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INTRODUCTION |
The Rab class of low molecular weight GTPases has been implicated
in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells (see Refs. 1-3 for reviews). Distinct
Rab proteins are associated with specific membranes and are necessary
for vesicle movement in the secretory (4-6) and endocytosis pathways
(7-9). For example, in yeast YPT1 is associated with the endoplasmic
reticulum and is necessary for endoplasmic reticulum to Golgi
transport; SEC4 is associated with post-Golgi vesicles and is required
for vesicle fusion with the plasma membrane. Recent studies of the
vacuole-vacuole fusion pathway in yeast implicate a Rab GTPase in a
reversible tethering step prior to SNAREmediated docking (10,
11).
We have been interested in Rab3a and related family members that play a
role in regulated secretion in mammalian cells. Rab3a is found in cells
including neurons and chromaffin cells with a highly differentiated
Ca2+-dependent secretory pathway (12-14). We
(15) and others (16) had previously demonstrated that Rab3a is a
negative regulator of exocytosis in experiments in bovine chromaffin
cells and PC12 cells. Examination of various mutants indicated that the
GTP-bound rather than GDP-bound Rab3a was the inhibitory form.
Experiments in permeabilized chromaffin cells suggest that Rab3a acts
upstream of the final fusion events triggered by Ca2+ and
may be involved in preparing the granules to undergo exocytosis (15).
The inhibition of secretion caused by overexpression of Rab3a is
consistent with the enhancement of secretion from hippocampal neurons
in Rab3a knockout mice (17) and the enhancement of secretion by
injection into chromaffin cells of antisense rab3a oligonucleotide (16).
The precise function of Rab proteins is unknown. It has been postulated
that Rab proteins may be important for the proper targeting of donor
membranes to the correct acceptor membrane. However, studies in yeast
with chimeras of YPT1 and SEC4 indicate that single constructs can
substitute for both YPT1 and SEC4 and maintain proper targeting of the
vesicles in the secretory pathway (18, 19).
The specific functions of the Rab GTPases are likely to be determined
by specific proteins with which they interact (2). For Rab3a, three
such proteins have been identified: Rabin3a (20), Rabphilin3 (21), and
Rim (Rab3-interacting molecule) (22). All three bind preferentially to
Rab3a-GTP. Rabin is predominately cytosolic with a widespread
distribution. Rabphilin3 co-localizes with Rab3a to synaptic vesicles
and chromaffin granules. Rim is localized to the plasma membrane of
nerve terminals, but not to synaptic vesicles.
Our studies have focussed on a possible pathway that modulates
regulated secretion through the coordinated interaction of Rab3a and
Rabphilin3. Rabphilin3 was initially identified as a 85-kDa protein,
which bound Rab3a with a much greater affinity for the GTP- than the
GDP-bound forms. Cloning revealed an open reading frame encoding 704 amino acids in bovine brain (21) and a 710 splice variant in bovine
chromaffin cells (23). Overexpression of Rabphilin3 in chromaffin cells
(23) and insulin-secreting cells (24) enhances stimulated secretion.
Both Rab3a and Rabphilin3 are found on synaptic vesicles and chromaffin
granules (14, 23, 25-28) where they can directly interact (29).
Rabphilin3 has two discrete domains (21, 30), an amino-terminal
Rab3a-GTP binding domain (Rp(51-190)) (31) and carboxyl-terminal
tandem C2 domains that confer Ca2+-dependent
binding to lipid vesicles containing phosphatidylserine and/or
phosphatidylinositol 4,5-bisphosphate (32, 33).
The opposite effects on secretion of Rab3a and Rabphilin3 suggest a
sequence of events in which the formation of a Rab3a-GTP·Rabphilin3 complex inhibits secretion and the subsequent release of Rabphilin3 from the complex (perhaps because of GTP hydrolysis) enhances secretion
(23, 31). Overexpressed Rab3a bound to GTP would inhibit secretion by
binding and reducing the amount of activated Rabphilin3 on the
chromaffin granule.
Rab3a is one of a subfamily of Rab3 GTPases. The four members of the
family have 219-228 residues with 85-90% amino acid identity from
amino acids 16 to 191 (34, 35). There is virtually complete identity in
the region (residues 51-59) that is homologous to the effector domain
(loop 2 or switch I) of Ras (Rab3c is the only member of the family
with a non-conservative amino acid in the consensus sequence). There is
much less identity (<30%) in the amino and carboxyl termini (residues
4-15 and 192-216) of the Rab3 family.
