From the Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, United Kingdom
Received for publication, August 2, 2000, and in revised form, December 4, 2000
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ABSTRACT |
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Rab proteins comprise a family of GTPases,
conserved from yeast to mammals, which are integral components of
membrane trafficking pathways. Rab3A is a
neural/neuroendocrine-specific member of the Rab family involved in
Ca2+ -regulated exocytosis, where it functions in an
inhibitory capacity controlling recruitment of secretory vesicles into
a releasable pool at the plasma membrane. The effector by which Rab3A
exerts its inhibitory effect is unclear as the Rab3A effectors
Rabphilin and RIM have been excluded from for this role. One putative
Rab3A effector in dense-core granule exocytosis is the cytosolic zinc finger protein, Noc2. We have established that overexpression of Noc2
in PC12 cells has a direct inhibitory effect upon
Ca2+-triggered exocytosis in permeabilized cells. We
demonstrate specific nucleotide-dependent binding of Noc2
to Rab3A and show that the inhibition of exocytosis is dependent upon
this interaction since Rab3A binding-deficient mutants of Noc2 do not
inhibit exocytosis. We propose that Noc2 may be a negative effector for
Rab3A in regulated exocytosis of dense-core granules from
endocrine cells.
The secretory pathway of eukaryotic cells is essential to the
maintenance of cellular architecture and, in certain specialized cell
types, serves as the basis for cell-cell communication. Intracellular transport occurs via the targeted passage of transport vesicles between
distinct cellular compartments and is dependent upon the complex
interplay of a large number of protein factors (1-4). Rab proteins
comprise a large family of monomeric GTPases that are intimately linked
to the secretory pathway in eukaryotes (5). Distinct Rabs appear to
operate at different transport steps within the cell, exerting an
essential or regulatory function on membrane fusion events (6)
including in the mediation of vesicle tethering to target membranes.
Rab3A is a mammalian, neural/endocrine-specific Rab protein present on
synaptic vesicle and secretory granules (7-10) that appears to be
involved in the control of Ca2+-regulated fusion of
vesicles with the plasma membrane. Experiments using Rab3A knockout
mice have demonstrated a nonessential role for this protein in
regulating the efficiency of neurosecretion, possibly by limiting the
recruitment of synaptic vesicles into the releasable pool at the plasma
membrane for subsequent fusion events (11, 12). Rab3 mutants in
Caenorhabditis elegans are also viable but have a reduction
in synaptic vesicles close to the active zone (13). This suggests a
role for Rab3 in vesicle tethering in common with other Rab proteins
(14, 15). It is likely that Rab3 has multiple functions in exocytosis
(16). Active forms of Rab3A inhibit exocytosis in various endocrine and
neuroendocrine cell types (8, 17-19) and in neurons (20) consistent
with an inhibitory function of Rab3A in regulated exocytosis.
The mechanism underlying the inhibitory effect of Rab3A on regulated
exocytosis is unknown. It is likely that one or more specific Rab3A
effector proteins that interact with the GTP-bound form of Rab3A are
responsible for this inhibitory phenotype, and several such factors
have now been identified and characterized. The first of these was
Rabphilin3A (21), a 78-kDa neurally expressed protein that specifically
binds to the GTP-bound state of Rab3A (22). The N-terminal portion of
Rabphilin3A contains a zinc finger domain, while the C-terminal half of
the protein harbors two C2 domains, which mediate
Ca2+-dependent binding to phospholipids
(23-25). In addition, Rabphilin3A also binds to Noc2 (No C2 domain) is a 38-kDa cytosolic
protein cloned on the basis of its similarity to Rabphilin3A (28). As
the name suggests, Noc2 lacks the C2 domains present in the C-terminal half of Rabphilin3A and yet it is highly homologous (77.9% sequence similarity) to the N-terminal Rab3A-binding domain of Rabphilin. Noc2
appears to be predominantly expressed in endocrine and neuroendocrine tissues, and, although it is also expressed at low levels in brain, it
may represent a dense core granule-specific Rab3A effector. Interestingly, despite its homology with Rabphilin3A, an interaction between Noc2 and Rab3A has not been demonstrated (28, 32). Overexpression of Noc2 was found, however, to enhance high
K+ induced secretion from transfected PC12 cells,
indicating a role for Noc2 in some aspect of regulated secretion from
endocrine cell types, although an indirect action on Ca2+
channels or the Ca2+ signal could not be ruled out.
