(Received for publication, December 6, 1995; and in revised form, January 11, 1996)
From the
Rab3A and Rab3B are highly homologous monomeric GTPases that are
putative regulators of exocytosis in those tissues in which they are
expressed. We have characterized and directly compared the targeting
and functional properties of these isoforms in PC12 neuroendocrine
cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing
large dense core vesicles (LDCVs) when stably expressed in PC12 cells,
as determined by immunofluorescence and membrane fractionation. Both
Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent
manner. The membrane association of rabphilin-3A was modestly enhanced
in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells.
In addition, overexpression of Rab3A modestly inhibited
Ca-evoked NE release, whereas Rab3B and a GTP binding
mutant (Rab3B N135I) markedly stimulated the efficiency of
[
H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of
Rab3B and Rab3B N135I increased not only the efficiency of NE secretion
but also the accumulation of [
H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of
these effects was attributable to changes in the numbers of LDCVs nor
the docking of LDCVs at the plasma membrane. Our results indicate that
Rab3A and Rab3B have similar membrane targeting properties and are
capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally
distinct monomeric GTPases with Rab3B stimulating a late step in
Ca
-evoked secretion when expressed in PC12 cells.
Members of the Rab family of Ras-related monomeric GTPases are major candidates for controlling membrane docking and/or fusion in a wide variety of cell types. According to current models, the docking and fusion of donor and acceptor membranes is specified by interactions between proteins unique to transport vesicles, termed v-SNAREs, with their cognate t-SNAREs located on the intended target membrane(1, 2) . Rabs are not found in purified SNARE complexes(3) ; however, genetic studies in yeast indicate that additional proteins, including members of the Rab and Sec1 families, serve to control SNARE assembly by proofreading the fidelity of SNARE interactions and/or by imposing an additional layer of specificity(4, 5) .
The Rab family represents a
large number (>30) of homologous proteins, most of which are
expressed in a wide variety of tissues where they probably regulate
protein traffic pathways that are common to all cells (e.g. endocytosis in the case of Rab5)(6) . However, members of
the Rab3 subfamily (Rab3A-Rab3D), which are 77-85%
identical at the amino acid level, are tissue-specific proteins that
may regulate cell type-specific secretory pathways. For example, Rab3A
is a brain-specific monomeric GTPase that binds to small synaptic
vesicles, from which it dissociates during neurotransmitter
release(7) . Rab3A also reportedly associates with the large
secretory granules characteristic of adrenal chromaffin cells and PC12
neuroendocrine cells(8) . Rab3B and Rab3D are primarily
expressed outside of the nervous system, with Rab3B being expressed in
a wide variety of epithelial tissues (9) and Rab3D enriched in
adipocytes(10) . Rab3C shares many properties with Rab3A
including expression in brain, targeting to synaptic vesicles, and
transient dissociation from synaptic vesicles during neurotransmitter
release(11) . Rab3A and Rab3C both bind in a GTP-dependent
manner to rabphilin-3A, a Ca and phospholipid binding
protein that is a putative downstream effector for Rab3A in
neurons(12, 13, 14) .
All Rab3 isoforms
have been proposed to regulate exocytosis in their respective tissues;
however, in most cases direct evidence for such regulation is lacking.
Moreover, there is evidence to suggest that these structurally related
proteins may in fact be functionally distinct. For example, on the
basis of the results of antisense RNA experiments performed on anterior
pituitary cells, Lledo et al.(15) concluded that
Rab3B is a positive regulator of Ca-evoked secretion
from these cells. Conversely, Holz et al.(16) have
reported that transient overexpression of wild type Rab3A and certain
mutants inhibited Ca
-dependent secretion from bovine
chromaffin cells. A similar conclusion was drawn by Johannes et al.(17) , who examined the effects of Rab3A mutants that were
microinjected or transiently expressed in PC12 neuroendocrine cells.