The Rab3 family members have been found in different tissues. Rab3a and
Rab3c are both associated with synaptic vesicles and secretory granules
in brain and neuroendocrine cells (13, 14, 25, 36, 37). Rab3b is
expressed in epithelial cells (38) and anterior pituitary cells (39).
Rab3d is expressed in adipocytes (35) and on zymogen granules in
pancreatic acinar cells (40, 41). Rab3a, -b, and -c have been shown to
interact with Rabphilin3 (21, 28, 42), although no quantitative
comparisons of the binding characteristics of the Rab3 proteins have
been reported.
Whereas a variety of different approaches indicate that Rab3a is an
inhibitory modulator of exocytosis, there is evidence that Rab3b and
Rab3d are positive regulators of secretion (39, 42, 43). To our
knowledge, there is no information concerning the direct functional
effects on secretion of Rab3c.
The diverse effects of different members of the Rab3 family in
different cell types observed using different techniques prompted us to
compare the localization and effects on secretion of transiently overexpressed Rab3 family members in chromaffin and PC12 cells. We
found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying
degrees in PC12 cells and in a fraction enriched in chromaffin granule
membranes. Overexpression of each of the four members of the Rab3
family inhibits secretion. Although the binding constants for the
interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d
with Rabphilin3 are comparable, investigation of Rab3a mutants with
impaired ability to interact with Rabphilin3 indicates that the
inhibition of secretion caused by overexpression of Rab3a is unrelated
to its ability to interact with Rabphilin3.
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MATERIALS AND METHODS |
Chromaffin Cell Preparation, Transfection, and Secretion
Experiments--
Chromaffin cell preparation, transient transfection
and secretion experiments were performed as described previously (15, 23, 44). Ca2+ phosphate precipitation was used for
transfections according to Wilson et al. (45) in 12-well
plates (22.6-mm well diameter) for secretion experiments and in 35-mm
diameter dishes for detection of expressed proteins. Secretion
experiments were generally performed 5-6 days after transfection at
27 °C in a physiological salt solution containing 145 mM
NaCl, 5.6 mM KCl,, 2.2 mM CaCl2,
0.5 mM MgCl2, 5.6 mM glucose, and
15 mM HEPES (pH 7.4). There were four wells or
dishes/group. Human growth hormone was measured with a high sensitivity
chemiluminescence assay from Nichols Institute (San Juan Capistrano,
CA). Endogenous catecholamine secretion was measured with a
fluorescence assay (60). Since only 1-4% of the cells are
transfected, catecholamine secretion mainly reflects secretion from
nontransfected cells and served as a control in the
hGH1 secretion experiments.
Secretion was expressed as the percentage of the total cellular human
GH (or catecholamine) that was released into the medium. There was
usually 0.5-2.0 ng of hGH and 20-40 nmol of catecholamine/22.6-mm
diameter well.
Isolation of P2 Fraction and Chromaffin Granule
Membranes from Bovine Adrenal Medullae--
Fresh bovine adrenal
medullae were homogenized in 0.29 M sucrose, 10 mM Hepes (pH 7.1), 0.5 mM EDTA, and 0.5 mM PMSF. The supernatant from a 10-min 800 × g centrifugation was re-centrifuged at 27,000 × g for 10 min to generate a large granule fraction (P2 fraction). The P2 pellet was resuspended in
homogenization buffer, and 8 ml was layered onto 26 ml of a solution
containing 1.7 M sucrose, 2 mM Hepes (pH 7.1),
and 0.1 mM EDTA. The tubes were centrifuged at 194,000 × g for 60 min in a 50.2 Ti ultracentrifuge rotor (Beckman
Instruments, Fullerton, CA). The pellet at the bottom of the tube
consisted of highly purified chromaffin granules. The chromaffin
granules in the pellet were lysed in 10 mM Hepes (pH 7.1),
0.2 mM EDTA, frozen, and thawed. The chromaffin granule membranes were pelleted by centrifugation at 30,000 × g for 20 min and resuspended in lysis buffer. They were
again centrifuged at 30,000 × g for 20 min, and the
chromaffin granule membrane pellet was resuspended in 10 mM
Hepes (pH 7.1) without EDTA, aliquoted, and stored at
70 °C.
Monolayers of PC12 cells plated in 60-mm dishes were grown for 2 days
and subcellular fractionation performed as described previously (46).