In this study, we have attempted to clarify the function of Noc2 in
regulated exocytosis. The data presented here indicate that
recombinantly expressed Noc2 is able to specifically associate with
Rab3A and that this interaction is sensitive to the guanine nucleotide
state of Rab3A. We have also provided evidence for a direct role of
Noc2 in regulated exocytosis due to an inhibitory effect of Noc2
overexpression on exocytosis triggered directly by Ca2+ in
permeabilized PC12 cells. To determine whether this inhibitory phenotype is dependent directly upon a Noc2-Rab3A interaction, we have
generated mutant forms of Noc2, which fail to bind Rab3A completely or
which do so with a reduced affinity. Overexpression of the mutant
constructs in PC12 cells did not inhibit exocytosis as had been seen
with wild type Noc2. We suggest that Noc2 directly associates with
Rab3A in neuroendocrine tissues and that this interaction may be
essential for an inhibitory action in
Ca2+-dependent exocytosis.
Cloning of Noc2--
Rat brain cDNA was purchased from
Origene (Rockville, MD). The full-length Noc2 sequence was amplified by
PCR1 using an Omne-E dryblock
thermocycler (Hybaid, Middlesex, United Kingdom (UK)). Primers based on
the rat Noc2 nucleotide sequence (GenBankTM accession no. AF022774)
containing restriction endonuclease sites (underlined) for
subsequent cloning were: sense,
5'-GGCGGGATCCATGGCTGACACCATCTTCAGCAG-3' (BamHI);
antisense, 5'-GCCGCTCGAGTCAGTAATGGGTAGTTGCCCAG-3'
(XhoI); and antisense,
5'-GCCGAAGCTTTCAGTAATGGGTAGTTGCCCAG-3'
(HindIII). For protein expression, BamHI and
XhoI primers were used for PCR, the product digested and
ligated into pGex4T-2 vector (Amersham Pharmacia Biotech) to generate
an N-terminally GST-tagged fusion construct. For expression in PC12
cells, BamHI and HindIII primers were used for
PCR and the product ligated into pcDNA3.1( Generation of Noc2 Mutants--
Mutations were generated in both
pGex4T-2-Noc2 and pcDNA3.1(
Nucleotides in parentheses indicate bases that were changed to generate
the appropriate amino acid substitutions. The underlined, mutated
sequence also generated a NotI restriction site used to screen mutant colonies. All mutant constructs were checked by automated
sequencing in both directions (Oswell, Southampton, UK).
Cell Culture and Transfection--
PC12 cells were cultured in
suspension in RPMI 1640 media supplemented with 10% (v/v) horse serum,
5% (v/v) fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml
streptomycin (37). Cells were maintained at 37 °C in a humidified
atmosphere of 95% air, 5% CO2. Freshly trypsinized cells
were plated onto 24-well trays at a density of 1 × 106 cells/well and transfected with 2 µg of test plasmid
along with 3 µg of human growth hormone (hGH) plasmid (pXGH5, Nichols
Institute Diagnostics) using LipofectAMINE transfection reagent (Life
Technologies, Paisley, UK) as described previously (38, 39). Three days following transfection, the cells were assayed for hGH release. Cells
were permeabilized with 20 µM digitonin for 6 min and
then challenged with buffer containing either 0 or 10 µM
free Ca2+ for 15 min (39). Secreted hGH was quantitated
using an enzyme-linked immunosorbent assay kit (Roche Molecular
Biochemicals, East Sussex, UK) according to manufacturer's instructions.
