Subcellular Distribution and Function of Rab3A, B, C, and D Isoforms in Insulin-Secreting Cells
Mariella Iezzi,
Gérard Escher,
Paolo Meda,
Anne Charollais,
Giulia Baldini,
François Darchen,
Claes B. Wollheim and
Romano Regazzi
Division de Biochimie Clinique (M.I., C.B.W.) Département
de Médicine Interne and Département de Morphologie
(P.M., A.C.) Université de Genève Geneva,
Switzerland 1211
Institut de Biologie Cellulaire et de
Morphologie (G.E., R.R.) Université de Lausanne Lausanne,
Switzerland 1005
Department of Anatomy and Cell Biology
(G.B.) Columbia University College of Physicians and
Surgeons New York, New York 10032
Institut de Biologie
Physico-Chimique (F.D.) Centre Nationale de la Recherche
Scientifique ERS 575 Paris, France 75005
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ABSTRACT
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Insulin-secreting cells express four GTPases of
the Rab3 family. After separation of extracts of INS-1 cells on a
sucrose density gradient, the bulk of the A, B, and C isoforms was
recovered in the fractions enriched in insulin-containing secretory
granules. Rab3D was also mainly associated with secretory granules, but
a fraction of this isoform was localized on lighter organelles.
Analyses by confocal microscopy of immunostained HIT-T15 cells
transfected with epitope-tagged constructs confirmed the distribution
of the Rab3 isoforms. Transfection of HIT-T15 cells with
GTPase-deficient mutants of the Rab3 isoforms decreased
nutrient-induced insulin release to different degrees
(D>B>A>>C), while overexpression of Rab3 wild types had minor or no
effects. Expression of the same Rab3 mutants in PC12 cells provoked an
inhibition of K+-stimulated secretion of dense
core vesicles, indicating that, in ß-cells and neuroendocrine cells,
the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C
chimera in which the carboxy-terminal domain of A was replaced with the
corresponding region of C inhibited insulin secretion as Rab3A. In
contrast, a Rab3C/A chimera containing the amino-terminal domain of C
was less potent and reduced exocytosis as Rab3C. This suggests that the
degree of inhibition obtained after transfection of the Rab3 isoforms
is determined by differences in the variable amino-terminal region.
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INTRODUCTION
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Pancreatic hormones released by the cells of the islets of
Langerhans play a pivotal role in the regulation of nutrient disposal
and metabolism. In fact, insulin secretion from pancreatic ß-cells is
an essential requirement for the achievement of blood glucose
homeostasis. Although the molecular details of the process of insulin
exocytosis remain to be fully clarified, during the last few years
several important components of the machinery permitting the targeting
of secretory vesicles to the plasma membrane have been identified
(1).
According to current models, the basic components controlling the
targeting and fusion of secretory vesicles with the plasma membrane are
largely conserved between cell types and between species (2, 3). The
docking of transport vesicles to the appropriate membrane is thought to
be specified by pairing of proteins located on the vesicle membrane
termed v-SNAREs (vesicular SNAP receptors) with their specific partners
on the acceptor membrane termed t-SNAREs (target SNAP receptors) (3).
Pancreatic ß-cells and clonal insulin-secreting cell lines express
the v-SNAREs, VAMP-2 and cellubrevin, and the t-SNAREs, SNAP-25 and
syntaxin-I (4, 5, 6, 7). Each of these proteins has been demonstrated to play
a role in insulin exocytosis using clostridial neurotoxins or with
inactivating antibodies (5, 6, 7, 8).
Rab GTPases represent a large family of homologous Ras-like GTP-binding
proteins that direct the vectorial movement of secretory vesicles.
These regulatory proteins act as molecular switches that flip between
two conformational states, the active GTP-bound and an inactive
GDP-bound form (9). The activated form of Rab GTPases has been proposed
to catalyze the formation of the v-SNARE/t-SNARE complex. Under resting
conditions, t-SNAREs are unable to interact efficiently with v-SNAREs
as they are bound to members of the Sec-1 protein family (10, 11).
Studies performed in yeast suggest that activated Ras-like GTPases
located on transport vesicles disrupt the association between t-SNAREs
and Sec-1-like proteins on the acceptor membrane (12). The displacement
of Sec-1-like proteins would result in the assembly of t-SNAREs and
v-SNAREs, leading to the docking of the vesicle to the target membrane
(12).
The members of the Rab3 subfamily are the best candidates for
controlling docking and fusion of Golgi-derived secretory vesicles with
the plasma membrane (9, 13). Four isoforms of Rab3 (Rab3A, -B, -C, and
-D) have been identified so far. At the protein level Rab3 isoforms
display about 80% amino acid identity and differ almost exclusively in
two short variable domains located at the amino- and at the carboxy
terminus. Rab3A and -C are primarily expressed in neuronal and
neuroendocrine cells, while Rab3B and -D are more abundant outside the
nervous system (9, 13). The precise function of the different Rab3
isoforms remains to be established. Differences in the subcellular
localization of Rab3 isoforms (14, 15, 16) and in their functional
involvement in the exocytotic process (17) have been reported. On the
other hand, the relatively mild phenotype observed in Rab3A-deficient
mice (18) and the ability of distinct isoforms to interact with the
same effectors (17, 19, 20) suggest at least some degree of redundancy
between Rab3 proteins.