PC12 cells were harvested into 1 ml of hypotonic lysis buffer (10 mM Tris, pH 7.4, 5 mM KCl, 2 mM
MgCl2, and 1 mM PMSF) and incubated on ice for
10 min. The cells were ruptured by passage through a 26-gauge needle
and centrifuged at 100,000 × g for 10 min. The
supernatant was removed, and the pellet was rinsed twice and
resuspended in 1 ml of hypotonic lysis buffer. Trichloroacetic acid was
added to a final concentration of 10% and the samples were incubated
on ice for 30 min and then centrifuged at 16,000 × g
for 15 min. The pellets were washed in acetone and redissolved in 0.1 N NaOH, 0.1% deoxycholate for SDS-PAGE and blotting.
SDS-PAGE, Western Analysis, and Immunocytochemistry--
The
expression of transiently transfected plasmids in bovine chromaffin
cells and PC12 cells was examined 6 and 2 days, respectively, after
transfection. Cells were harvested into sample buffer and subjected to
12% or 15% SDS-PAGE, followed by immunoblotting with anti-HA1
antibody (1:5000 dilution) and protein detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Rab3a Binding Assays--
[
-32P]Rab3a, Rab3b,
Rab3c, and Rab3d binding to GST-Rabphilin3(1-206) was investigated as
in previous experiments (46).
Experiments investigating the binding of Rabphilin3 to Rab3a constructs
with mutants in the putative effector domain of Rab3a were performed as
follows. HA-tagged Rab3a, Rab3a(F51L), Rab3a(T54A), Rab3a(F59S), and
Rab5 (as a control) proteins were expressed by transient transfection
in COS cells. Cell lysates in 50 mM HEPES, pH 7.4, 1%
Triton X-100, 2 mM MgCl2, 150 mM
NaCl, 1 mM DTT, and 1 mM PMSF were centrifuged
2 min at 500 × g to remove nuclei, and cell debris and
supernatants were spun at 100,000 × g for 45 min.
Concentrations of the supernatants were adjusted to 50 mM
HEPES, pH 7.4, 0.5% Triton X-100, 1 mM MgCl2,
150 mM NaCl, 1 mM DTT, 5 mM EDTA,
and 1 mM PMSF. In half of the supernatants, 1 mM GTP
S was added. After 30 min at 4 °C,
MgCl2 was added to a final concentration of 10 mM and extracts were incubated for 1 h at 4 °C with
5 mg of GST-Rabphilin(1-206) bound to glutathione-Sepharose beads.
Beads were washed twice in 50 mM HEPES, 0.5% Triton X-100, 5 mM MgCl2, 150 mM NaCl, and 1 mM DTT, and then twice in the same buffer minus Triton
X-100. Denaturing loading buffer was added to the beads and to the
second half of the supernatant from the ultracentrifugation. Samples
were submitted to SDS-PAGE and then transferred to nitrocellulose
membranes. HA-tagged proteins were detected by incubation with anti-HA1
antibody, followed by enhanced chemiluminescence as described
previously (46). The amount of HA-tagged Rab protein bound to Rabphilin
was quantitated from the blots and related to the total amount of the
protein present in the supernatant.
Immunocytochemistry and confocal microscopy were performed as described
previously to determine the localization of transiently expressed hGH
and the HA1 epitope in PC12 cell (26, 31, 46). Overlap in the
fluorescein isothiocyanate channel (hGH) and the rhodamine channel
(HA1-Rab3) was determined by multiplication of the images pixel by
pixel in the two channels using NIH Image (1.59). The resulting pixel
intensities were scaled to fit an eight-bit scale.
Antibodies and Plasmids--
Human GH and the HA1 epitope
(YPYDVPDYA) were detected by confocal microscopy with rabbit anti-human
pituitary GH polyclonal antibody (1:1000, National Hormone and
Pituitary Program, NIDDKD, National Institutes of Health, Bethesda, MD)
and mouse anti-HA1 (monoclonal antibody 12CA5, 1:500-1:2000, Berkeley
Antibody Co.) with appropriate secondary anti-rabbit and anti-mouse
antibodies tagged with Oregon Green 488 or lissamine rhodamine
(Molecular Probes, Eugene, OR). Anti-Rab3a was a rabbit peptide
antibody to a unique 13-amino acid stretch near the carboxyl terminus
(47). Rabbit polyclonal antibody to human Rab3b (38) was a gift from Dr. K. L. Kirk (University of Alabama, Birmingham, AL). Anti-Rab3c was a rabbit peptide antibody directed against amino acids 195-209 of
the bovine sequence and was obtained from Dr. Ahmed Zahraoui (Institut
Curie, Paris, France). Anti-Rab3d anti-serum (40) was obtained from Dr.