Transfection of HeLa Cells--
Freshly trypsinized HeLa cells
were plated at a density of 100 × 105/35-mm plate and
cultured in Dulbecco's modified Eagle's medium + 5% fetal bovine
serum. After 24 h, the cells were transfected with 1 µg of
plasmid (pcDNA3, or plasmids encoding Noc2, Noc2(V58A), or
Noc2(AAA)) using 3 µl of FuGENE transfection reagent (Roche). After
an additional 48 h, the cells were lysed in 200 µl of SDS dissociation buffer and samples separated by SDS-PAGE. Nitrocellulose blots were probed with affinity-purified anti-Noc2 (1:100) and visualized using ECL (Amersham Pharmacia Biotech).
Expression and Purification of Recombinant
Proteins--
pGex4T-2 Noc2 plasmid constructs encoding wild type,
Noc2(V58A), or Noc2(AAA) sequences were used to transform BL-21 RIL
codon plus Escherichia coli (Stratagene). Protein expression
was induced with 50 µM
isopropyl-1-thio- GST-Noc2 Binding to Recombinant His6-Rab3A--
1
µM GST, GST-Noc2, or GST-Noc2 mutant proteins were
immobilized onto 20 µl of GST-Sepharose by incubation for 30 min at
4 °C in 100 µl of binding buffer (20 mM HEPES, 150 mM NaCl, 1 mM dithiothreitol, 2 mM
MgCl2, 1 mM ATP, 0.5% (v/v) Triton X-100, pH
7.4). This was supplemented with either 50 µM GDP or
GTP Analysis of Rat Brain/PC12 Cell Protein Binding to
GST-Noc2--
1 µM GST or GST-Noc2 was immobilized onto
20 µl of GST-Sepharose by incubation for 30 min at 4 °C. 200-µl
aliquots of rat brain/PC12 cell cytosolic or membrane proteins,
prepared as previously described (40, 41), were then added to
incubations and supplemented with either 50 µM GDP or 50 µM GTP Production of Noc2 Antiserum--
GST-Noc2 (100 µg) in
complete Freund's adjuvant (Pierce, Chester, UK) was injected
subcutaneously into rabbits, followed after 4 weeks by a similar
injection with incomplete Freund's adjuvant (Pierce). Bleeds were
taken thereafter at 2-4-week intervals following boosts with GST-Noc2
(100 µg) in incomplete Freund's adjuvant.
Immunofluorescence--
PC12 cells (1 × 106/well) were plated onto glass coverslips and transiently
cotransfected with 2 µg of enhanced green fluorescent protein (GFP)
plasmid in combination with 2 µg of pcDNA3.1( Noc2 overexpression has been found to modify
K+-induced secretion in PC12 cells but it was not clear if
this was a direct effect on exocytosis. We were interested, therefore,
to determine whether overexpression of Noc2 would have a direct effect
on exocytosis rather than via ion channels, by examining the effect of
overexpression on exocytosis in permeabilized cells. The approach used
involved cotransfection with a plasmid encoding hGH to allow assay of
exocytosis from transfected cells (39, 42). When Noc2 was overexpressed in PC12 cells, we observed a reproducible reduction in hGH release relative to controls (Fig. 1) following
stimulation with 10 µM Ca2+ in
digitonin-permeabilized cells. It appears, therefore, that Noc2 can
exert a direct inhibitory effect on Ca2+-stimulated
exocytosis. We also found that Noc2 overexpression led to an inhibition
of K+-induced hGH release in intact cells (data not shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actinin, a
cytoskeletal protein involved in actin bundling, and hence may serve to
integrate Rab3A activation with cytoskeletal rearrangements necessary
for vesicle dynamics (26). Other Rab3A-specific effectors include RIM
(27) and Noc2 (28). RIM is a large multidomain protein that localizes
to pre-synaptic active zones and associates specifically with the
GTP-bound form of Rab3A via a zinc finger motif. The function of RIM is
as yet undetermined, although its limited homology to putative
tethering proteins, along with its plasma membrane localization may
link it to a pre-docking role via Rab3A (29). Overexpression of
Rabphilin 3A (30) or an N-terminal fragment of RIM stimulates
exocytosis (27). The use of mutagenesis and analysis of gene knockouts has ruled out a requirement for Rab3A interaction for these effects and
have eliminated Rabphilin 3A or RIM as the inhibitory effectors of
Rab3A (31-34). Another candidate Rab3A-interacting protein (35) that
has been suggested to be involved in the inhibition of exocytosis (32)
is calmodulin. This interpretation is problematic, however, as
calmodulin has been shown to have a stimulatory effect on exocytosis (36). In addition, the binding of calmodulin to Rab3A is of low
affinity and is not dependent on the nucleotide state of Rab3A (35).
