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INTRODUCTION |
Rab proteins are Ras-like GTPases that play important roles in
mediating vesicular membrane trafficking in eucaryotic cells (1-3).
More than 40 mammalian and 11 yeast Rab proteins have been
characterized. They are localized in different compartments and control
distinct transport systems. The Rab3 subfamily is associated with
secretory granules and vesicles and plays a role in regulated
secretion. Rab3a and Rab3c are associated with synaptic vesicles and
chromaffin granules (4-8). Rab3a inhibits secretion when overexpressed
in chromaffin cells or PC12 cells (9, 10). Experiments with various
protein mutants indicate that the GTP-bound rather than the GDP-bound
Rab3a is the inhibitory form. Mice with Rab3a genetically removed show
a variety of alterations in synaptic transmission consistent with Rab3a
modulating vesicular trafficking in the nerve terminal (11-13).
Like other GTP-binding proteins, Rab3a is thought to regulate vesicular
trafficking by interacting with effector proteins. To date, three
possible Rab3a effectors have been identified: Rabin (14), Rabphilin3
(15-17), and Rim family members (18, 19). All preferentially bind to
GTP-bound Rab3a. Rabin has no apparent effect on secretion. Rabphilin3
and Rim1 both enhance secretion and possess similar domains. Both
contain an N-terminal zinc finger domain and two C-terminal C2 domains.
The C2 domains of Rabphilin3 bind calcium and acidic phospholipids (20,
21) with especially strong and specific interactions with
phosphatidylinositol 4,5-bisphosphate (20). The C2 domains of
Rabphilin3 and Rim1 are not highly homologous, suggesting that the
lipid and Ca2+ binding characteristics are different. Rim,
but not Rabphilin3, contains a PDZ domain that may contribute to its
reported localization to presynaptic release sites (18).
Rabphilin3 is present on synaptic vesicles and secretory granule
membranes and binds Rab3a in situ (22). Overexpression of
full-length Rabphilin3 increases secretion by 30% in chromaffin cells
(23) and 2-fold in insulin-secreting cells (24, 25). Binding of
Rab3a-GTP stabilizes Rabphilin3 in chromaffin cells (23) and probably
in brain (17) but is not necessary for the enhancement of secretion
(23, 25).
Although Rim1 and Rabphilin3 have structural homologies, the
proteins have different functions and characteristics in
situ. Rim1 is stable in Rab3a knockout mice, and its localization
in nerve terminals is reported to be at active zones and not on
synaptic vesicles (18). N-terminal Rim1, which contains the Rab3a
binding domain, enhances secretion in PC12 cells (18), in contrast to the homologous domain of Rabphilin3, which inhibits secretion (23, 27).
The zinc finger-containing region in Rabphilin3 is also required for
binding to Rab3a-GTP (22, 23, 28).
In this study, we determined the minimal domains of Rim1 necessary for
Rab3a binding and for the enhancement of secretion. We found that the
domains are distinct, are both located in the N terminus of Rim1, and
can function independently. Surprisingly, the Rab3a binding domain of
Rim1 does not include the zinc finger domain but requires only a short
segment of ~30 amino acids N-terminal to the zinc finger domain.
Finally, we determined the step in the secretory pathway at which
N-terminal Rim1 enhances secretion.
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MATERIALS AND METHODS |
N-terminal Rim1 Cloning and Sequencing--
According to the
Rim1 sequence (18), two primers (5'-cgg gat
cca tgt cct cgg ccg tg-3'; 5'-gga
att cct aca tgc gca tct gct gct c-3') were
designed to amplify Rim(1-399) from rat brain mRNA (CLONTECH) using reverse transcription-polymerase
chain reaction. Polymerase chain reaction products were digested with
BamHI and EcoRI and then cloned into pCMV-HA, a
mammalian expression vector that contains the hemagglutinin epitope
(PYDVPDYA). Sequencing analysis showed that residues 56-105 were
missing in splice site #1 (19).
Construction of Full-length and N-terminal Deletion
Mutants--
Deletion mutants of Rim1 were made by polymerase chain
reaction from Rim(1-399). Primers were designed to generate
5'-BamHI and 3'-EcoRI restriction sites.
Polymerase chain reaction products were digested with BamHI
and EcoRI and subcloned into pGEX-2T (Amersham Pharmacia
Biotech) and pCMV-HA. Full-length Rim1 cDNA (from Dr. Thomas
Sudhof) was found to have an unexpected N-terminal stop codon. A
plasmid encoding full-length Rim1 was constructed by digesting the
plasmid from Dr. Sudhof with KpnI and EcoRI to obtain a C-terminal fragment. The fragment was then ligated to the
BamHI/KpnI fragment from Rim(1-399). The
resulting construct was subcloned into pCMV-HA to obtain full-length
Rim1 (residues 1-1553). Because of the various splice sites, the
full-length Rim did not contain residues 56-105 in splice site #1,
residues 1032-1106 in splice site #4, and residues 1145-1316 in
splice site #5.
Chromaffin Cell Preparation, Transfection, and Secretion
Experiments--
Chromaffin cell preparation, transient transfection,
and secretion experiments were performed as described previously (9, 29). Calcium phosphate precipitation was used for transfections according to Wilson (30) in 12-well plates (22.6-mm well diameter). Secretion experiments were performed 4-5 days after transfection at
30 °C in a physiological salt solutions containing 145 mM NaCl, 5.6 mM KCl, 2.2 mM
CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, 15 mM HEPES, pH 7.4, and 0.5 mM ascorbate. Permeabilized cell experiments were performed
in potassium glutamate solution (KGEP) containing 139 mM
potassium glutamate, 20 mM
PIPES,1 pH 6.6, 2 mM MgATP, 5 mM EGTA with various concentrations
of Ca2+ that resulted in free Ca2+
concentrations between 0 and 30 µM Ca2+ (31).
