-dependent NPY secretion
(Refs. 4 and 5 and data not shown), both Slp4-a and Slp3-a are likely
to be involved in a late step of dense-core vesicle exocytosis (i.e. postdocking steps) rather than dense- core vesicle
biogenesis, transport step, or docking step. Consistent with this,
Rab27A has been shown to be required for a late step in granule
exocytosis in cytotoxic T-lymphocytes (14, 15).
To further verify my hypothesis, I expressed loss-of-function type
Slp4-a mutants that interact only with the GTP-bound form of Rab27A and
not the GDP-bound form of Rab27A, in PC12 cells (Fig. 3C,
lanes 1, 2, 5, and
6), and I tested their effect on NPY secretion. As expected,
expression of Slp4-a(A4) or Slp4-a(3-SHD), a chimera between Slp3-a and
Slp4-a, had no significant effect on NPY secretion (Fig. 4B,
hatched bars).
Interaction of Slp4-a with SNARE-related Proteins in PC12
Cells--
In the final set of experiments, I investigated the
interaction of Slp4-a with SNARE-related proteins, essential components of regulated exocytosis (34, 35), in PC12 cells, because Slp4-a has
recently been proposed to inhibit insulin secretion in pancreatic Since the effectors for Rab small GTP-binding proteins are
generally believed to bind the GTP-bound, activated form of Rab and not
the GDP-bound, inactivated form, my finding that Slp4-a interacts with
the dominant negative form of Rab27A(T23N) (mimics the GDP-bound form)
was unexpected and surprising, with other Rab27 effectors (Slp1,
Slp2-a, Slp3-a, Slp5, Slac2-a, Slac2-b, Slac2-c, and rabphilin) only
recognizing the dominant active form of Rab27A(Q78L) (Figs. 1 and 2).
It should be noted that binding of Slp4-a to GDP-Rab is specific to
Rab27A but not to Rab3A and Rab8A, indicating that Slp4-a recognizes
Rab3A, Rab8A, and Rab27A in a different fashion. Consistent with this
notion, the SHD1, zinc finger motifs, and SHD2 of Slp4-a contribute
differently to Rab3A, Rab8A, and Rab27A recognition. The SHD1 of Slp4-a
functions as a specific GTP-Rab27A binding site, whereas the SHD2
(i.e. TGDWFY motif) is involved in GDP-Rab27A recognition,
although the SHD2 alone is not an autonomous Rab27A-binding site.
Recognition of Rab3A by Slp4-a requires the SHD1 and zinc finger
motifs, whereas recognition of Rab8A by Slp4-a requires the whole SHD.
Despite sharing a similar (T/S)(G/L)WF(Y/F) motif in the SHD2 of other Slps and Slac2s (7, 10), they are unable to interact with Rab27A(T23N).
Why only Slp4-a recognizes Rab27A(T23N) is currently unknown. The
nonconserved sequences around the TGDWFY motif of the Slp4-a SHD2 may
also be involved in Rab27A(T23N) recognition, and
three-dimensional structural analysis of the Slp4-a·Rab27a complex
will be necessary to answer this question.
Since I also found that the Slp4-a mutants that specifically interacted
with GTP-Rab27A had no effect on dense-core vesicle exocytosis in PC12
cells (Fig. 4B) and since expression of Rab27A(T23N) or
Rab27A(N133I) (mimics GDP-bound form) in melanocytes has been shown to
inhibit melanosome transport from the perinuclear region to cell
periphery (i.e. melanosomes accumulate in the perinuclear region) (36, 37), the most straightforward explanation of the
inhibitory effect of the wild-type Slp4-a expression on dense-core vesicle exocytosis is that Slp4-a interacts with GDP-bound form of
Rab27A. Trapping of the GDP-bound form of Rab27A by Slp4-a may reduce
the availability of the GTP-bound form of Rab27A that participates in
enhancement of dense-core vesicle exocytosis, although I cannot
completely rule out the possibility that interaction of Slp4-a with
Munc18-1 (or syntaxin IA) also exerts an inhibitory effect on
dense-core vesicle exocytosis (see below).