This is, therefore, not consistent with calmodulin acting as a Rab3A effector.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) (Invitrogen, The
Netherlands). All constructs were checked in both directions by
automated sequencing (Oswell, Southampton, UK).
)-Noc2 by site-directed mutagenesis
using the Quickchange system (Stratagene). For the V58A single
amino acid mutant, the primers were: sense,
5'-GGTGGAGGTCATCCTTCAGG(T/C)CATCCAGAGAGCAGAGC-3'; and antisense,
5'-GCTCTGCTCTCTGGATG(A/G)CCTGAAGGATGACCTCCACC-3'. The AAA triple amino
acid mutant (residues 154-156) used the following primers: sense,
5'-GGAAGAGGTCAGGGGCCG(T/G)(G/C)G(T/G)(T/C)C(T/G) (A/C)CAAAGGGCTCCCCAAGTAC-3'; and antisense,
5'-GTACTTGGGGAGCCCTTTG(T/G)(A/C)G(A/G)(A/C)C(C/G)(A/C)GGCCCCTGACCTCTTCC-3'.
-D-galactopyranoside for 1 h.
Protein was purified from cytosolic fractions on glutathione-Sepharose 4B (GST-Sepharose, Amersham Pharmacia Biotech) and eluted with 10 mM reduced glutathione. E. coli expressing
His6-Rab3A (a gift from Dr. M. Clague, University of
Liverpool, UK) were induced with 400 µM
isopropyl-1-thio-
-D-galactopyranoside for 2 h and protein purified from cytosolic fractions on nickel-nitrilotriacetic acid-agarose (Qiagen). Bound protein was eluted using 400 mM imidazole.
S. Recombinant His6-Rab3A was then added to
incubations at a final concentration of 1 µM and binding
allowed to proceed for 2 h at 4 °C with agitation.
GST-Sepharose pellets were collected and washed three times in 1 ml of
the appropriate binding buffer containing GDP or GTP
S. Final pellets
were extracted by addition of 50 µl of SDS dissociation buffer (4%
(w/v) SDS, 2 mM EDTA, 10% (w/v) sucrose, 1% (v/v)
-mercaptoethanol, 10% (v/v) glycerol, 125 mM HEPES, pH
6.8) and boiling for 5 min. Bound proteins were analyzed on SDS-PAGE
(12.5% gel) and proteins transferred to nitrocellulose membranes for
immunoblotting via transverse electrophoresis. Protein was detected
with anti-Rab3A (1:400, Transduction Laboratories) and visualized using
enhanced chemiluminescence reagents (ECL). In some experiments, the
concentration of Rab3A was varied over the range of 0.2-1.0
µM and bound Rab3A quantified using Imagequant densitometry software (Molecular Dynamics).
S. Binding was allowed to proceed for 2 h
at 4 °C with agitation. GST-Sepharose pellets were then collected
and washed three times in 1 ml of either GDP- or GTP
S-containing binding buffer. Bound proteins were analyzed on SDS-PAGE (12.5% gel)
and transferred to nitrocellulose membranes for immunoblotting. Protein
was detected using anti-Rab3A (1:400, Transduction Laboratories) or
anti-Rab5B (1:100, Santa Cruz Biotechnology) followed by development with ECL.