Initial evidence for the possible implication of Rab3 proteins in
insulin secretion came from studies performed in permeabilized cells.
In this cell preparation, the introduction of peptides mimicking the
putative effector-binding domain of Rab3 proteins stimulates insulin
release (21, 22, 23). Subsequently, it was found that Rab3A is associated
with the membrane of secretory granules and that the overexpresssion of
a GTPase-deficient mutant of this isoform inhibits nutrient-stimulated
insulin secretion (24). Insulin-secreting cells also express Rab3B, -C,
and -D (24). Consequently, fine tuning of insulin secretion may result
from the interplay between different isoforms.
In this study, we compared the subcellular distribution and the
functional role of the four Rab3 isoforms in exocytosis. Our results
indicate that a fraction of each Rab3 isoform is associated with
dense-core insulin-containing secretory granules and that all of them,
if kept in a GTP-bound form, inhibit exocytosis.
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RESULTS
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We have previously demonstrated that the ß-cell line INS-1
expresses four Rab3 isoforms and that Rab3A is associated with
dense-core insulin-containing secretory granules (24). In a first
attempt to evaluate the localization of Rab3B, -C, and -D in
insulin-secreting cells, we determined the subcellular distribution of
each Rab3 isoform using a continuous sucrose density gradient (0.452
M) (24, 25). INS-1 cells contain a large number of
secretory granules (26) and are, therefore, well suited for this type
of biochemical characterization. As demonstrated in the top
panel of Fig. 1
, when homogenates of
INS-1 cells are centrifuged at equilibrium, dense-core
insulin-containing secretory granules are recovered in the fractions
corresponding to 1.31.8 M sucrose, while synaptophysin, a
marker of
-aminoisobutyric acid-containing synaptic-like
microvesicles, is recovered at 0.81 M sucrose. The
distribution of plasma membranes, endoplasmic reticulum, and the Golgi
complex is shown in the lower panel of Fig. 1
. The plasma
membrane marker Na+/K+-ATPase was recovered
in the fractions containing 0.91.2 M sucrose; BHKp23, a
protein associated with the cis-side of the Golgi complex
(27), was concentrated in the fractions containing 1.21.3
M sucrose; calreticulin, a resident protein of
the endoplasmic reticulum (28) was found in the fractions containing
1.31.4 M sucrose.

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Figure 1. Separation of INS-1 Organelles on a Sucrose Density
Gradient
A postnuclear supernatant of INS-1 cells disrupted by nitrogen
cavitation was loaded on a continuous sucrose density gradient (0.452
M sucrose) and centrifuged for 18 h at 110,000 x
g. The distribution of the position of the organelles
was assessed by analyzing aliquots of each fraction of the gradient by
Western blotting followed by densitometric scanning of the films
(synaptophysin, Na+/K+, ATPase, BHKp23,
calreticulin) or by RIA (insulin). Sucrose concentration was calculated
from the refractive index of the fractions. The top
panel shows the distribution of synaptic-like microvesicles
(synaptophysin) and dense-core insulin-containing granules. The
lower panel illustrates the distribution of plasma
membranes (Na+/K+ ATPase), Golgi complex
(BHKp23), and endoplasmic reticulum (calreticulin).
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The distribution of the four Rab3 isoforms in the sucrose gradient is
given in Fig. 2
. A fraction of Rab3A, -B,
and -C was recovered between 1.3 and 1.8 M sucrose
consistent with an association with insulin-containing granules. These
GTPases were also detected in lighter fractions (0.50.6 M
sucrose), most likely reflecting the presence of a soluble pool (29).
These Rab3 isoforms were barely detectable in fractions enriched in
synaptic-like microvesicles and plasma membrane (Fig. 2
). The
distribution of Rab3D was, in part, different from that of the other
Rab3 isoforms. As was the case for Rab3A, -B, and -C, Rab3D was mainly
associated with organelles displaying the same density as
insulin-containing secretory granules (Fig. 2
). However, in contrast to
the other isoforms, a significant portion of Rab3D was recovered in
fractions containing 0.81.1 M sucrose (Fig. 2
). In
addition, Rab3D was almost absent from light fractions, suggesting that
the soluble pool of this GTPase is very small.

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Figure 2. Subcellular Distribution of Rab3 Isoforms
Subcellular fractions of INS-1 cells were obtained using the sucrose
density gradient described in Fig. 1 . Aliquots of fractions 316 were
analyzed by Western blotting using antibodies directed against the four
Rab3 isoforms. The figure shows the distribution of Rab3 isoforms after
densitometric scanning of the autoradiographic films. Sucrose
concentration was calculated from the refractive index of the
fractions.
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The subcellular localization of Rab3A, -B, -C, and -D was also
investigated after transient transfection of the hamster ß-cell line
HIT-T15. To follow the expression of the transfected proteins, a myc
epitope was inserted at the amino terminus of each Rab3 isoform.