Very recently, Slp4-a has been shown to interact with a closed
conformation of syntaxin IA in vitro, and this interaction has been suggested to inhibit insulin secretion in pancreatic In summary, I have discovered that Slp4-a is an exception and interacts
with both the GTP- and GDP-bound forms of Rab27A and that the SHD1 and
SHD2 of Slp4-a contribute to GTP- and GDP-Rab27A recognition in a
different manner. I also found a strong correlation between the
GDP-Rab27A binding activity of Slp4-a and the inhibitory effect of
Slp4-a on dense-core vesicle exocytosis in PC12 cells. Based on these
findings, I propose that expression of Slp4-a inhibits dense-core
vesicle exocytosis (possibly postdocking steps) through interaction
with the GDP-bound form of Rab27A. Since Slp4-a interacts only with the
GTP-bound form of Rab3A and Rab8A, it is possible that Rab3A·Slp4-a
and Rab8A·Slp4-a complexes positively control dense-core vesicle
exocytosis, although the proportions of these complexes are much
smaller than that of Rab27A·Slp4-a complex in PC12 cells (5). Further
work is necessary to elucidate this issue.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells (1), and overexpression studies have shown that it modulates
dense-core vesicle exocytosis in pancreatic
-cell lines, AtT20
cells, and PC12 cells through interaction with Rab27A (or Rab27B), one
of the small GTP-binding proteins (2-5). Granuphilin-a was
subsequently reported to be the fourth member of the Slp
(synaptotagmin-like protein) family
and was therefore renamed Slp4-a (6, 7). The Slp family consists of
five members (Slp1-5) in humans and mice (1, 7-9), and a Slp protein
is defined as a protein having an N-terminal Rab27-binding domain (also
called the Slp homology domain
(SHD))1 and C-terminal tandem
C2 domains (called the C2A domain and the C2B domain) (7, 10) (reviewed
in Ref. 11). Since patients with human type I Griscelli syndrome and
the corresponding model mice, ashen (i.e. both of
which have mutations in the rab27A gene) (12, 13) exhibit
defects in granule exocytosis in cytotoxic T lymphocytes (14, 15), a
great deal of attention has been focused on involvement of the Slp
family in granule exocytosis (5, 11, 16). However, the Slp(s) that
functions in the cytotoxic T lymphocytes has never been identified. The
SHD has also been found in the Slac2 (Slp homologue
lacking C2 domains) family without tandem C2
domains at the C terminus (Slac2-a/melanophilin, Slac2-b, and
Slac2-c/MyRIP) (17-21). Slac2-a regulates melanosome transport in
melanocytes through interaction with Rab27A and myosin Va (18, 22-24),
and Slac2-c has been suggested to regulate retinal melanosome transport
through interaction with Rab27A and myosin VIIa (20, 21).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C2AB primer,
antisense), 5'-GGATCCAGAAGTCCAGGGTCTGAAGA-3' (Slp5
SHD-5' primer, sense), and 5'-TCAGAGCCTACATTTCGCCA-3' (Slp5 stop primer, antisense). The rat Slp5 cDNA was
similarly amplified from rat testis cDNA from rat MTC panel I
(Clontech) by using the above oligonucleotides for
mouse Slp5. The 5'-untranslated region of mouse Slp5 cDNA was
obtained by 5'-rapid amplification of cDNA ends from Marathon-Ready
adult mouse spleen cDNA (Clontech) essentially
as described previously (25). cDNA encoding the open reading
frame of mouse Munc18-1 was also amplified by reverse transcriptase-PCR from Marathon-Ready adult mouse brain cDNA
(Clontech) by using the following oligonucleotides
designed on the basis of the published sequences in the data base
(GenBankTM accession number XM_130124):
5'-GCGGATCCATGGCCCCCATTGGCCTCAA-3' (Munc18-1 Met
primer, sense) and 5'-TTAACTGCTTATTTCTTCAT-3' (Munc18-1
stop primer, antisense).