) plasmid
(control) or 2 µg of the appropriate pcDNA-Noc2 plasmid construct
using LipofectAMINE treatment (Life Technologies, Paisley, UK). Three
days following transfection, cells were fixed in 4% formaldehyde in
PBS for 30 min, washed twice in PBS, and incubated for 30 min in PBTA
(0.1% Triton X-100, 0.3% bovine serum albumin in PBS). Cells were
subsequently incubated with anti-Noc2 serum (1:100) in PBTA for 1 h, and washed three times with 1 ml of PBTA. The cells were then
incubated with anti-rabbit-biotinylated IgG (1:100, Amersham Pharmacia
Biotech) for 1 h and washed three times in 1 ml of PBTA, followed
by incubation with streptavidin-Texas Red (1:50, Amersham Pharmacia
Biotech) for 30 min. Cells were washed three times with 1 ml of PBTA
and coverslips blotted and allowed to air-dry prior to mounting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of Noc2 has a direct
inhibitory effect upon Ca2+-regulated exocytosis in
digitonin-permeabilized PC12 cells. Control vector or plasmid
encoding wild-type Noc2 were cotransfected into PC12 cells in
combination with a plasmid encoding hGH. Three days following
transfection, the cells were permeabilized with digitonin, challenged
directly with 0 or 10 µM Ca2+, and released
hGH assayed as a percentage of the total hGH expressed. The data shown
are combined from three independent transfection experiments with a
total n = 18 per condition (mean ± S.E.).
To determine the mechanism by which Noc2 overexpression inhibited
secretion we re-analyzed its potential association with Rab3A, as
previous studies had apparently failed to show binding between these
proteins (28, 32). First, we analyzed the distribution of both Rab3A
and the endosome-specific, Rab5B in rat brain protein extracts by
immunoblotting, which demonstrated that we could detect Rab3A in the
membrane fraction and Rab5B in both membrane and cytosol fractions
(Fig. 2A). A series of binding
assays were then performed using immobilized GST-Noc2 as an affinity
column over which rat brain or PC12 cell protein extracts were passed.
In the first of these experiments, rat brain cytosol and membrane protein fractions were incubated with the GST-Noc2 affinity resin, or a
GST control column. Incubations were supplemented with either GDP or
GTPS, and bound proteins analyzed by immunoblotting. In this
experiment (Fig. 2B), GST-Noc2 effectively bound Rab3A from rat brain membrane samples only in the presence of GTP
S. As a specificity check, samples were also blotted for the endosomal Rab5B
protein. No binding of Rab5B to Noc2 was observed in any sample
although Rab5B was easily detectable in the rat brain extracts (Fig.
2A). This result suggests that the Noc2-Rab3A interaction is
highly specific since a second closely related Rab protein failed to
bind to Noc2 under identical experimental conditions. No detectable
binding of Rab3A to GST controls was observed (data not shown). We next
used the same affinity binding approach to analyze association of
proteins from PC12 cell extracts with immobilized GST-Noc2 (Fig.
2C). In this system, as with rat brain, GST-Noc2 specifically bound Rab3A from membrane protein samples, again in a
GTP
S-dependent fashion. This interaction was confirmed
with purified recombinant Noc2 and Rab3A, where we observed binding that was substantially greater in the presence of GTP
S compared with
GDP (Fig. 2D). No binding of Rab3A to GST-loaded control beads was detected in the presence of GTP
S or GDP (Fig.
2D). This experiment demonstrates that the Rab3A must bind
directly to Noc2. To determine the efficiency of binding in the assay, the signal from bound Rab3A was compared with that from known amounts
of input Rab3A. From a quantitative analysis, it appeared that around
0.3% of the input was bound. This low efficiency may be due to the low
affinity of the Noc2-Rab3A interaction (see below) that would not be
well preserved during washing of the beads in the assay.