Mark McNiven (Mayo Clinic, Rochester, MN). The specificity of these
antibodies has been documented in the literature. We verified that
anti-Rab3b detected only Rab3b by SDS-PAGE and immunoblots of COS cells
transiently expressing equivalent amounts of Rab3a, Rab3b, Rab3c, or
Rab3d. Rat Rab3a, Rab3b, Rab3c, mouse Rab3d, and human Rab8 plasmids
were constructed under transcriptional control of the immediate-early
enhancer-promoter region of the human cytomegalovirus (CMV) and usually
contained the HA1 epitope (YPYDVPDYA) in frame at the amino terminus
(48). Rab3a, Rab3b, and Rab3c were amplified from reverse transcribed
rat brain mRNA, and sequences were confirmed. Mouse Rab3d was
obtained from Drs. H. Ohnishi and J. A. Williams (University of
Michigan, Ann Arbor, MI). The plasmid encoding human Rab3b (under
control of the CMV promotor) (42) was a gift from Dr. K. L. Kirk.
The cDNA encoding Rab8 was obtain from A. Zahraoui (INSERM, U-248,
Paris, France). For the immunocytochemistry in Fig. 6, three
consecutive HA epitopes were present at the amino terminus of the Rab3
family members. The construction of GFP-Rabphilin was described
previously (46).
Data are generally expressed as mean ± standard error of the mean.
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RESULTS |
Members of the Rab3 Family Are Detected in Chromaffin Granule
Membranes and PC12 Cells--
Immunoblots of equal amounts of proteins
in a P2 fraction and a highly enriched chromaffin granule
membrane fraction prepared from fresh adrenal medulla detected Rab3a,
Rab3b, Rab3c, and Rab3d (Fig. 1). The
primary antibodies that were used for detection of the various Rab3
proteins were highly specific with virtually no cross-reactivity (see
"Materials and Methods"). Significantly more of each of the Rab
isoforms were detected in the chromaffin granule membrane fraction than
in the P2 fractions consistent with their localization on
chromaffin granules.

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Fig. 1.
Endogenous Rab3a, Rab3b, Rab3c, and Rab3d are
detected in a chromaffin granule membrane fraction and in PC12
cells. CC, chromaffin cells; s, soluble
fraction; p, particulate fraction;
P2, pellet; CG, chromaffin granule
membrane fraction.
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The expression of the various Rab3s was also investigated in PC12
cells, a clonal cell line from a rat adrenal medullary tumor (Fig. 1).
The four different Rab3 GTPases could be detected to varying degrees
with Rab3b only weakly detected. Equal proportions of the soluble and
particulate fractions were applied to the gels. Rab3a, Rab3b, and Rab3d
were mainly particulate. Rab3c was found to greater extent in the
soluble fraction.
It had been previously reported that PC12 cells do not express Rab3b
(42). The low level of expression was detected with the same anti-Rab3b
antibody used in the previous study. The difference in results could
reflect a difference in the two PC12 cell lines.
HA-tagged Rab3 Family Members Can Be Transiently Expressed in
Bovine Chromaffin Cells and PC12 Cells and Localize to Secretory
Granules--
HA-tagged members of the Rab 3 family could be readily
expressed in bovine chromaffin cells (Fig.
2A) and PC12 cells (Fig. 2B) by transient transfection.

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Fig. 2.
Transiently transfected HA-Rab3a, Rab3b,
Rab3c, and Rab3d are expressed in bovine adrenal chromaffin cells and
PC12 cells. s, soluble fraction; p,
particulate fraction.
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Confocal microscopy was performed on PC12 cells transiently
cotransfected with a plasmids encoding hGH and one of the HA-Rab3 GTPases. PC12 cells have far fewer secretory granules than bovine chromaffin cells and have a characteristic peripheral localization near
the plasma membrane that facilitates resolution of the granules (49).
HGH-containing granules tended to be localized adjacent to the plasma
membrane (Fig. 3, Control).
Often a perinuclear staining was also observed suggestive of Golgi.
Transfected HA-Rab3a, -b, -c, and -d all had a punctate, peripheral
localization that overlapped with the cotransfected hGH in the cell
periphery (right side of each panel in Fig. 3).
The transfected Rab3 GTPases were not observed in a perinuclear
region.

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Fig. 3.
Transfected HA-tagged Rab3a, Rab3b, Rab3c,
and Rab3d colocalize with hGH-containing granules in the periphery of
PC12 cells. PC12 cells were cotransfected with a plasmid
expressing hGH and with plasmids expressing HA-Rab3a, HA-Rab3b,
HA-Rab3c, or HA-Rab3d or with a control plasmid, pCMVneo. Two days
later, cells were fixed and the expression of hGH and the HA-tagged
proteins were detected by confocal microscopy. The left side
of each panel shows hGH; the right side shows HA-Rab. Notice
the tendency for peripheral localization of hGH-containing granules.