Such data have led to the proposal that Rab3A participates in the
formation of a multimeric prefusion complex that must be dissociated
prior to membrane fusion and secretion. This proposal seems consistent
with the phenotype exhibited by Rab3A-negative transgenic mice, which
display no obvious decrement in synaptic transmission except during
repetitive stimulation, i.e. when synaptic vesicle recruitment
to the presynaptic membrane becomes rate-limiting(18) .
The
preceding results imply that Rab3 isoforms, in particular Rab3A and
Rab3B, may not be functionally interchangeable molecules, despite the
fact that they are highly homologous proteins (80% amino acid
identity). The goal of the present study was to test this notion by
characterizing the targeting and functional properties of Rab3A and
Rab3B in PC12 neuroendocrine cells. PC12 cells were utilized because
they have a well characterized, regulated secretory pathway (i.e. the norepinephrine-containing LDCVs) (
)and because they
normally express Rab3A but not Rab3B. Our results indicate that both of
these monomeric GTPases target to LDCVs when stably expressed in PC12
cells and that both are capable of binding to rabphilin-3A, a putative
downstream effector for Rab3A. Moreover, our functional data confirm
that Rab3A overexpression inhibits secretion, albeit modestly and to
varying degrees. Conversely, we observed that wild type Rab3B and a
GTP-binding mutant (i.e. Rab3B N135I) are potent stimulators
of catecholamine release. Interestingly, Rab3B stimulated secretion
both by increasing the efficiency of radiolabeled catecholamine release (i.e. release normalized to cell-associated radioactivity) and
by increasing the uptake of exogenous catecholamine into LDCVs (i.e. increasing the cargo available for secretion). Thus,
Rab3A and Rab3B are functionally distinct molecules, with Rab3B
enhancing secretion by PC12 cells at multiple levels of regulation.
The GTP binding mutant Rab3B N135I was generated as described(23) . Briefly, the Rab3B coding region was subcloned into M13 mp19 phage, and a point mutation was generated using the mutagenic oligonucleotide primer 5`-GTCACACTTGATCCCCACCAG-3`. The mutation was confirmed by manually sequencing the entire coding region in single-stranded M13 templates using the dideoxy chain termination method (Sequenase, U.S. Biochemical Corp.). For expression in PC12 cells, all cDNAs were ligated into pMEP4 (a gift of M.L. Tykocinski, Case Western Reserve University, Cleveland, OH), an expression vector containing the human metallothionein promoter, SV40 polyadenylation site and a hygromycin B resistance element(24) .
For Western blot analysis PC12 cells were grown in
poly-L-lysine-coated 100-mm Petri dishes, washed in PBS, and
lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Hepes (pH 7.0), 1 mM EDTA, 1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Lysates were clarified by
centrifugation at 16,000 g for 20 min. Protein
concentrations of cell lysates were determined using the micro-BCA
assay kit (Pierce). Proteins were resolved on 8 or 10%
SDS-polyacrylamide gels, transferred to polyvinylidene difluoride
membranes, rinsed in Tris-buffered saline, and then blocked in 5% dry
milk plus 0.1% Tween 20 in Tris-buffered saline (10 mM Tris-base, 150 mM NaCl, pH 7.5). Blots were incubated
with primary and secondary antibodies at room temperature for 1 h in 1 M glucose, 0.5% Tween 20, 10% glycerol and 5% dry milk in
Tris-buffered saline. The blots were developed by ECL (Amersham Corp.).
For quantitative Western blotting the blots were analyzed by
densitometry. In pilot experiments we calibrated the ECL signal by
blotting serial dilutions of Rab3B-containing cell lysates and observed
a linear relationship between density and protein amount.
For membrane
fractionation, postnuclear supernatants that were prepared from cells
grown on two 75-90% confluent 100-mm Petri dishes were layered
onto a 0.6-1.8 M linear sucrose gradient in 10 mM Hepes, pH 7.4, with a 2.25 M sucrose pad (27) .