Immunostaining followed by conventional fluorescence microscopy
revealed that 7075% of the transfected HIT-T15 cells contained
detectable levels of insulin in the four series of experiments (Table 1
). A minority of these cells expressed
levels of Rab3 isoform that were clearly detectable at the
immunofluorescence level (Table 1
). In virtually all cells transfected
with myc-tagged Rab3A, Rab3B, and Rab3C, the GTPases were found in
the cellular compartment containing immunoreactive insulin (Table 1
).
In most of the cells transfected with myc-tagged Rab3D, the GTPase
colocalized with insulin. However, Rab3D was also observed within
cytoplasmic compartments of insulin-positive cells that did not contain
detectable levels of the hormone or in cells in which insulin was not
detectable (Table 1
). Similar results were obtained after transfection
of INS-1 cells with Rab3A, -B, -C, and -D (not shown). These results
confirm the biochemical data obtained with the sucrose gradient and
suggest that Rab3D has a subcellular distribution different, at least
in part, from the other isoforms of Rab3.
The subcellular localization of Rab3 isoforms in HIT-T15 cells was
analyzed by high-resolution confocal microscopy. Using this technique
the myc-tagged Rab3 isoforms (Fig. 3
, AD) as well as insulin (Fig. 3
, EH) were localized on punctate
structures in the cytoplasmic compartment. Mathematical comparison of
the images obtained in the fluorescein and the rhodamine channels
demonstrates that myc-tagged Rab3 proteins colocalize with
insulin-containing secretory granules (Fig. 3
, IL). As a negative
control, HIT-T15 cells were also transfected with myc-tagged Rab5,
another member of the Rab family that is normally associated with early
endosomes (30). As expected, myc-tagged Rab5 did not colocalize with
insulin (data not shown). In contrast to the other isoforms and in
agreement with the results in Table 1
, in some cells myc-tagged Rab3D
was also detected in cellular compartments that did not contain
detectable levels of insulin (Fig. 4
).

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Figure 3. Immunolabeling of Transfected Clones for Insulin
and Rab3 Isoforms
HIT-T15 cells were transiently transfected with myc-tagged Rab3A
(panels A, E, and I), Rab3B (panels B, F, and J), Rab3C (panels C, G,
and K), and Rab3D (panels D, H, and L). The cells were analyzed by
confocal microscopy after double immunofluorescence with a monoclonal
antibody against the myc epitope (revealed using fluorescein-conjugated
antibodies) (panels AD) and with a polyclonal antibody against
insulin (detected using rhodamine-conjugated antibodies) (panels EH).
Panels IL show the regions of colocalization between transfected Rab3
proteins and insulin granules. The colocalization images were obtained
as described in Materials and Methods by plotting all
pixels whose intensities in both channels were simultaneously above the
40% level (representing <10% of the total pixel count).
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Figure 4. Myc-Tagged Rab3D Is Not Exclusively Associated with
Secretory Granules
HIT-T15 cells transiently transfected with myc-tagged Rab3D were
analyzed by confocal microscopy after double immunofluorescence with a
monoclonal antibody against the myc epitope (panel A) and with a
polyclonal antibody against insulin (panel B). The
arrows indicate examples of subcellular regions
containing high levels of myc-tagged Rab3D and undetectable levels of
insulin.
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We have previously determined the role of Rab3A in insulin exocytosis
by transiently coexpressing this GTPase and human proinsulin in
the hamster ß-cell line HIT-T15 (24). This line displays a higher
transfection efficacy than INS-1 cells, which makes it more suitable
for transient transfection experiments. The release of human C-peptide
by the transfected hamster cells was used as a reporter for exocytosis.
Using this system we found that the overexpression of a
GTPase-deficient mutant of Rab3A, in which Glu81 is
replaced by Leu (Q81L), leads to the inhibition of
nutrient-induced secretion (24). To assess the involvement of
Rab3B, -C, and -D in exocytosis, HIT-T15 cells were cotransfected with
human proinsulin and with the wild-type or with the Q81L mutants of the
four Rab3 isoforms. As shown in the upper panel of Fig. 5
, the isoforms were expressed at
comparable levels. None of the constructs affected the content or the
basal release of human C peptide (not shown). The overexpression of
wild-type Rab3A, -B, and -C had no significant effect on stimulated
human C peptide release, while exocytosis triggered by nutrients and
bombesin was slightly decreased by the overexpression of Rab3D (Fig. 5
, lower panel). In contrast to the wild type, the mutants of
the four Rab3 isoforms deficient in GTPase activity (Q81L) inhibited
exocytosis elicited by secretagogues (Fig. 5
, lower panel).