The HA-Rab proteins tend to colocalize with hGH granules. All but one
of the images were optical sections through the middle of the cells.
The middle right panel presents an
optical section tangent to the surface of a HA-Rab3b-transfected cell.
The calibration bar corresponds to 5 µm.
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The colocalization of hGH and the four members of the rab3 family was
apparent both in optical sections through the center of cells and in
optical sections tangent to the cell surface (e.g. see
Rab3b, right panel). The colocalization was
confirmed by computer-generated maps of pixels with significant
intensities in both channels (dark spots and areas in Fig.
4). The peripheral localization of the secretory granules was not disrupted by the transient expression of any
of the Rab3 family members.

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Fig. 4.
Computer-determined overlap of hGH and HA-Rab
immunofluorescence. The dark spots and areas correspond to pixels
with significant intensities due to expression of hGH and either
HA-Rab3a or HA-Rab3b. The analysis was performed on the images in Fig.
3.
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Members of the Rab3 Family of GTPases Interact Similarly with
Rabphilin3--
Rabphilin3 interacts with Rab3a-GTP and has been
postulated to be part of a Rab3a/Rabphilin3 pathway that regulates
exocytosis. The abilities of the Rab3-GTP isoforms to bind to the Rab3a
binding domain of Rabphilin3 were compared (Fig.
5). Various concentrations of
Rab3-[
-32P]GTP were incubated with
GST-Rabphilin3(1-206) bound to glutathione-Sepharose. The binding was
saturable with Kd values of 10-20 nM for all the Rab3 isoforms (Table I). The
amount of GST-Rabphilin3(1-206) in the assay was 1.34 nmol. The
Bmax values of the isoforms were similar with a
range of 0.8-1.5 nmol, indicating that the stoichiometry of the
binding was approximately 1:1. The binding studies suggest that Rab3a,
Rab3b, Rab3c, and Rab3d interact similarly with Rabphilin3.

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Fig. 5.
Rab3a, Rab3b, Rab3c, and Rab3d bind to
Rabphilin3 in a similar manner. Rabphilin3 (1.34 pmol) was present
in each assay (100 µl). Data points are the means of duplicates with
the range in all cases less than 5%. Lines were plotted with a
non-linear curve fitting algorithm (Levenberg-Marquardt) for a simple,
one-site binding model (B = Bmax/(Kd + S)),
where Bmax = total amount of bound Rab3 (pmol),
Kd = dissociation constant, and S = free concentration of Rab3. See Table I for binding parameters.
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Table I
Rab3a, -b, -c, and -d bind to Rabphilin3 with similar binding constants
Rabphilin3 (1.34 pmol) was present in each assay. See Fig. 5 legend for
experimental details and binding curves.
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Colocalization of HA-tagged Rab3 Family Members with Cotransfected
GFP Rabphilin3 in PC12 Cells--
The similar ability of the members
of the Rab3 family to interact with Rabphilin3 in vitro
suggested that members of the Rab3 family would similarly co-localize
with Rabphilin3 in situ. Confocal microscopy of transfected
cells indicates that this prediction is correct (Fig.
6). PC12 cells were transfected with
plasmids encoding GFP-Rabphilin3 and one of either HA-Rab3a, -b, -c, or -d. Cells were subsequently treated with nerve growth factor to cause
process formation. Cells were fixed and processed for HA1 immunocytochemistry. Rabphilin3 was detected by the fluorescence of
GFP. Both the GFP-Rabphilin3 and all of the Rab3 GTPases tended to
accumulate in the processes. A pixel-by-pixel analysis demonstrated substantial overlap of GFP-Rabphilin3 and each of the Rab3
proteins.

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Fig. 6.
Colocalization of HA-Rab3 proteins with
GFP-Rabphilin3 in nerve growth factor-treated PC12 cells. PC12
cells were transfected with plasmids expressing GFP-Rabphilin3 and the
indicated HA-Rab3. The localization of the HA-tagged proteins was
determined by HA immunocytochemistry. The localization of
GTP-Rabphilin3 was determined by the intrinsic fluorescence of GFP. The
pixel-by-pixel colocalization of the HA-Rab3 proteins and
GFP-Rabphilin3 is shown in the bottom
panel.