In order to label catecholamine-containing secretory granules, two
100-mm Petri dishes were also labeled with 40 µCi of
[H]norepinephrine/dish for 1 h at 37 °C and
then chased for 1 h at 37 °C prior to fractionation (details
described below for NE release assay). All gradients contained the
protease inhibitor mixture described above. Equilibrium sedimentation
was achieved by centrifugation in a SW40 rotor (Beckman) at 30,000 rpm
for 6 h at 4 °C. Fractions were collected from the bottom of the
tube (450-500 µl/fraction; 20 or 21 fractions). The protein
content of each fraction from unlabeled cells was determined with the
micro-BCA assay kit. The sucrose concentration of each fraction was
determined by measuring the refractive index. The radioactivity of each
fraction from cells preloaded with [
H]NE was
measured by scintillation counting and corrected for quenching at high
sucrose concentrations (as determined by counting
[
H]NE standards in 0.6-1.8 M sucrose). For immunoblot analysis fractions were diluted 1:20 in
water, and proteins were precipitated by incubation with 10%
trichloroacetic acid and 0.1 mg/ml deoxycholate overnight at 4 °C.
Protein pellets were washed twice with acetone and then solubilized in
5
SDS sample buffer prior to immunoblot analysis.
Figure 1: Immunoblot analysis of Rab3A, Rab3B, and Rab3B N135I protein in PC12 cells. Total cell lysates (A) and membrane (pellet) and cytosol (supernatant) fractions of cell homogenates (B) are shown. All lanes were loaded with 50 µg of protein, except Rab3A clone 17 (pellet and supernatant each 35 µg). Samples were blotted with monoclonal Rab3A (1:2,500) or polyclonal Rab3B (10 µg/ml) specific antibodies. P, high speed pellet (membrane); S, supernatant (cytosol).
Figure 2: Immunofluorescence analysis of the expression and targeting of Rab3A and Rab3B in PC12 cells. Left column, mock-transfected cells, middle column, PC12 cells transfected with Rab3B coding region; right column, PC12 cells transfected with Rab3A cDNA. Top row, monoclonal Rab3A-specific antibody (1:100 dilution); bottom row, polyclonal anti-Rab3B antibody (1:50 dilution). No staining was observed in parallel control experiments using isotype-matched control and preimmune IgG, respectively (data not shown). Bar, 10 µm.
To further characterize the
subcellular locations of Rab3A and Rab3B in PC12 cells, we fractionated
selected PC12 clones by equilibrium sedimentation through sucrose
density gradients ( Fig. 3and Fig. 4). Homogenates of
stably transfected PC12 cells were layered onto 0.6-1.8 M sucrose gradients and centrifuged to equilibrium. Individual
fractions were collected and immunoblotted with antibodies against
Rab3A, Rab3B, secretogranin II (i.e. a marker of dense
secretory granules(30) ) and synaptophysin (i.e. a
marker of endosomes and small secretory vesicles(26) ). The
dense core granule fraction was also identified by preloading the cells
with [H]norepinephrine prior to fractionation. Fig. 3summarizes the results of a representative experiment
performed on a clone expressing wild type Rab3B (3B 10; see Fig. 1, A and B). Rab3B and synaptophysin were
enriched in distinct membrane fractions, with membrane-associated Rab3B
present in relatively dense fractions (Fig. 3, A and B). Moreover, the majority of
[
H]NE-labeled membranes resided in the same
heavier sucrose fractions that were enriched in Rab3B and secretogranin
II. Thus, membrane-associated Rab3B cofractionates with dense,
catecholamine-containing secretory granules in PC12 cells. Fig. 4summarizes the results of a representative experiment
performed on a clone that overexpresses Rab3A protein (clone 3A 17; see Fig. 1, A and B). Like Rab3B, Rab3A
cofractionated with norepinephrine-containing PC12 membranes. These
results, in combination with our immunofluorescence analysis, indicate
that Rab3A and Rab3B have similar membrane targeting properties in PC12
cells.