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To determine whether the Noc2-mediated inhibition of
Ca2+-stimulated exocytosis in PC12 cells depends upon its
association with Rab3A, we took a mutagenesis approach, whereby key
residues in Noc2 were altered in an attempt to create mutant forms of
the protein, which could no longer support binding to Rab3A. Previous studies with Rabphilin3A mutants highlighted a number of residues essential for Rab3A association (31). Based on the high degree of
similarity between Noc2 and the Rab3A binding domain of Rabphilin3A (Fig. 3A), we reasoned that
mutations at corresponding positions in Noc2 might also generate
mutants defective in Rab3A binding. The mutation V61A in Rabphilin3A
completely abolishes Rab3A binding and so the first Noc2 mutant
generated altered the corresponding valine at position 58 to alanine,
to produce Noc2(V58A). The crystal structure of Rab3A complexed to the
N-terminal domain of Rabphilin3A has recently been solved (23). From
this structural analysis, it is clear that Val-61 of Rabphilin3A
(Val-58 of Noc2) contacts the switch region of Rab3A, an important
point of interaction between the two proteins. The crystal structure
also highlighted the potential importance of a conserved six-amino acid
motif, which is found in various Rab3A effector proteins (23). This SGAWF(F/Y) sequence (Fig. 3A) resides in a second important
contact area between Rabphilin3A/Rab3A. Mutants in this motif have
not previously been examined, but we reasoned from this structural information that mutation of residues in the SGAWF(F/Y) motif should
also generate Noc2 mutants with defective Rab3A binding. The second
Noc2 mutant generated, Noc2(AAA), therefore changed SGAWFY
in Noc2 to SGAAAA. The recombinant Noc2 mutant proteins, fused to GST, were expressed, and their binding to recombinant Rab3A in
the presence of GTPS was analyzed and compared with wild type
protein (Fig. 3B). Wild type Noc2 effectively associated with Rab3A, as did the Noc2(V58A) single amino acid mutant at high
concentrations. Noc2(AAA), conversely, exhibited a complete loss of
Rab3A binding in the same assay. Control GST protein gave no detectable
Rab3A binding.
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To further characterize the binding of the V58A Noc2 mutant, an assay
was performed with a constant concentration of Noc2 protein and varying
concentrations of wild type or mutant Rab3A in the presence of GTPS
(Fig. 3C). In these assays, GST controls, along with
GST-Noc2 (AAA) exhibited no detectable binding even at the highest
concentration of Rab3A. Wild type Noc2 exhibited saturable binding over
a range of Rab3A concentrations. This binding was of lower affinity
than observed for Rabphilin3A binding to Rab3A (43) and was
half-maximal at around 500 nM Rab3A. Noc2(V58A) also bound
Rab3A over this concentration range, although with a lower affinity
than the wild type protein and with ~40% less Rab3A bound at the
maximal concentration tested (Fig. 3C).
Having generated Noc2 mutants defective in Rab3A association, we next
analyzed the effects of overexpression of these proteins on
Ca2+-triggered exocytosis from PC12 cells. It was necessary
to first ensure that all of the Noc2 constructs were overexpressed in
transfected cells, and, for this purpose, an anti-Noc2 antiserum was
raised and affinity-purified. On blots with equal loading of
recombinant Noc2, Noc2(V58A) and Noc2(AAA), the anti-Noc2 serum
recognized each of the proteins with no apparent bias to any particular
construct (Fig. 4A). The
preimmune antiserum at the same concentrations did not give any signal.
To determine that the Noc2 wild-type and the Noc2(V58A) and Noc2(AAA)
mutants were expressed at comparable levels and that the full-length
protein was expressed in all cases, transfected cells were examined by
immunoblotting. The low level of transfection of PC12 cells (around
5%) coupled with the endogenous expression of Noc2 in these cells
precludes demonstration of overexpression in this cell type by this
technique. We, therefore, adopted a previously used strategy (44) of
examining whether the proteins were expressed similarly in an easily
transfectable cell type. As shown in Fig. 4B, no endogenous
Noc2 was detected in HeLa cells transfected with control vector, and
wild-type and mutant Noc2s were present at essentially the same levels,
showing that these proteins are expressed and are stable in eukaryotic
cells.
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PC12 cells transfected with each of the Noc2 constructs or control
vector, in combination with a plasmid encoding enhanced GFP as a marker
for transfection, were then processed for immunofluorescence with
anti-Noc2. In control conditions with vector, cells expressing GFP
displayed only background levels of staining with anti-Noc2 (Fig.
4C, top two panels). Each
of the Noc2 expression constructs, by contrast, exhibited anti-Noc2
immunostaining far above background levels in non-GFP-expressing cells
(Fig. 4C, bottom six
panels). These data confirmed that all Noc2 constructs were
efficiently overexpressed in PC12 cells. The effect of overexpression
of Noc2(V58A) and Noc2(AAA) on Ca2+-regulated exocytosis
was then examined. Overexpression of Noc2(V58A) in PC12 cells had a
minor inhibitory effect upon release due to 0 or 10 µM
Ca2+ but Ca2+-dependent release was
not statistically significantly different (Fig.