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Members of the Rab3 Family Inhibit Secretion from Bovine Chromaffin
Cells and PC12 Cells--
The effects of overexpression of HA-tagged
Rab3 family members on secretion were compared in bovine chromaffin
cells and PC12 cells (Fig. 7). Cells were
transiently co-transfected with plasmids encoding hGH and one of the
HA-Rab3 family members. Two days later (PC12 cells) or 6 days later
(bovine chromaffin cells), hGH secretion was stimulated with either
elevated K+ (PC12 cells) or the nicotinic agonist, DMPP
(bovine chromaffin cells). All of the Rab3 family members inhibited
secretion (Fig. 7). In both chromaffin and PC12 cells, overexpression
of Rab3a, Rab3b, Rab3c, and Rab3d inhibited stimulated secretion by
50% or more. These effects were specific since transfection with a plasmid encoding HA-Rab8 resulted in expression of the GTPase in
chromaffin cells (data not shown) but did not alter secretion (Fig.
7A).

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Fig. 7.
The Rab3a family of GTPases inhibit secretion
from bovine chromaffin cells (A) and PC12 cells
(B). A, bovine chromaffin cells were
transfected with pXGH5 and a control plasmid (pCMVneo, parent plasmid)
or one expressing one of the indicated HA-tagged Rab GTPases. Six days
later, secretion was stimulated with the nicotinic agonist DMPP (20 µM) for 2 min. Three separate experiments are shown.
B, PC12 cells were transfected similarly to bovine
chromaffin cells. Three days later cells were stimulated with elevated
K+ for 15 min. [3H]NE secretion induced by
elevated K+ was 17.5-20% for all groups. There were four
wells per group. Two separate experiments are shown. The inhibition of
hGH secretion from both chromaffin cells and PC12 cells by all the Rab3
plasmids was statistically significant (p < 0.01 for
all Rab3 GTPases except for the inhibition of secretion from PC12 cells
by Rab3c where p < 0.05). Ctrl,
control.
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It has been reported that ionomycin (calcium ionophore)-induced
secretion is enhanced from PC12 cell lines stably expressing human
Rab3b (42). Ionomycin-induced hGH secretion from PC12 cells was
inhibited 53% (mean of 47% and 58% inhibition in two experiments) by
transient expression of HA-Rab3b. Because stable transfectants may
express less protein than transient transfectants, the effects of
reducing the amount of the HA-Rab3b-encoding plasmid during
transfection on the subsequent inhibition of secretion was
investigated. Decreasing the amount of plasmid encoding rab3b by
10-fold (keeping the total DNA in the transfection constant with
control DNA) did not cause an enhancement of secretion (data not
shown). The Rab3b construct was derived from the rat cDNA sequence.
Transient transfection with the human rab3b construct used in the
earlier study similarly inhibited elevated K+ (secretion
relative to CMV.neo control, 0.491 ± 0.039, n = 3 experiments). The experiments do not reveal an ability of Rab3b to
enhance secretion in PC12 or chromaffin cells.
Inhibition of Secretion Caused by Mutations in Rab3a That Reduce or
Eliminate Rabphilin3 Binding--
A number of point mutations in Rab3a
were created and investigated for their ability to inhibit binding to
Rabphilin3 and to inhibit secretion. The binding of Rabphilin3 to
Rab3a(T54A) or Rab3a(F59S) was reduced by 82% or 98%, respectively,
compared with binding to wild type Rab3a (Fig.
8). It has been previously demonstrated
in PC12 cells that Rab3a(T54A) partitions between soluble and
particulate fractions and colocalizes with GFP-Rabphilin3 in
immunocytochemistry experiments identically to wild type Rab3a (46). We
found that Rab3a(T54A) colocalized to hGH-containing granules in
transfected PC12 cells identically to wild type Rab3a, consistent with
these findings (data not shown). Rab3a(F59S) also colocalized to
hGH-containing granules. The images suggested that there was also a
significant amount of cytosolic Rab3a(F59S) (data not shown). Both
these mutations were as effective as wild type Rab3a in inhibiting
secretion in chromaffin cells and PC12 cells (Fig.
9). Rab3a (F51L) bound Rabphilin3
similarly to wild type Rab3a and also inhibited secretion. The data
suggest that, although all members of the Rab3 family bind Rabphilin3,
the inhibition of secretion caused by Rab3 does not require this
interaction.

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Fig. 8.
Binding of different Rab3a effector mutants
to GST-Rabphilin(1-206). Rab3a wild type (wt),
effector domain mutants Rab3a(F51L), Rab3A(T54A), Rab3A(F59S), or Rab5
were expressed in COS cells, solubilized with Triton X-100 and
incubated with GTP S and GST-Rabphilin bound to beads as described
under "Materials and Methods." A, immunodetection after
SDS-PAGE and immunoblotting using monoclonal anti-HA antibody.