Figure 3:
Rab3B cofractionates with LDCVs as
determined by equilibrium density gradient centrifugation. A,
equal volumes of fractions of wild type Rab3B-expressing PC12 clone
(Rab3B-WT10) were blotted with monoclonal anti-synaptophysin (1:2,000)
and polyclonal anti-Rab3B antibodies (10 µg/ml). Total cell lysates
of nontransfected PC12 cells (nPC12) and of clone WT10 were also
blotted as controls (far right). B, top
panel, plot of sucrose concentration, as determined by refractive
index measurements and plot of protein concentration for each fraction. Bottom panel, plots of [H]NE
radioactivity and of Rab3B, synaptophysin, and secretogranin II
immunoreactivity (see panel A) for each fraction.
Immunoreactivity for each fraction was determined by densitometry and
normalized to total immunoreactivity summed over all fractions.
Fractions were collected from the bottom of the tube, with the first
fraction representing the heaviest sucrose fraction. Rab3B
immunoreactivity in the lightest fractions presumably represents
cytosolic Rab3B.
Figure 4:
Rab3A cofractionates with
catecholamine-containing secretory granules as determined by
equilibrium density centrifugation. Top panel, plots of
sucrose concentration and protein concentration for each fraction. Bottom panel, plots of [H]NE
radioactivity and of Rab3A and synaptophysin immunoreactivity for each
fraction. Shown are the results for a Rab3A-overexpressing clone (clone
3A 17). Levels of endogenous Rab3A were insufficient for such an
analysis of untransfected or mock-transfected cells (data not
shown).
Figure 5:
Rab3B as well as Rab3A binds to
rabphilin-3A. A, binding of Rab3A and Rab3B to recombinant
rabphilin-3A. Bacterially expressed GST or a GST-rabphilin fusion
protein containing full-length rabphilin-3A (GST-rabphilin-3A) were
immobilized on glutathione-agarose beads and incubated with PC12 or
Ht29 Cl19A lysates in the presence or absence of 50 µM GTPS. Bound proteins were resolved and immunoblotted with a
monoclonal antibody recognizing multiple Rab3 isoforms. Note that the
GST control beads contained 10 times more recombinant protein (i.e. GST) than the corresponding GST-rabphilin beads, which accounts
for the diffuse, nonspecific band in the GST samples. Note also that
the binding of either Rab3A or Rab3B to GST-rabphilin-3A was
GTP-dependent. B, binding of rabphilin-3A to recombinant Rab3A
and Rab3B. Bacterially expressed GST, GST-Rab3A, and GST-Rab3B fusion
proteins were immobilized on glutathione-agarose beads and incubated
with PC12 or Ht29 Cl19A supernatants. Bound proteins were resolved by
SDS-polyacrylamide gel electrophoresis and immunoblotted with an
antibody raised against an N-terminal fragment of rat rabphilin-3A (I 374(14) ). The band denoted by the arrow precisely comigrates with rabphilin-3A observed in total cell
lysates (not shown). The diffuse higher molecular weight band is of
unknown origin and was occasionally observed to bind to GST
alone.
On the basis of the results of these in vitro binding experiments, we examined the levels and membrane association of rabphilin-3A in Rab3A- and Rab3B-transfected PC12 cell lines. This analysis was motivated in part by the proposal that Rab3A potentiates the stability and/or membrane targeting of rabphilin-3A in brain(14) . We found no apparent differences between mock-transfected, Rab3A-transfected, and Rab3B-transfected clones in the total amount of rabphilin-3A protein present in cell lysates or homogenates (data not shown). However, two potentially interesting differences between the Rab3B-expressing clones and all other clones did emerge when we analyzed the relative distribution of rabphilin-3A between membranes and cytosol (see Fig. 6). First, both Rab3B-expressing clones that were examined (WT8 and WT9) exhibited a 40-kDa immunoreactive band in the cytosol that was much more prominent than in the nontransfected, mock-transfected, or Rab3A-overexpressing cells. This band presumably represents a proteolytic fragment of rabphilin-3A, which is highly sensitive to proteases(14) . Second, the relative amount of rabphilin-3A that was membrane-associated was higher in these Rab3B-expressing clones as compared with Rab3A-transfected clones (e.g. in clone Rab3B-WT8, 50% of the total rabphilin signal (i.e. the sum of the 80- and 40-kDa signals in the membrane pellet and supernatant) was membrane-associated, as compared with 24, 30, and 39% for the Rab3A-transfected, mock-transfected, and untransfected cells, respectively). Thus, the stable expression of Rab3B in PC12 cells has consequences on the membrane association and stability of rabphilin-3A that are not evident in mock-transfected or Rab3A-transfected cells.