Despite the fact that all Rab3 Q81L constructs were expressed at a
similar level, the Rab3 isoforms inhibited exocytosis with different
efficacy. On average the most potent isoform was Rab3D followed in
order by Rab3B, and -A. C Peptide secretion in cells transfected with
Rab3C Q81L was also significantly different from the secretion measured
in control cells (P < 0.05; n = 20; paired
t test). However, the inhibitory effect of Rab3C Q81L was
significantly smaller (P < 0.05; n = 9; paired
t test) than the effect of the other Rab3 isoforms. A
similar variation in the degree of inhibition was observed if secretion
was triggered by depolarizing potassium concentrations (not shown). In
HIT-T15 cells, differences exist between the level of expression of
endogenous Rab3 isoforms (24). Thus, we examined whether the efficacy
with which the four isoforms inhibit exocytosis correlates with the
ratio between endogenous and transfected proteins. As estimated by
immunoblotting compared with the endogenous protein, Rab3A was
overexpressed about 100-fold (Rab3A is barely detectable in HIT-T15
cells), while Rab3B, -C, and -D were overexpressed about 5- to 7-fold.
Thus, the ratio between the amount of endogenous and transfected
proteins cannot explain the differences between the four Rab3
isoforms.

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Figure 5. Effect of Wild-Type and GTPase-Deficient Mutants of
Rab3 Isoforms on Insulin Secretion
HIT-T15 cells were transiently cotransfected with human proinsulin and
with wild-type or Q81L mutants of the four Rab3 isoforms. Two days
after transfection, the cells were incubated in the presence or in the
absence of 10 mM glucose, 5 mM leucine, 5
mM glutamine, and 100 nM bombesin for 30 min.
The cells were then collected and analyzed by Western blotting using an
antibody against the myc epitope. The top panel shows
the results of a representative experiment. The amount of human
C-peptide secreted by the cells in response to the stimuli was measured
by ELISA. The lower panel shows the mean ±
SE of three to five representative experiments. In control
cells the mixture of nutrients and bombesin elicited a 4- to 8-fold
increase in C peptide release.
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We also tested for the effect on exocytosis of another mutant of Rab3
that is deficient in guanine nucleotide binding. This mutant, in which
Asn135 is replaced by Ile (N135I), displays a very high
dissociation rate for both GDP and GTP (31). Rab3A N135I and Rab3D
N135I were well expressed in HIT-T15 cells (Fig. 6
, upper panel) and inhibited
exocytosis triggered by nutrients and bombesin with an efficacy similar
to Rab3A Q81L and Rab3D Q81L, respectively (Fig. 6
, lower
panel). We also attempted to investigate the effect on exocytosis
of Rab3B N135I and Rab3C N135I. However, the level of expression of
these two mutants achieved in transfected HIT-T15 cells was too low
to permit the interpretation of the results (data not shown).

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Figure 6. Effect of the Rab3 Mutants Deficient in Guanine
Nucleotide Binding on Insulin Secretion
HIT-T15 cells were transiently cotransfected with human proinsulin and
with the N135I mutants of Rab3A and Rab3D. Two days after transfection,
the cells were incubated as described in Fig. 5 . At the end of the
incubation the cells were collected and analyzed by Western blotting
using an antibody against the myc epitope (top panel).
The amount of human C peptide secreted by the cells in response to the
stimuli was measured by ELISA. The lower panel shows the
mean ± SE of three representative experiments. In
control cells the mixture of nutrients and bombesin elicited a 4- to
6-fold increase in C-peptide release.
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Permeabilized cells have been used as a model system to measure effects
on exocytosis that are independent of membrane depolarization, channel
activity, and soluble second messengers (32). We found that the release
of C peptide, resulting from the incubation of
Streptolysin-O-permeabilized HIT-T15 cells with 100 µM
GTP
S or with 10 µM Ca2+, was strongly
reduced by the overexpression of the Q81L and N135I mutants of Rab3A
(Fig. 7
). Thus, the inhibition caused by
the overexpression of Rab3 Q81L and Rab3 N135I is likely to reflect an
effect of the mutants on the exocytotic process rather than an effect
on the signaling pathway that triggers hormone secretion.

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Figure 7. Effect of Rab3A Q81L and Rab3A N135I on Exocytosis
in Streptolysin-O Permeabilized HIT-T15 Cells
HIT-T15 cells were transiently cotransfected with human preproinsulin
and with the vector alone (C), with Rab3A Q81L (Q81L), or with Rab3A
N135I (N135I). Two days after transfection the cells were permeabilized
with Streptolysin-O. The medium was then removed and the cells were
incubated for 7 min in the presence of 0.1 µM free
Ca2+, 0.1 µM Ca2+ plus 100
µM GTP S, or 10 µM Ca2+. The
amount of human C peptide secreted by the cells was measured by ELISA.
The results represent the mean ± SE of three
independent experiments.
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Stable expression of wild-type Rab3B or of Rab3B N135I has been
reported to enhance Ca2+-evoked secretion in the
neuroendocrine cell line PC12 (17). Differences between neuroendocrine
cells and HIT-T15 cells could potentially explain the discrepancy
between our data and the results obtained with PC12 cells. To assess
whether this was the case, we analyzed exocytosis in PC12 cells
transiently cotransfected with the Q81L mutants of the four Rab3
isoforms and with human GH (hGH). Exocytosis from the fraction of cells
expressing the Rab3 constructs was determined by measuring hGH release
(33). As was the case in HIT-T15 cells, the GTPase-deficient mutants of
the four Rab3 isoforms inhibited stimulus-induced secretion (Fig. 8
).