SHD1, and pEF-T7-Slp4-
SHD2 were essentially
constructed by PCR using the following oligonucleotides with
appropriate restriction enzyme sites (underlined) and/or stop codons
(in boldface type) as described previously (25-27):
5'-GCACTAGTCACAGGAGCTCATTCTTCAGTC-3' (Slp4-SHD1-3' primer, antisense),
5'-CGGATCCGAGATCAAAAGAAAAGGGGC-3' (Slp4-
SHD1 primer,
sense), and 5'-GCACTAGTCATATCTCCTTCGAGCACACCT-3' (Slp4-
SHD2 primer, antisense). Other expression constructs
(pEF-T7-Slps, pEF-T7-Slac2s, pEF-T7-Rabphilin, pEF-FLAG-Rabs,
pEF-FLAG-sytaxin IA, pEF-FLAG-SNAP-25
(synaptosome-associated
protein of 25 kDa), and pEF-FLAG-VAMP-2
(vesicle-associated membrane
protein-2)/synaptobrevin-2) were prepared as
described previously (9, 10, 21, 29).
S, and protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A) in a glass-Teflon Potter homogenizer with
10 strokes at 900-1000 rpm, and proteins were solubilized
with 1% Triton X-100 at 4 °C for 1 h. After the removal of
insoluble materials by centrifugation at 15,000 rpm for 10 min, the
supernatant was incubated with either anti-Slp4-a-C2B IgG (5) or
control rabbit IgG (20 µg/ml) for 1 h at 4 °C and then
incubated with Protein A-Sepharose beads (wet volume 15 µl; Amersham
Biosciences) for 1 h at 4 °C. After washing the beads five
times with 50 mM HEPES-KOH, pH 7.2, 150 mM
NaCl, 0.2% Triton X-100, and protease inhibitors, the
immunoprecipitates were subjected to 12.5% SDS-PAGE, followed by
immunoblotting with anti-syntaxin I (anti-HPC-1; 1:100 dilution; Santa
Cruz Biotechnology, Inc., Santa Cruz, CA), anti-SNAP-25 (1:1000
dilution; Upstate Biotechnology, Inc., Lake Placid, NY), anti-VAMP-2
(1:100 dilution; StressGen Biotechnologies Corp., Victoria,
Canada), anti-Munc18-1 (1:250 dilution; Transduction Laboratories,
Lexington, KY), anti-Rab27A mouse monoclonal antibodies (1:250
dilution; Transduction Laboratories), or anti-Slp4-a antibody (1 µg/ml dilution) as described previously (5, 25). Immunoreactive bands
were visualized with enhanced chemiluminescence (Amersham
Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical regions (named SHD1 and SHD2), separated by two zinc finger
motifs (Fig. 1A), and SHD1
alone (but not SHD2 and the zinc finger motifs) functions as an
autonomous specific GTP-dependent Rab27A/B-binding site in
Slac2-a (33). The only exception is the Slp4-a SHD, because it can also
interact with Rab3A and Rab8A (4, 5, 10, 22). Why and how the Slp4-a
SHD recognizes Rab3A and Rab8A in addition to Rab27A, however, had
never been elucidated, although this information is important to
understanding the physiological function of the Rab3A·Slp4-a and the
Rab8A·Slp4-a complex. Deletion and mutation analyses clearly showed
that three subdomains of the Slp4-a SHD (SHD1, SHD2, and zinc finger
motifs) contribute differently to the recognition of Rab3A, Rab8A, and
Rab27A (Fig. 1, A and B). The SHD1 of Slp4-a
(i.e. GST-SHD1) functions as a specific Rab27A-binding site,
the same as the Slac2-a SHD (33), whereas the SHD1 + zinc finger motifs
(i.e.
SHD2) function as a Rab3A-binding site, and the
whole SHD is required for recognition of Rab8A. Since deletion of the
SHD1 of Slp4-a (i.e.
SHD1) completely abrogated binding activity toward all three Rabs (Fig. 1B), the zinc finger
motifs and the SHD2 of Slp4-a themselves are not an autonomous Rab
binding domain, the same as the Slac2-a SHD (33).
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Fig. 1.
Different contributions of the SHD1, zinc
finger motifs, and SHD2 of Slp4-a to Rab binding specificity.