Figure 6: Immunoblot analysis of rabphilin-3A protein in membranes and cytosol of Rab3B- and Rab3A-transfected PC12 clones. All lanes were loaded with 50 µg of protein. Samples were blotted with polyclonal rabphilin-3A antibody (1:2,000). P, high speed pellet (membrane); S, supernatant (cytosol).
Figure 7:
Regulated catecholamine release from
Rab3A- and Rab3B-transfected PC12 cells. A, time course of NE
release induced by 1 µM ionomycin. Shown are results
averaged for nontransfected cells (nPC12) and selected clones of
mock-transfected, Rab3B-transfected, and Rab3A-transfected PC12 cells.
Secretion was induced at time 0. Media were collected and replaced
every second minute. Media counts were normalized to total cell counts. B, ionomycin-induced [H]NE release by
Rab3B-expressing cells is dependent on extracellular
Ca
. Ionomycin was present from time 0 throughout the
experiment. Secretion was induced at time 0 and then again at 6 min by
adding Ca
for 2 min at various concentrations (1
µM to 1.8 mM CaCl
). C,
summary of NE secretion assays performed on numerous mock-transfected
clones and clones expressing recombinant Rab3A, Rab3B, and Rab3B N135I.
Secretion was induced by 1 µM ionomycin. Plotted is the
mean ionomycin-induced release of [
H]NE over a
14-min time period normalized to total cell counts. Clones transfected
with the same construct are presented as follows: nontransfected cells (dark checked bars), mock clones (white bars), wild
type Rab3B-expressing clones (black bars), Rab3B
N135I-expressing clones (dotted bars), and Rab3A-expressing
clones (striped bars). Error bars represent
S.E.
Fig. 7C summarizes the results of NE secretion
assays performed on numerous mock-transfected clones and clones
expressing recombinant Rab3A, Rab3B, or Rab3B N135I. Plotted is the
mean ionomycin-induced release of [H]NE over a
14-min time period normalized to total cell counts. Mock-transfected
and nontransfected cells exhibited very similar secretory responses,
indicating that the transfection procedure and growth in antibiotic
selection media had minimal effects on the efficiency of secretion.
Conversely, the Rab3B-expressing clones consistently exhibited greater
secretory responses than the mock-transfected cells. Shown here are the
results obtained for five clones that were expressing the highest
levels of wild type Rab3B. We also assayed the secretory responses of
two additional Rab3B clones that expressed considerably less
recombinant Rab3B. These lower expressing clones exhibited secretory
responses that were intermediate between the high expressers and
mock-transfected clones (data not shown). Clones expressing Rab3B N135I
had variable secretory responses, with three of four clones showing
responses greater than mock-transfected cells. Note that the Rab3B
N135I clone that showed no difference in secretion efficiency as
compared with mocks is the one clone for which we detected virtually no
membrane association of Rab3B N135I (clone 3B N135I 32 in Fig. 1B). Rab3A-overexpressing clones displayed
dramatically different secretory responses than those clones that were
expressing wild type Rab3B. In general, the Rab3A-overexpressing clones
exhibited lower secretory responses than mock-transfected or
nontransfected cells, although this inhibitory effect was more subtle
and variable than the stimulatory effect of Rab3B. This relatively
small inhibitory effect of Rab3A overexpression on secretory efficiency
is partially obscured when data from experiments that were performed on
different days and different seedings are averaged together (as for the
data reported in Fig. 7C).