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Figure 8. Effect of the GTPase-Deficient Mutants of Rab3
Isoforms on Exocytosis in PC12 Cells
PC12 cells were transfected by electroporation with hGH and with the
Q81L mutants of Rab3A (A), Rab3B (B), Rab3C (C), or Rab3D (D). Four
days after transfection the cells were stimulated with depolarizing
concentrations of K+ for 2 min. The figure shows the amount
of hGH released in response to the stimulus. The results correspond to
the mean ± SE of three independent experiments.
K+ depolarization of control cells caused an increase of 4-
to 6-fold in hGH release.
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The GTPase-deficient mutant of Rab3C is less efficient than the
corresponding mutants of Rab3A, -B, and -D. Rab3 isoforms contain two
short variable domains at the amino- and at the carboxy terminus of the
protein. To address whether either one of these domains is responsible
for the differences observed between isoforms, we generated chimeric
constructs in which the amino- or the carboxy terminus of Rab3A Q81L
was replaced by the corresponding region of Rab3C (Fig. 9
). After transfection, all the chimeric
proteins were expressed at comparable levels (Fig. 10
, upper panel). The Rab3A
chimera containing the carboxy terminus of Rab3C (Rab3A/C) inhibited
exocytosis to the same extent as Rab3A Q81L (Fig. 10
, lower
panel). Similar results were obtained with two Rab3A chimeric
constructs containing, respectively, the carboxy terminus of Rab3B and
Rab3D (data not shown). In contrast, the Rab3A chimera with the amino
terminus of Rab3C (Rab3C/A) was much less efficient, decreasing
stimulated secretion like Rab3C. As a control, we also tested the
corresponding Rab3A chimeric constructs containing the amino terminus
of Rab3B and Rab3D. The inhibition of exocytosis obtained with these
constructs was not significantly different from that of Rab3A, -B, or
-D (data not shown). The localization of all the chimeric constructs
analyzed by immunofluorescence was identical to that of Rab3A (data not
shown).

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Figure 9. Schematic Representation of the Chimeric Constructs
between Rab3A and Rab3C
The Rab3A/C chimera was generated by inserting the carboxy-terminal
fragment of Rab3C (downstream to amino acid 179) in the corresponding
region of Rab3A. To produce the amino-terminal Rab3C/A chimera, the
first 19 amino acids of Rab3A were replaced by the corresponding
residues of Rab3C.
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Figure 10. Effect on Exocytosis of Protein Chimeras between
Rab3A and Rab3C
HIT-T15 cells were transiently cotransfected with human proinsulin and
with Rab3A Q81L (A), Rab3C Q81L (C), a Rab3A Q81L construct with the
carboxy-terminus of Rab3C (A/C), and a Rab3A Q81L construct with the
amino terminus of Rab3C (C/A). Two days after transfection the cells
were incubated in the presence or in the absence of 10 mM
glucose, 5 mM leucine, 5 mM glutamine, and 100
nM bombesin for 30 min. The cells were then collected, and
the expression of the exogenous proteins was analyzed by Western
blotting using an antibody against the myc epitope. The top
panel shows the results of a representative experiment. The
amount of human C peptide secreted by the cells in response to the
stimuli was measured by ELISA. The lower panel shows the
mean ± SE of three independent experiments. In
control cells the mixture of nutrients and bombesin elicited a 4- to
6-fold increase in C peptide release.
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DISCUSSION
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The members of the Rab3 family are thought to control the
targeting and/or the fusion of Golgi-derived vesicles with the plasma
membrane (9, 13). However, the precise subcellular distribution and the
respective role of the different Rab3 isoforms in exocytosis are still
unclear. In bovine chromaffin cells, antibodies against Rab3A and Rab3C
were found to stain vesicle structures, whereas an antibody against
Rab3B stained the plasma membrane (16). In polarized epithelial cells,
Rab3B was localized to the apical pole of the cells near the tight
junctions (34). However, the same investigators observed that
transfected epitope-tagged Rab3B colocalized with dense-core granules
of PC12 cells (17). We have previously demonstrated that Rab3A is
associated with insulin-containing secretory granules of pancreatic
ß-cells (24). In this study, using double immunofluorescence
experiments analyzed by confocal microscopy, we demonstrate that
myc-tagged Rab3B, -C, and -D are also associated with
insulin-containing granules and that, clearly, none of the isoforms are
localized on the plasma membrane. It is very unlikely that our
morphological observations are biased by the overexpression of the
proteins. In fact, first, these findings are corroborated by
subcellular fractionation studies indicating that the endogenous Rab3
isoforms display a similar distribution. Second, overexpression of
about 100-fold of Rab3A (that is expressed at low level in HIT-T15
cells) does not affect the targeting of the protein to the secretory
granules. In addition, we found the same localization in cells
expressing low levels of the exogenous protein and in cells producing
high levels of the transfected Rab3 isoforms. Third, when Rab5, another
member of the Rab family known to be associated with early endosomes,
was overexpressed in HIT-T15 cells, the protein was not associated with
secretory granules.