A, schematic representation of deletion mutants of the
Slp4-a SHD. The Rab binding activity of each mutant ( , ±, +, or, ++)
is indicated. The amino acid positions are indicated on both
sides. B, different Rab binding activity of the
Slp4-a SHD mutant. Note that the SHD1 of Slp4-a alone was essential for
Rab27A binding but not for Rab3A or Rab8A binding (33). C,
GTP/GDP-independent interaction of the Slp4-a SHD with Rab27A and
GTP-dependent interaction with Rab3A or Rab8A. Note that
the whole SHD bound both Rab27A(Q78L) and Rab27A(T23N), but the Slp4-a
SHD mutants lacking the SHD2 (GST-SHD1 and
SHD2)
specifically bound Rab27A(Q78L). pEF-T7-Slp4-a deletion mutants and
pEF-FLAG-Rab were cotransfected into COS-7 cells, and associations
between the T7-Slp4-a mutant and FLAG-Rab were evaluated by
co-immunoprecipitation assay as described previously (10, 18).
Co-immunoprecipitated FLAG-Rab and the immunoprecipitated T7-Slp4-a
mutant were visualized with HRP-conjugated anti-FLAG tag antibody
(1:10,000 dilution; Sigma) (Blot, anti-FLAG) and
HRP-conjugated anti-T7 tag antibody (1:10,000 dilution)
(Blot: anti-T7), respectively. Input,
one-eightieth volume of the reaction mixture used for
immunoprecipitation (top panels). The positions
of the molecular mass markers (×10
3) are shown on the
left.
SHD2) recognizes
only the dominant active form (Fig. 1C, compare
lanes 5 and 6). By contrast, however,
the Slp4-a SHD specifically recognized the GTP-bound form of both Rab3A
and Rab8A (i.e. dominant active form) but not their
GDP-bound forms (i.e. dominant negative forms), the same as
rabphilin (Figs. 1C (lanes 1-4) and
2B).
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Fig. 2.
GTP-dependent binding of Slps and
Slac2s to Rab27A. A, Rab27A(T23N), a dominant negative
form that mimics the GDP-bound form, interacts with Slp4-a but not
other Slps, Slac2s, and rabphilin (asterisk). Note that,
with the exception of Slp4-a, all of the proteins bound Rab27A in a
GTP-dependent manner (i.e. Rab27A(Q78L))
(lane 1). B, GTP-dependent
interaction of Slp4-a and rabphilin with Rab3A and Rab8A. T7-Slps
(T7-Slac2s or T7-Rabphilin) and FLAG-Rab were coexpressed in COS-7
cells, and their associations were determined by co-immunoprecipitation
assay as described previously (10, 18). Co-immunoprecipitated FLAG-Rab
and immunoprecipitated T7-tagged proteins were visualized with
HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG) and
HRP-conjugated anti-T7 tag antibody (Blot: anti-T7),
respectively. Input, one-eightieth volume of the reaction
mixture used for immunoprecipitation (top
panels). The positions of the molecular mass markers
(×10 3) are shown on the left.
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Fig. 3.
The SHD2 of Slp4-a is involved in
binding of GDP-Rab27A. A, schematic representation of
the mutant Slp4-a SHD. The Rab27A binding activity of each Slp4-a
mutant is indicated ( or ++). ND, not determined.
B, effect of the single amino acid substitution in the SHD1
of Slp4-a on Rab27A binding activity (see the arrowheads in
A). C, effect of the mutations in the zinc finger
motifs (C102A/C105A) or the TGDWFY motif in the SHD2 of Slp4-a on
Rab27A binding. Note that the Slp4-a-SHD(A4) and Slp4-a(3-SHD) carrying
the SHD of Slp3-a specifically bound the GTP-bound form of Rab27A(Q78L)
and did not bind the GDP-bound form of Rab27A(T23N) (lanes
1, 2, 5, and 6). T7-Slp4-a
mutants and FLAG-Rab27A were coexpressed in COS-7 cells, and their
associations were determined by co-immunoprecipitation assay as
described previously (10, 18). Co-immunoprecipitated FLAG-Rab and
immunoprecipitated T7-Slp4-a mutants were visualized with
HRP-conjugated anti-FLAG tag antibody (Blot: anti-FLAG,
middle panels) and HRP-conjugated anti-T7 tag
antibody (Blot: anti-T7, bottom
panels), respectively. Input, one-eightieth
volume of the reaction mixture used for immunoprecipitation
(top panels). The positions of the molecular mass
markers (×10
3) are shown on the left.