Fig. 8shows that
the expression of Rab3B and Rab3B N135I had effects, not only on the
efficiency of secretion (i.e. release normalized to total cell
radioactivity) but also on the accumulation of
[H]NE (i.e. secretory cargo) by PC12
cells. Shown is the amount of [
H]NE that was
accumulated from the media by mock-transfected cells and cells that
were expressing Rab3A, 3B, and Rab3B N135I. Most striking are the
markedly higher levels of [
H]NE uptake exhibited
by the Rab3B and Rab3B N135I-expressing clones, which were on average
approximately 8-fold (wild type Rab3B) and 50-fold (Rab3B N135I)
greater than the uptake exhibited by mock-transfected cells. The uptake
of NE by these clones was saturable and reserpine-sensitive (Table 1), as expected if the majority of the
[
H]NE had accumulated within secretory granules
(see also the results of our density gradient analysis shown in Fig. 3). These dramatic increases in granule uptake of
[
H]NE cannot be explained by differences in cell
number or cell density, nor can they be explained by increased numbers
of secretory granules in Rab3B-expressing and Rab3B N135I-expressing
cells (see below). Interestingly, Rab3B N135I clone 32, which exhibits
virtually no detectable membrane association of Rab3B N135I (Fig. 1B) and no alteration in secretory efficiency as
compared with mock-transfected cells (Fig. 7C), does
exhibit a markedly increased capacity for [
H]NE
uptake (range: 1.4
10
to 3.7
10
cpm/35-mm Petri dish). Thus, the effect of Rab3B N135I on
[
H]NE uptake appears to be independent of its
ability to stimulate the efficiency of Ca
-induced NE
release and requires little or no membrane association of the mutant
protein.
Figure 8:
Summary
of total [H]NE uptake by mock-transfected clones
and clones expressing recombinant Rab3A, Rab3B, and Rab3B N135I. Total
cell cpm per 35-mm dish was determined by summing all media counts and
the radioactivity remaining in the cells at the end of the experiment.
Shown for each construct is the mean ± S.E. for eight dishes,
two each for four different clones. Note the logarithmic
scale.
Our results indicate that two highly homologous Rab3 isoforms (i.e. Rab3A and Rab3B) have similar targeting properties but
opposite effects on regulated catecholamine secretion in PC12 cells.
Heterologous expression of Rab3B markedly increased, whereas Rab3A
overexpression modestly inhibited, the efficiency of
Ca-triggered NE secretion by PC12 cells. Rab3B N135I
also potentiated NE release, provided that it was detectably
membrane-associated. Interestingly, the expression of Rab3B and Rab3B
N135I increased not only the efficiency of [
H]NE
secretion (i.e. secretion normalized to total cell
radioactivity), but also the accumulation of
[
H]NE (i.e. secretory cargo) by PC12
cells. Neither of these functional consequences of Rab3B and Rab3B
N135I expression can be accounted for by altered numbers or
distributions of secretory granules. Thus, the stimulation of secretory
efficiency by Rab3B and its mutant is most likely attributable to an
increase in the fusion competence of secretory granules at the plasma
membrane. Conceivably, this effect of Rab3B is related to its
interaction with the putative downstream effector rabphilin, as
discussed below.
As has been observed for other Rab
proteins(6, 32) , Rab3A overexpression did not lead to
mislocalization, even when this protein was overexpressed severalfold.