In contrast to the other isoforms, Rab3D was also found in cells
containing undetectable levels of insulin or within cytoplasmic
compartments of insulin-positive cells not containing detectable levels
of the hormone. It has been previously reported that Rab3A and Rab3D
are associated with different vesicular compartments (14, 15, 35). The
present data confirm a partial difference in the localization between
the two isoforms but indicate that a considerable fraction of Rab3D is
associated with insulin-containing granules. At present, the precise
nature of the vesicular structures containing Rab3D but devoid of
insulin is unknown. In CHO cells, Rab3D cofractionates with a
population of post-Golgi storage vesicles slightly larger than synaptic
vesicles that have been identified with labeled glycosaminoglycan
chains (36). When combined with our results, it is tempting to
speculate that the fraction of Rab3D not associated with
insulin-containing secretory granules may be located on a similar type
of storage vesicles.
The presence of more than one isoform within the same cell raises the
question whether Rab3 proteins are redundant or whether they play a
specific role in the exocytotic process. Transient overexpression of a
constitutively active mutant of Rab3A reduces stimulus-induced
secretion in neuroendocrine (33, 37) and in insulin-secreting cells
(24). These observations have been taken as an indication that Rab3A is
a negative modulator of exocytosis (33, 37). In agreement with this
model, Rab3A-deficient mice display an increased number of synaptic
vesicle fusion events shortly after the arrival of the nerve impulse
(38). An alternative interpretation of the results would be that the
hydrolysis of GTP is a prerequisite for the fusion of
secretory vesicles with the plasma membrane. In this case, the
GTPase-deficient mutant of Rab3A associated with secretory vesicles
would have a dominant-negative effect and would inhibit exocytosis. The
latter view is favored by the results obtained in yeast with the
corresponding GTPase-deficient mutant of Sec4 (39).
Rab3B has been suggested to have functional properties distinct from
Rab3A. Thus, in pituitary cells, inhibition of Rab3B expression was
found to attenuate Ca2+-dependent exocytosis (40) and in
PC12 cells stable expression of wild-type Rab3B or of Rab3B N135I has
been reported to potentiate the efficiency of Ca2+-evoked
secretion and to markedly increase the accumulation of norepinephrine
in secretory granules (17). Here we show that transient overexpression
of wild-type Rab3B in HIT-T15 cells has no significant effect on
stimulated secretion, and that Rab3B Q81L strongly inhibits exocytosis.
Thus, the data are consistent with a similar role for Rab3A and Rab3B
in insulin-secreting cells. Our findings cannot be attributed to
differences between insulin-secreting cells and neuroendocrine cells,
since we demonstrate that transient expression of Rab3B Q81L diminishes
stimulated secretion also in PC12 cells. Unfortunately, in HIT-T15
cells, Rab3B N135I was poorly expressed and it was, therefore,
impossible to evaluate its effect on insulin release. A possible
explanation for the low level of Rab3B N135I and Rab3C N135I detected
in transfected cells is that the high dissociation rate for guanine
nucleotides is affecting the turnover rate of the protein.
We have analyzed the effect of Rab3B mutants on exocytosis after
short-term expression (2 days). In contrast, Weber et al.
(17) analyzed the impact of Rab3B expression on secretion using stable
cell lines. After long-term expression of Rab3B, the stability of
Rabphilin, a putative Rab3 effector, was found to decrease (17). Thus,
it is possible that long-term expression of Rab3B leads to secondary
alterations in the secretory phenotype that are not evident in
transient transfection experiments.
The overexpression of a GTPase-deficient mutant of each of the four
Rab3 isoforms tested in this study inhibited exocytosis. However, the
decrease in nutrient-induced secretion observed in the presence of
Rab3C was much less pronounced than the decrease caused by the other
isoforms. Exchange of the carboxy-terminal domain of Rab3A with those
of any of the other Rab3 isoforms did not alter the extent of
inhibition measured after overexpression of the chimeric constructs.
Substitution of the amino terminus of Rab3A with the corresponding
domain of Rab3B or Rab3D was also without effect, but replacement of
the amino terminus of Rab3A with the residues of Rab3C reduced the
effect of the GTPase on exocytosis. The amino termini of Rab5 and of
Rab2 have been shown to be required for the function of these GTPases
and for the interaction with components of the transport machinery (41, 42). The variable domain at the carboxy terminus of Rab proteins
appears to dictate the localization of the GTPases to specific cellular
compartments (43, 44). Our results suggest that the amino terminus of
the Rab3 isoforms may be involved in the interaction with an, as yet
unidentified, component of the secretory machinery. Differences in the
amino-terminal domain of the Rab3 isoforms may affect the affinity for
this putative component and could determine the efficacy with which
they regulate insulin secretion.