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Fig. 4.
Differential effects of Rab27A effectors (Slp
and Slac2) on dense-core vesicle exocytosis in PC12 cells.
A, effect of expression of the mouse Slp or Slac2 on high
KCl-dependent NPY secretion in PC12 cells. Note that only
Slp4-a expression significantly inhibited
high-KCl-dependent NPY secretion (cross-hatched
bar in A). B, effect of expression of Slp4-a
mutants on high KCl-dependent NPY secretion in PC12 cells.
Note that the Slp4-a mutants lacking the Rab27A(T23N)-binding activity
had no effect on high KCl-dependent NPY secretion in PC12
cells (hatched bars in B). The
NPY-T7-GST secretion assay was performed as described previously (31).
The results are expressed as percentages of NPY-T7-GST secretion in
control samples (control, closed bar)
without expression of recombinant proteins. Bars, means ± S.E. of three determinations. The results shown are representative
of at least three independent experiments. **, p < 0.01; *, p < 0.05, Student's unpaired t
test. Insets in A and B show expressed
recombinant proteins visualized with HRP-conjugated anti-T7 tag
antibody. Similar amounts of the wild-type and mutant Slp4-a were
detected in the total cell lysates by immunoblotting (data not shown),
indicating that the lack of effects of the Slp4-a mutants is not
attributable to lower protein expression levels.
-cell lines through interaction with syntaxin IA (2). As shown in
Fig. 5, only a small amount of syntaxin I
and Munc18-1, but not SNAP-25 or VAMP-2, were co-purified with the
anti-Slp4-a antibody (lane 3) but not with the
control antibody (lane 2). By contrast, however,
Rab27A was dramatically more abundant in the anti-Slp4-a IgG
immunoprecipitates (Fig. 5, top panel). To
further determine whether syntaxin IA and Munc18-1 interact with Slp4-a
or Slp4-a·Rab27A complex, I then tested the interaction between
T7-tagged Slp4-a and FLAG-tagged SNARE-related proteins in the presence
or absence of HA-tagged Rab27A by cotransfection assay in COS-7 cells,
where neuronal SNARE-related proteins are not endogenously expressed (29). As shown in Fig. 6C
(lanes 4 and 8), Munc18-1 interacted with Slp4-a irrespective of the presence of Rab27A, but syntaxin IA did
not interact with Slp4-a even in the presence of Rab27A under my
experimental conditions (lanes 1 and
5). SNAP-25 and VAMP-2 also did not interact with Slp4-a,
consistent with the immunoprecipitation experiments described above.
Similar results were obtained for Slp3-a, but the interaction of Slp3-a
with Munc18-1 was relatively weak. I also mapped the Munc18-1 binding
site to the C-terminal domain of Slp4-a, but not the N-terminal SHD
(Fig. 6D, lanes 3 and 6 in
the third panel), consistent with the fact that
the Slp4-a·Munc18-1 interaction is Rab27A-independent. The interaction between Slp4-a and Munc18-1 should be direct, because purified components formed a complex even in vitro (Fig.
6E).
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Fig. 5.
In vivo interaction of Slp4-a with
SNARE-related proteins in PC12 cells. Anti-Slp4-a IgG
(lane 3), but not control IgG (lane
2), efficiently immunoprecipitated Rab27A and a small amount
of syntaxin I and Munc18-1 (arrowheads), but no SNAP-25 or
VAMP-2 was detected under my experimental conditions.
Immunoprecipitation (IP) and immunoblotting were performed
as described under "Materials and Methods." Input,
one-eightieth volume of the reaction mixtures used for
immunoprecipitation (lane 1).