Upper panel, Rab proteins bound to GST-Rabphilin;
lower panel, expression of HA-tagged Rab in COS
cells. B, binding of Rab3a effector mutant proteins to
GST-Rabphilin relative to the binding of the wild type protein (100%).
Data are the mean of two to four independent experiments.
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Fig. 9.
Rab3a mutants defective in Rabphilin3 binding
inhibit secretion from chromaffin cells (A) and PC12
cells (B). Cells were transfected and secretion
stimulated as described in Fig. 7. Panels A and
B each show two experiments. WT, wild type;
Ctrl, control.
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DISCUSSION |
The similar primary sequences of the Rab3 family, and the
virtually identical sequences of the putative effector domains
suggested that they would bind similarly to Rabphilin3 and would have
similar effects on secretion. All members of the Rab3 family were
expressed in PC12 and chromaffin cells, and bound Rabphilin3 similarly. When overexpressed in chromaffin or PC12 cells they all inhibited secretion. However, analysis of the effects of Rab3a mutants indicated that the binding to Rabphilin3 was unnecessary for the inhibition of
secretion. These issues are discussed below.
Members of the Rab3 Family Are Enriched into Chromaffin Granule
Fraction and Are Expressed in PC12 Cells--
Antibodies that were
specific for each of the Rab3 GTPases demonstrated that Rab3a, Rab3b,
Rab3c, and Rab3d are enriched in a chromaffin granule membrane fraction
(compared with a P2 fraction) from the adrenal medulla and
are expressed to varying degrees in PC12 cells. Although it is possible
that some of the Rab3 GTPases in the chromaffin granules fraction are
contaminants from adrenal cortical, endothelial or fibroblast cells,
their expression in PC12 cells, a cell line derived from tumor of rat
chromaffin cells, supports the notion that all four isoforms can be
expressed in chromaffin cells. There is evidence that Rab3b may also be
associated with the plasma membrane in chromaffin cells (50).
Secretory granules in PC12 cells tend to be peripherally localized
(49). Transiently expressed, HA-tagged Rab3a, Rab3b, Rab3c, and Rab3d
all co-localized with hGH in granules in the periphery of the cells.
This observation is consistent with previous work that demonstrated by
immunocytochemistry or subcellular fractionation the presence of
endogenous or transfected Rab3a, Rab3b, and Rab3c on chromaffin
granules from the adrenal medulla or on secretory granules in PC12
cells (14, 26, 42). Rab3d is associated with zymogen granules in
pancreatic acinar cells (40, 41) and perhaps with Glut4-containing
vesicles in adipocytes (35). Overexpression of none of the
isoforms had noticeable effects on the distribution of the
hGH-containing granules in PC12 cells detected by immunocytochemistry.
The Similar Inhibition of Secretion by Rab3 Family Members Does Not
Involve Binding to Rabphilin3--
Rab3a, Rab3b, Rab3c, and Rab3d all
inhibited secretion from PC12 and chromaffin cells. Direct in
vitro binding assays demonstrated similar affinities and
Bmax values for Rab3a, Rab3b, Rab3c, and Rab3d
for binding to recombinant Rabphilin3. Since the overexpression of
Rabphilin3 enhances secretion in chromaffin cells and PC12 cells, it
was possible that the inhibition of secretion caused by overexpression
of each of the members of the Rab3 family could have been caused by
binding endogenous Rabphilin3. However, a mutant with reduced ability
to bind Rabphilin3 (Rab3aT54L) and a mutant devoid of measurable
Rabphilin3 binding (Rab3aF59S) inhibited secretion from chromaffin and
PC12 cells similarly to wild type Rab3a. Both of the mutations are in a
region (the putative switch 1 region of Rab3a) that that has been
recently demonstrated to interact with Rabphilin3 (51). Phenylalanine
59 directly contacts Rabphilin3. The experiments indicate that the
inhibition of secretion caused by overexpression of Rab3a does not
involve direct interaction with Rabphilin3.
In an earlier study we provided evidence that the enhancement of
secretion induced by overexpression of Rabphilin3 does not require
binding to Rab3a-GTP (31). Expression of an amino truncated mutant of
Rabphilin3 that did not bind Rab3a-GTP enhanced secretion similarly to
wild type Rabphilin3. A similar conclusion was drawn from experiments
with insulin-secreting cells (24). Thus, although Rab3a and Rabphilin3
are located on the same secretory granule or vesicle and interact both
in vitro (21) and in situ (29), the
inhibition of secretion by Rab3a and the enhancement of secretion by
Rabphilin3 are each independent of the direct interaction of the two proteins.