In conclusion, we have demonstrated that the secretory granules of
pancreatic ß-cells contain four Rab3 isoforms with similar roles in
the regulation of insulin secretion. Further experiments are needed to
determine the step(s) and the precise mechanism by which the GTPases of
the Rab3 family control the exocytotic process.
 |
MATERIALS AND METHODS
|
---|
Materials
The cDNA coding for human wild-type Rab3B was kindly provided by
Dr. K. Kirk (University of Alabama at Birmingham, AL). Bovine wild-type
Rab3C cDNA and polyclonal antibodies directed against Rab3A, Rab3B, and
Rab3C were generously provided by Dr. A. Zahraoui (Curie Institute, UMR
144 CNRS, Paris, France). Plasmid pBRhins, derived from pBR322 and
containing the human preproinsulin gene under the control of the
cytomegalovirus promoter), was kindly provided by Dr. J.-C. Irminger
(University of Geneva). The generation of rabbit polyclonal antibodies
against the amino-terminal region of Rab3D was described previously
(14). The mouse monoclonal antibody specific for human c-myc
(9E10) was produced from myeloma SP2/0; it was affinity purified from
the hybridoma medium using sheep antimouse IgG antibodies coupled to
Protein A Sepharose CL-4B (Sigma P-3391, Sigma Chemical Co., St. Louis,
MO). A polyclonal rabbit antibody raised against the
-subunit of
Na+/K+-ATPase was kindly supplied by Dr. E.
Ferraille (University of Geneva).
The polyclonal antibody against BHKp23 was obtained from Dr. J.
Gruenberg (University of Geneva); the polyclonal against calreticulin
was provided by Dr. K.-H. Krause (University Hospital, Geneva). The
monoclonal antibody against synaptophysin was purchased from Sigma.
Cell Culture
The insulin-secreting cell lines HIT-T15 and INS-1 were cultured
in RPMI 1640 medium supplemented with 10% FCS and other additions as
described for each cell line (26, 45). PC12 cells were cultured in DMEM
supplemented with 6% FCS and 6% horse serum.
Subcellular Fractionation
Subcellular fractionation of approximately 108 INS-1
cells was performed as described (24). Briefly, a postnuclear
supernatant obtained after disruption of the cells by nitrogen
cavitation was loaded on a continuous sucrose density gradient (8 ml;
0.452 M sucrose). After centrifugation for 18 h at
110,000 x g, 16 fractions of 0.5 ml were collected
from the top of the tube. The concentration of sucrose in the fractions
was determined by measuring the refractive index of the solution. The
amount of insulin present in the fractions was measured by RIA. The
distribution throughout the gradient of BHKp23, calreticulin,
Na+/K+ ATPase, synaptophysin, and Rab3A/B/C and
D was assessed by Western blotting (24) followed by densitometric
scanning of the autoradiographic films.
Generation of DNA Constructs
The generation of myc-tagged human wild-type Rab3A and of the
mutant at positions 81 (Q81L) and 135 (N135I) has been described
previously (37). Myc-tagged wild-type Rab3A and the corresponding Q81L
and N135I mutants were subcloned in the mammalian expression vector
pcDNA3 (Invitrogen, San Diego, CA). Wild-type Rab3B, Rab3C, Rab3D, and
the Q81L mutant of Rab3D were subcloned into pcDNA3 containing the
N-terminal myc epitope. To construct the Rab3A/C chimera containing the
carboxy terminus of Rab3C, we generated a XhoI restriction
site in Rab3A and Rab3C at the level of amino acid 179; the fragment of
Rab3C downstream to the XhoI site was then excised and
inserted in the corresponding XhoI site of Rab3A Q81L. A
similar approach was used to produce the amino-terminal Rab3C/A
chimera. In this case, a XhoI site was introduced in the
sequence of Rab3A and Rab3C at the level of amino acid 19; the fragment
downstream to the XhoI site of Rab3A Q81L was inserted in
the corresponding site of Rab3C.
Site-Directed Mutagenesis
The mutants of Rab3B and Rab3C were generated by site-directed
mutagenesis according to the method of Kunkel (46). The mutations
generated were confirmed by DNA sequencing of the plasmids.
Transfection
For transient transfection experiments, HIT-T15 cells were
seeded in 24-multiwell plates (4 x 105 cells per
well). After 3 days of culture, the cells were cotransfected using the
lipopolyamine Transfectam (Promega) with 2.5 µg of the vector
encoding human preproinsulin and with 5 µg of the plasmids containing
the cDNAs under study (24, 47). Transient transfection of PC12 cells
was performed by electroporation; 4 x 106 cells were
resuspended and electroporated in 400 µl of serum-free DMEM in the
presence of 40 µg of a plasmid vector encoding hGH (Nichols
Institute, San Juan Capistrano, CA) and 40 µg of pcDNA3
encoding the Rab3 proteins. Immediately after transfection the cells
were diluted in culture medium and seeded in 24-multiwell plates.
Secretion from Transfected HIT-T15 Cells
Forty-eight hours after transfection the cells were preincubated
for 30 min in modified Krebs-Ringer bicarbonate buffer (24). They were
then incubated for 30 min in Krebs-Ringer bicarbonate buffer in the
presence or in the absence of 10 mM glucose, 5
mM leucine, 5 mM glutamine, and 100
nM bombesin, a mixture known to strongly stimulate insulin
secretion in these cells. Secretion from transfected cells was assessed
by measuring the amount of human C peptide released into the medium
during the incubation period by enzyme-linked immunosorbent assay
(ELISA) (Dako Corp., Carpenteria, CA). Secretion experiments with
permeabilized cells were performed as described (48). Forty-eight hours
after transfection the cells were permeabilized with Streptolysin-O for
8 min. The medium was then removed, and exocytosis was triggered by
adding 100 µM GTP
S or by increasing the free
Ca2+ concentration from 0.1 µM (basal) to 10
µM.