View larger version (17K):
[in a new window]
Fig. 6.
Munc18-1, but not target SNAREs (syntaxin IA
and SNAP-25) or vesicle SNARE (VAMP-2), interacts with Slp4-a in a
Rab27A-independent manner. T7-Slp4-a, HA-Rab27A, and each of the
FLAG-tagged SNARE-related proteins (Munc18-1, syntaxin IA, SNAP-25, or
VAMP-2) were coexpressed in COS-7 cells. After solubilizing cells with
1% Triton X-100, T7-Slp4-a (or T7-Slp3-a) was immunoprecipitated with
anti-T7 tag antibody-conjugated agarose (Novagen) as described
previously (10, 18). Co-immunoprecipitated FLAG-tagged protein and
HA-Rab27A were first detected with HRP-conjugated anti-FLAG tag
antibody (Blot: anti-FLAG, top panels
in B and C) and HRP-conjugated anti-HA tag
antibody (1:20,000 dilution; Sigma) (Blot: anti-HA,
middle panels in B and C),
respectively. The same blots were then stripped and reprobed with HRP-conjugated anti-T7 tag
antibody to ensure that equivalent amounts of T7-tagged proteins had
been loaded (1:10,000 dilution) (Blot: anti-T7,
bottom panels in B and C).
Input, means one-eightieth volume of the reaction mixture
used for immunoprecipitation (IP) (A). The
positions of the molecular mass markers (×10 3) are shown
on the left. Note that Slp4-a (or Slp3-a) only interacted
with Munc18-1, irrespective of the presence of Rab27A. D,
Slp4-a interacts with Munc18-1 through the C-terminal domain but not
with the N-terminal SHD, irrespective of the presence of Rab27A
(lanes 3 and 6 in the third
panel). E, direct interaction of T7-tagged Slp4-a
with FLAG-Munc18-1 visualized by HRP-conjugated anti-T7 tag antibody
(top panel) and Amido Black staining
(bottom panel), respectively (10). Note that the
purified T7-GST-Slp4-a bound only the FLAG-Munc18-1 beads
(lane 1), not the beads alone (lane
2). Input, one-fifth volume of the reaction
mixture used for the assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cell
lines (2). Although small amounts of syntaxin I and Munc18-1 were
co-purified with Slp4-a even in PC12 cells, the cotransfection assay in
COS-7 cells in the present study clearly showed that syntaxin IA does
not directly interact with Slp4-a even in the presence of Rab27A,
whereas Munc18-1 interacts with the C-terminal domain of Slp4-a
independent of Rab27A (Fig. 6). The most likely explanation for the
apparent discrepancy is that syntaxin IA indirectly binds Slp4-a
through interaction with Munc18-1, because Munc18-1 is known to
directly bind syntaxin IA (38-40) and the Munc18-1·syntaxin IA
interaction was prominent even in the cotransfection assay in COS-7
cells (data not shown). Alternatively, the co-immunoprecipitated
syntaxin detected with anti-HPC-1 (anti-syntaxin I) may correspond to
other syntaxin isoforms (e.g. syntaxin IB), because the
anti-HPC-1 antibody also recognizes other syntaxin isoforms.2 Since I have
already shown that the C-terminal portion of Slp4-a (i.e.
Slp4-a-
SHD) that interacts with Munc18-1 failed to inhibit NPY
secretion in PC12 cells (5), I concluded that the SHD of Slp4-a is the
primary reason for the inhibitory effect on dense-core vesicle exocytosis.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Science and Technology Agency of Japan (to M. F.) and Grant 13780624 from the Ministry of Education, Science, and Culture of Japan (to M. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB098160-3.
To whom correspondence may be addressed: Fukuda Initiative
Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: 81-48-462-4994; Fax: 81-48-462-4995; E-mail:
mnfukuda@brain.riken.go.jp.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M213090200
2 M. Fukuda, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
SHD, Slp
homology domain;
GST, glutathione S-transferase;
GTPS, guanosine 5'-O-(3-thiotriphosphate);
HRP, horseradish
peroxidase;
NPY, neuropeptide Y;
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor.
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