Although we have been unable to demonstrate a direct function of the
binding of Rab3a and Rabphilin3 in the secretory pathway, there are
other known consequences to their interaction. The binding of
Rabphilin3 to Rab3 stabilizes Rabphilin3 in chromaffin cells (31) and
neurons (52) and may play a role in transporting Rabphilin3 to the
nerve terminal (28). In addition, the interaction of Rab3a to the amino
terminus of Rabphilin3 prevents the interaction of Rabphilin3 with
-actinin (53), an actin-binding protein, and with Rabaptin5 (54), a
protein important in the endocytic pathway. Thus, the association of
Rab3a and Rabphilin3 may regulate the cytoskeleton and/or endocytosis
in secretory cells.
Members of the Rab3 Family Have Diverse Effects on
Secretion--
Experiments in chromaffin cells, PC12 cells, an
insulin-secreting cell line, and hippocampal neurons suggest a
consistent role for Rab3a as an inhibitory modulator of exocytosis.
Injection of Rab3a antisense oligonucleotides into chromaffin cells
(16, 55) enhances exocytosis. Similarly, removal of the Rab3a gene in
mice enhances vesicular release in cultured hippocampal neurons (17).
Conversely, overexpression of Rab3a inhibits exocytosis in PC12,
chromaffin cells (15, 16) and insulin-secreting cells (37). Secretory
granules in PC12 cells, chromaffin cells, and insulin-secreting cells
are derived from the trans-Golgi network of the protein secretory
pathway, whereas synaptic vesicles participate in an
exocytotic-endocytotic cycle. The common negative modulation of both
pathways by Rab3a is consistent with a common step in exocytosis being
modulated, perhaps related to the function of SNARE proteins
(56-58).
Experiments investigating the roles of Rab3b and Rab3d in secretion
give a less consistent picture. The introduction of antisense oligonucleotides to Rab3b into pituitary cells inhibits exocytosis (39)
and overexpression in mice of Rab3d using a transgenic approach
enhances exocytosis, suggesting that both Rab3b and Rab3d normally
enhance secretion. On the other hand, we determined in this study that
overexpression of Rab3b or Rab3d inhibits secretion in PC12 and
chromaffin cells. In addition, expression of Rab3dN135I inhibits
regulated secretion in AtT-20 cells (59). A comparable mutation in
Rab3a greatly increases GTP and GDP exchange rates and is predicted to
render the protein predominantly GTP-bound within cells (48). The N135I
mutation in Rab3a inhibits secretion in PC12 cells and chromaffin cells
(15, 16). Thus, it is likely that the same Rab protein in different
cells can have different overall effects on the secretory pathway.
One possible explanation for the different effects of the same Rab3
isoform in different cells is that different cells have different
effector pathways. The present study indicates that differences in the
expression of one possible effector, Rabphilin3, are unlikely to be the
basis for the difference of effects in the different cells types. In
addition to the three proteins that bind Rab3-GTP, Rabin, Rabphilin3,
and Rim, there are likely to be other effectors for the Rab3 family.
Genetic experiments indicate that the single Rab3 homologue in
Caenorhabditis elegans enhances but is not essential for
synaptic transmission (60). This GTPase may act through AEX-3, which is
homologous to segments in DENN, a human protein of unknown function
(61).
Effects of Rab3b in Secretion--
A complication in understanding
the role of Rab3b in exocytosis is the finding that when Rab3b is
expressed in stably transfected PC12 cells, it enhanced
ionomycin-induce [3H]norepinephrine secretion (42). We
found that overexpression of Rab3b by transient transfection inhibited
protein secretion induced by elevated potassium and ionomycin in PC12
cells and by DMPP in chromaffin cells. Since transient transfectants
may express more protein than stable transfectants, we attempted
without success to enhance secretion by reducing the amount of
Rab3b-encoding plasmid used in the transfections. A possible
explanation for the different effects is that, in the previous
experiments with stably transfected PC12 cells, there was an average
8-fold increase in [3H]norepinephrine stored
intracellularly in the various clonal lines. Although there was no
change in the number or distribution of large dense core vesicles in
the clones, there may be another vesicular compartment that expanded
and accounts for the change. It need not be a protein-secreting
compartment since [3H]norepinephrine enters the
compartment from the cytosol. The large increase in storage in the
stable Rab3b-expressing cell lines compared with control or
Rab3a-overexpressing cell lines creates uncertainties in the
comparisons of secretory responses of the various clones, despite
secretion being normalized to the total
[3H]norepinephrine content in the cells. In the present
study, immunocytochemistry of Rab3b-transfected cells did not reveal a
qualitative change in the number of the growth hormone-containing
granules or their distribution in the periphery of the cell.