These findings are consistent with the notion that Rab proteins
associate with a relatively abundant downstream target protein on
membranes(33) . We observed no obvious competition between
Rab3A and Rab3B for membrane targeting, in spite of the fact that both
proteins associate with LDCVs. The Rab3B N135I mutant exhibited poor
membrane association as compared with wild type Rab3B when expressed
either in PC12 cells (present study) or in Madin-Darby canine kidney
epithelial cells. The corresponding mutant of Rab1B (i.e. 1B N121I) is also less efficient at associating with
membranes than wild type protein when transiently expressed in HeLa
cells(34) . Such Rab mutants, which correspond to an oncogenic
Ras mutant (Ras N116I), have higher dissociation rates for both GDP and
GTP(35) . Given that the association of Rabs with membranes
involves dissociation from guanine nucleotide dissociation inhibitor
(GDI) and GDP/GTP exchange(36) , it is possible that the
altered nucleotide binding properties of Rab3B N135I are responsible
for its less efficient membrane association(37) . Another
possibility is that Rab3B N135I is not as efficiently isoprenylated as
wild type Rab3B in PC12 cells (as shown in vitro for the
corresponding Rab5 mutant N133I(38) , which could also reduce
membrane binding. However, nonprenylated monomeric GTPases typically
have lower apparent mobilities on SDS-polyacrylamide gel
electrophoresis(39) , whereas Rab3B N135I exhibits a higher
mobility than wild type protein, as does Rab3A N135I when expressed in
chromaffin cells(16) . Interestingly, in preliminary
experiments we have observed that phosphatase treatment of cell
homogenates eliminates this difference in mobility,
which
implies that Rab3B and Rab3B N135I are phosphorylated to different
degrees. It will be of interest to determine if such posttranslational
modifications modulate the membrane targeting and functions of Rab3
isoforms.
In our studies Rab3A-overexpressing clones showed modest
but variable reductions in the efficiency of secretion as compared with
mock-transfected and nontransfected PC12 cells. These results are
consistent with the inhibitory effect of Rab3A on secretion when
transiently overexpressed in chromaffin cells(16) , and the
results of the Rab3A antisense and microinjection studies reported by
Johannes et al.(17) . This negative regulation by
Rab3A also seems to be consistent with the phenotype exhibited by
Rab3A-minus transgenic mice(18) , which has been interpreted to
indicate that Rab3A participates in the formation and/or maintenance of
a prefusion docking complex(40) . Overexpression of Rab3A may
inhibit the dissociation of such a complex and thereby inhibit
Ca-evoked fusion and secretion.
In contrast to Rab3A, Rab3B is capable of functioning as a positive regulator of secretion when stably expressed in PC12 cells. It seems highly unlikely that the stimulatory effect of Rab3B on regulated secretion by PC12 cells is due to some nonspecific effect on the availability of accessory proteins such as guanine nucleotide dissociation inhibitor and guanine nucleotide exchange factor for other Rabs, in particular Rab3A. The activities of such accessory proteins are not typically limiting under conditions of moderate Rab overexpression(16, 41) . Moreover, we noted no effect of Rab3B expression on the membrane targeting of endogenous Rab3A, which might otherwise be expected if Rab3B were competing with Rab3A for accessory proteins such as guanine nucleotide dissociation factor and guanine nucleotide exchange factor. On the other hand, we cannot rule out the possibility that long-term Rab3B expression leads to secondary alterations in the secretory phenotypes of PC12 neuroendocrine cells that might not to be evident in transient transfection experiments (e.g. changes in rabphilin stability, see below).
It seems plausible that the stimulatory effect of Rab3B
on secretion in PC12 cells is related to its interaction with
rabphilin-3A, a putative downstream effector for
Rab3A(31, 42) . Our results indicate that rabphilin-3A
is also capable of interacting with Rab3B in a GTP-dependent fashion.
Chung et al.(43) have recently reported that
rabphilin-3A is a positive regulator of secretion in chromaffin cells,
possibly by regulating interactions between secretory granules and the
actin cytoskeleton. Rab3B could enhance secretion through rabphilin-3A,
by competing with endogenous Rab3A for rabphilin-3A binding (which may
release rabphilin-3A from an inactive state) and/or by activating
rabphilin-3A in a way that Rab3A cannot. The apparently lower stability
of rabphilin in Rab3B-expressing PC12 cells could be related to a
disruption of Rab3A-rabphilin binding, similar to the reduced rabphilin
stability that was reported for the Rab3A knockout mouse(14) .