Secretion from Transfected PC12 Cells
Four days after transfection the cells were preincubated during
30 min in 20 mM HEPES, pH 7.4, 128 mM NaCl, 5
mM KCl, 2.7 mM CaCl2, 10
mM glucose, and 1 mM MgCl2. The
medium was then aspirated and the cells were stimulated for 2 min with
the same buffer but containing 53 mM NaCl and 80
mM KCl. Exocytosis from the subpopulation of transfected
cells was determined by measuring by ELISA (Boeringer Mannheim,
Indianapolis, IN) the amount of hGH secreted into the medium during the
incubation period.
Immunofluorescence
For immunofluorescence labeling, the cells were grown on glass
coverslips coated with extracellular matrix produced by A431 epidermoid
cells (49, 50). Subconfluent monolayers were fixed 30 min in a 4%
paraformaldehyde-0.1 M phosphate buffer solution, pH 7.4.
After rinsing for 10 min in PBS supplemented with 0.5% BSA (PBS) and
10 mM NH4Cl, the cultures were simultaneously
exposed for 2 h at room temperature to a guinea pig polyclonal
serum against porcine insulin (diluted 1:200) (51) and to a purified
mouse monoclonal antibody against human c-myc (clone 9E10; diluted
1:600). After rinsing in PBS, the cultures were incubated again for
1 h at room temperature in the presence of both a
rhodamine-labeled goat serum against guinea pig Ig (Cappel, Organon
Teknika AG, Switzerland; diluted 1:200) and a fluorescein-conjugated
sheep antimouse Ig (Biosys, Compiègne, France; diluted 1:200).
After careful rinsing, the coverslips were mounted with 0.02%
paraphenylenediamine in PBS-glycerol (1:2, vol/vol).
Two independent experiments were run for quantitative analysis. In each
experiment and for each of the four Rab3 isoforms, 10 fields were
randomly photographed at the fixed magnification of 100x. Counts were
performed on these photographs, which were projected on a screen at the
final magnification of 370x. The total number of cells was evaluated
by scoring the number of nuclei present per field. The number of
insulin-containing cells was established by scoring those cells that
showed a granular, rhodamine labeling of cytoplasm clearly above
background level. The number of Rab3-transfected cells was established
by scoring those cells that showed a fluorescein labeling of cytoplasm
clearly above background level. Colocalization of the two
immunoreactivities was assessed by evaluating the insulin labeling of
all Rab3-positive cells.
Two separate experiments were made for confocal microscopy (Leica
Lasertechnik, Heidelberg, Germany model TCS NT) analysis, to
discriminate intracellular granular staining from diffuse cytoplasmic
or membrane staining. Excitation was obtained with an Argon-Krypton
laser, with line set at 488 nm for fluorescein excitation and 568 nm
for rhodamine excitation, and the emitted light was filtered through
appropriate filters (BF 530/30 for fluorescein, LP 590 for rhodamine).
Images of 512 x 512 pixels (RabA, -B, and -C) and 1024 x
1024 pixels (RabD) were taken with a 63x objective, NA 1.32. A typical
pixel size was 35 nm (RabD, with a zoom of 2.3). For each field,
digitized series of optical sections at different planes of focus were
collected on the host computer; care was taken to use the full dynamic
range of the photomultipliers by using a special look up table
(glowover-glowunder, Leica); the sections were processed using Imaris
software (Bitplane AG, Zurich, Switzerland) on a Silicon Graphics
computer. No filters were applied, but background noise was reduced. To
calculate colocalization between transfected Rabs and insulin granules,
a correlogram of all intensities was generated, normalizing the
intensities from 0 to 100%; all pixels whose intensities in both
channels were simultaneously above the 40% level (representing <10%
of the total pixel number) were then plotted.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Dr. A. Zahraoui (Paris) for providing Rab3C
cDNA and antibodies against Rab3A, Rab3B, and Rab3C, to Dr. K. Kirk
(Birmingham, AL) for providing the Rab3B cDNA, to Dr. Gruenberg
(Geneva) for supplying the BHKp23 antibodies, and to Dr. Krause
(Geneva) for supplying the antibody against calreticulin. We are also
indebted to Miss D. Duhamel and Miss S. Lüthi for technical
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Romano Regazzi, Institut de Biologie Cellulaire et de Morphologie, Rue du Bugnon 9, 1005 Lausanne, Switzerland. E-mail: Romano.Regazzi{at}ibcm.unil.ch
Supported by grants from the Juvenile Diabetes Foundation International
(197124) PM, (196100) RR, the Swiss National Science Foundation
(3234086.95) PM, (3232376.91 and 3249755.96) CBW,
(3100050640.97) RR and the European Union (BMH4-CT961427).
Received for publication June 1, 1998.
Revision received October 9, 1998.
Accepted for publication October 13, 1998.
 |
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