5A). Overexpression of Noc2
(AAA) had no distinguishable effect upon
Ca2+-dependent hGH release compared with
control cells (Fig. 5B). It is apparent that, therefore,
these mutants impaired in Rab3A binding are no longer able to support
the inhibition of Ca2+-induced secretion elicited by the
wild type protein.
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DISCUSSION |
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The results presented in this paper have provided the following new insights. First, Noc2 interacts in a nucleotide-dependent manner with Rab3A. Second, Noc2 has a direct regulatory role in Ca2+-dependent exocytosis in permeabilized cells. Third, the demonstration of residues required for Noc2 binding to Rab3A including amino acids within the conserved SGAWFY motif. Fourth, the inhibitory effect of Noc2 overexpression on exocytosis may require interaction with Rab3A as Rab3A binding-deficient mutants were no longer inhibitory.
The importance of Rab GTPases in membrane trafficking processes has long been appreciated (5), although their precise function has remained elusive. Rab3A is unusual in that, unlike Rabs acting at other steps of the secretory pathway (45-47), it appears dispensable for membrane fusion (11, 12). Rab3A knockout mice are viable and display no obvious behavioral abnormalities but display enhanced neurotransmission in response to a nerve impulse (11, 12). An inhibitory role for Rab3A has also been observed in experiments involving endocrine or neuroendocrine cells (8, 17-19). Overexpression of constitutively active, GTPase-deficient, Rab3A mutants inhibits Ca2+-triggered secretion from bovine chromaffin cells (18, 19). In the same cell type, micro-injection of antisense Rab3A oligonucleotides to deplete Rab3A elicited an increased potential to respond to repetitive stimulations, in contrast to wild type cells, which underwent desensitization (18) again consistent with an inhibitory role for Rab3A. In the insulin-secreting HIT-T15 cell line, transfection with GTPase defective Rab3 mutants similarly caused an inhibition of nutrient-triggered insulin release (8, 48).
The regulatory roles of Rab3A in exocytosis are likely to be mediated by one or more specific effector proteins (5). Rab3A effectors appear likely to have a variety of roles, from vesicle tethering and docking with acceptor membranes (14, 29), to cytoskeletal interactions, which perhaps permit vesicles close access to the target membrane, allowing them to subsequently tether and dock (26, 28) as well as inhibitory functions (16). Rab3A interacts with at least two specific effectors, Rabphilin3A and Noc2, which in turn are able to interact with cytoskeletal proteins. Rabphilin3A overexpression enhances secretory responses (30, 49). This effect is not directly mediated by the interaction with Rab3A, as point mutants in Rabphilin3A having defective Rab3A binding still enhance secretion from an endocrine cell line (31). In a reciprocal study (33), Rab3A mutants defective in Rabphilin3A binding retained the wild type ability to inhibit secretion. Further evidence for a Rab3A-independent effect of Rabphilin 3A comes from studies using Rabphilin 3A knockout mice (34). These animals fail to exhibit any of the synaptic aberrations observed in Rab3A knockouts. In addition, Rab3A mutants that fail to bind RIM also retain the ability to inhibit Ca2+-triggered secretion (32). It would therefore seem that there is no link between the negative regulatory effect of Rab3A and these effectors. Rab3A also interacts with Ca2+/calmodulin (35), which has been evoked to explain the inhibitory effect of Rab3A on secretion (32). The Ca2+/calmodulin-Rab3A interaction is, however, difficult to interpret as it displays a number of peculiarities. Ca2+/calmodulin fails to discriminate between GDP and GTP bound forms of Rab3A, arguing against a Rab3A effector function, and its association occurs with much lower affinity than for other specific Rab3A effectors being half-maximal at 20 µM calmodulin (35). In addition, calmodulin has been shown to have positive rather than inhibitory effects on regulated exocytosis (36).