In this case, the effects of heterologous Rab3B expression in PC12
cells could be considered to be a nonphysiological maneuver to disrupt
Rab3A function in these cells. However, competitive interactions
between Rab3A and Rab3B for such downstream effectors also could be
physiologically relevant, given a report that some neural cells express
both isoforms(44) . We also think it is likely that Rab3B can
regulate exocytosis independently of any such competitive interactions
it may have with Rab3A. For example, inhibition of Rab3B expression in
anterior pituitary cells (which do not express Rab3A) reportedly leads
to an inhibition of Ca-dependent
exocytosis(15) . In addition, our identification of a
rabphilin-like protein in colonic epithelial cells (i.e. cells
that express Rab3B but not Rab3A) indicates that Rab3B-rabphilin
interactions may be functionally relevant in a Rab3A-negative setting.
Rabphilin may be an important component of the exocytic machinery in a
variety of cell types where its functional activity may be regulated
differentially by various Rab3 isoforms.
We found that Rab3B N135I
also stimulates the efficiency of NE secretion in PC12 cells, provided
that it is membrane-associated. On the basis of the relative rates of
GTP and GDP dissociation for Rab3A N135I, it has been argued that this
mutant would likely be preferentially GTP-bound in
vivo(35) . Our results are consistent with this notion,
given that the GTP-bound form of a Rab protein is presumably the
functionally active form. However, we note that apparently conflicting
results have been obtained in studies examining the functional
properties of this corresponding mutation in Rabs other then Rab3
isoforms. Specifically, we observed that Rab3B and Rab3B N135I have
qualitatively similar effects on Ca-induced regulated
secretion in PC12 cells. Johannes et al.(17) have
reported a similar finding for Rab3A and Rab3A N135I; namely, both
inhibit Ca
-evoked secretion. In contrast, the
corresponding mutant of Rab1B and Rab5 function as dominant negative
mutants; e.g. Rab5 N133I inhibits endocytosis, whereas the
overexpression of wild type Rab5 stimulates endocytosis. As argued by
Holz et al.(16) , this disparity may be attributable
to the different dynamics of regulated secretion (e.g. Ca
-evoked catecholamine release) and
constitutive membrane traffic (e.g. endocytosis). For example,
in the context of a constitutive membrane traffic pathway where the
Rabs must continuously cycle on and off the relevant donor membrane,
such mutants may sequester certain accessory proteins (e.g. guanine nucleotide dissociation inhibitor or guanine nucleotide
exchange factor) in nonproductive conformations. However, the
corresponding Rab3 mutants, once targeted to a secretory granule where
they may remain until secretion is evoked, appear fully capable of
mimicking the corresponding wild type protein during a round of
stimulated secretion.
Perhaps the most surprising result of the
present study was the dramatic stimulation by Rab3B and Rab3B N135I of
the accumulation of [H]NE into PC12 secretory
granules. The approximately 8-fold (wild type Rab3B) and 50-fold (Rab3B
N135I) greater uptake cannot be explained by increased numbers of
secretory granules in Rab3B-expressing and Rab3B N135I-expressing
cells. Moreover, the effects of Rab3B and Rab3B N135I on NE secretory
efficiency and on NE uptake into granules may be separable;
specifically, whereas the stimulation of secretory efficiency appears
to require membrane association, catecholamine uptake was stimulated in
the virtual absence of any membrane association of Rab3B N135I (see Fig. 1B and 7; clone Rab3B N135I 32). The implication
of these results is that Rab3 isoforms are capable of regulating the
secretory pathway at multiple levels including the amount of cargo
available for secretion. We presently do not understand the mechanism
by which Rab3B and Rab3B N135I stimulate NE uptake into granules, nor
do we know if these Rab3 isoforms (including Rab3A) regulate other
aspects of catecholamine metabolism. In this regard we note that
rabphilin-3A binds GTP cyclohydrolase I, the rate-limiting enzyme for
catecholamine synthesis (45) . This observation, in combination
with the present results, argues for a detailed examination of the
roles of Rab3 isoforms in regulating the various steps in catecholamine
metabolism (i.e. biosynthesis, transporter activity, etc.) in
neuroendocrine cells. The various stably transfected PC12 clones that
we have generated should be useful for such an analysis.