In this study, we have examined the properties of the putative Rab3A
effector, Noc2 (28). Noc2 is highly homologous to the Rab3A N-terminal
binding domain of Rabphilin3A; however, previous studies have
surprisingly failed to detect binding of Noc2 to Rab3A, although no
data were shown (28, 32). We have demonstrated a reproducible binding
of recombinant Noc2 to Rab3A and furthermore have demonstrated a strict
nucleotide dependence of this interaction consistent with an effector
function for Noc2 in combination with GTP-bound form of Rab3A. This
interaction was also observable when recombinant Rab3A was exchanged
for tissue or cell extracts containing a complex mixture of proteins
including Rab3A. Noc2 binding was specific for Rab3A, as the
endosome-specific Rab5B failed to associate in the presence of GTPS.
The affinity of the interaction (~500 nm) was lower than that
reported for Rabphilin binding to Rab3A of 150 nM (43) or
20 nM (33). This may explain the low efficiency of binding
in the in vitro assay that we observed. The affinity of the
interaction of Noc2 with Rab3A was still considerably higher than that
reported for Rab3A interaction with calmodulin (35). The reasons for
the difference between this and previous studies in detecting Noc2
binding to Rab3A is unclear but may be due to the low affinity of the
interaction and to the low stoichiometry of interaction being preserved
under in vitro binding conditions.
Noc2 overexpression in PC12 cells resulted in a significant (~50%) inhibition of evoked secretion from permeabilized cells. This differs from previous work showing that overexpression of Noc2 increased K+-induced release from PC12 cells (28). In our hands, K+-induced release of hGH was also inhibited in cells overexpressing Noc2. To test the possibility that this difference was due to a higher level of overexpression in our studies, the plasmid concentration was titrated to search for stimulation at lower plasmid concentrations. The only effect we observed, however, was a reproducible inhibition due to overexpression. The effect of transfection with the Noc2 plasmid appeared to be specific, as we have not seen such a partial inhibition following transfection and use of the GH assay with several other constructs. For example, overexpression of nSec-1 or complexin (38) had no effect on exocytosis, and overexpression of NCS-1 (39) led to an increase in stimulated exocytosis. The only constructs that led to an inhibition in our hands were plasmids encoding clostridial neurotoxin light chains that would be expected to be inhibitory and indeed almost abolished exocytosis (50, 51).
The inhibitory effect of Noc2 overexpression appeared to be dependent
upon the Noc2-Rab3A interaction, since mutant forms of Noc2, which
lacked the ability to associate with Rab3A, correspondingly lost their
ability to inhibit Ca2+-stimulated secretion. The two
mutations were in distinct structural regions that interact with Rab3A
in Rabphilin 3A (23). We cannot formally rule out, however, that the
two mutated domains in Noc2 may also be involved in interactions with
some other protein and that the inactivity of the Noc2 mutants could be
due to an alternative interaction. One of the tested mutants,
Noc2(AAA), also highlights the essential requirement for a conserved
Rab effector motif in binding to Rab3A. This motif has been suggested
from determination of the crystal structure of the Rab3A/Rabphilin3A
complex to be crucial for the interaction. Our mutagenesis of this
motif confirms its requirement for Rab3A binding in Noc2. Overall,
these data suggest that Noc2 may be a significant Rab3A inhibitory
effector in Ca2+-regulated exocytosis of dense-core granules.
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FOOTNOTES |
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* This work was supported in part by grants from the Wellcome Trust and the Medical Research Council (to R. D. B. and A. M.).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.
Recipient of a Wellcome Trust Prize studentship award.
§ To whom all correspondence should be addressed. Tel.: 44-151-794-5305; Fax: 44-151-794-5337;E-mail: burgoyne@liverpool.ac.uk.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M006959200
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ABBREVIATIONS |
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The abbreviations used are:
PCR, polymerase
chain reaction;
GST, glutathione S-transferase;
hGH, human
growth hormone;
GFP, green fluorescent protein;
PAGE, polyacrylamide
gel electrophoresis;
PBS, phosphate-buffered saline;
PBTA, Triton X-100
and bovine serum albumin in phosphate-buffered saline;
GTPS, guanosine 5'-O- (thiotriphosphate).
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