Human growth hormone (hGH) was measured with a high sensitivity chemiluminescence assay from Nichols Institute (San Juan Capistrano, CA). Endogenous catecholamine secretion was measured with a
fluorescence assay (32). Stimulated release was calculated as the
fraction of total hGH or catecholamine released into the incubation
medium. Data were expressed as mean ± S.E. Significance was
determined by Student's t test. There was usually 0.5-1.0
ng of hGH and 30-60 nmol of catecholamine/22.6-mm diameter well.
GST Fusion Protein Expression and Rab3a Binding
Assays--
GST-Rim1 fusion proteins were expressed in
Escherichia coli BL21-CodonPlusTM(DE3)-RP cells
(Stratagene) and HB101 cells by
isopropyl-
-D-thiogalactoside induction. Bacterial
lysates were incubated with glutathione-Sepharose 4B beads (Amersham
Pharmacia Biotech) for 1 h at 4 °C and washed 3 times with 1×
phosphate-buffered saline.
Rab3a binding assays were performed as described previously (33). Human
embryonic kidney 293 cells in 35-mm dishes (5 × 105
cells/dish) were transfected with HA-tagged Rab3a using LipofectAMINE (Life Technologies). 1 ml of ice-cold lysis buffer containing 50 mM HEPES, pH 7.4, 1% Triton X-100, 2 mM
MgCl2, 150 mM NaCl, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride was added 48-60 h after transfection. Cell lysates were spun at
100,000 × g for 15 min to remove debris. Supernatants
were adjusted to 50 mM HEPES, pH 7.4, 0.5% Triton X-100, 1 mM MgCl2, 150 mM NaCl, 1 mM dithiothreitol, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride and then incubated with 1 mM GTP
S or 1 mM GDP for 30 min at 4 °C.
MgCl2 was added to a final concentration of 10 mM, and extracts were incubated for 1.5 h at 4 °C
with 5 µg of GST-Rim1 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 dithiothreitol and once in the same buffer minus Triton
X-100. Proteins were boiled in 1× SDS sample buffer and subjected to 10% SDS-polyacrylamide gel electrophoresis followed by immunoblotting with the 12CA5 anti-HA antibody (1:2000 dilution). Rab3a was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Immunocytochemistry and Confocal
Microscopy--
Chromaffin cells were plated on glass coverslips
(Fisher, No.1 thickness) fastened to the bottom of punched-out wells on
12-well plates (diameter 22.6 mm). Coverslips were sequentially coated with poly-D-lysine and calf skin collagen to promote cell
adhesion. Cells were cotransfected with ANP-GFP and HA-tagged Rim1
constructs, fixed in paraformaldehyde, and permeabilized with methanol
4-5 days after transfection. Fluorescence microscopy was performed as
previously described (9). ANP-GFP was visualized directly by the
intrinsic fluorescence of the GFP in the fluorescein isothiocyanate channel. HA-tagged Rim1 was visualized with the 12CA5 anti-HA antibody
(1:1000 dilution) and goat anti-mouse Alexa 568 (Molecular Probe)
secondary antibody (1:1000 dilution) in the lissamine-rhodamine channel. Cells were imaged on a Bio-Rad MRC600 laser-scanning confocal
microscope with a 60× oil immersion lens. In none of the original
images in either channel were the intensities saturated.
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RESULTS |
N-terminal and Full-length Rim1--
We cloned N-terminal Rim1
(Rim(1-399)) from rat brain mRNA. Its nucleotide sequence was
identical to the published sequence (18) except for a larger deletion
(residues 56-105) in splice site #1. It encoded ~44-kDa protein
(data not shown). Recombinant full-length Rim1, amino acids 1-1553,
was constructed as described under "Material and Methods." It
lacked splice sites #1, #4, and #5 and encoded a ~160-kDa protein
(data not shown). The HA-tagged proteins were detected both with
anti-HA and anti-Rim1 directed against the zinc finger domain.
A schematic of the two constructs is shown in Fig.
1A. Unlike Rabphilin3 (27), no
endogenous Rim1 mRNA could be detected in either chromaffin cells
or PC12 cells (Northern blot data not shown), consistent with Rim1
being a brain-specific gene.

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Fig. 1.
Schematic diagram Rim1 and
Rabphilin3. A, Rim1. Full-length Rim1 contains numerous
domains including a zinc finger domain, a highly charged region
(++/ ), an alanine and proline-rich domain (A/P), a PDZ
domain, and two C2 domains. There are also three splice sites (numbered
according to Wang et al. (19)). The expanded view of
N-terminal Rim1 shows the Rab3a binding domain (amino acids 19-50) and
the minimal domain that enhances secretion (amino acids 51-190), as
demonstrated in this study. B, Rabphilin3, amino acids are
numbered from the bovine brain sequence (27).
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Full-length and N-terminal Rim1 Enhance Secretion When Transiently
Expressed in Chromaffin Cells--
Wang (18) reports that the
N-terminal region of Rim1 enhanced secretion in PC12 cells. We examined
the effects on secretion of transiently expressed full-length and
N-terminal Rim1 in primary cultures of bovine chromaffin cells.
Secretion stimulated by nicotinic agonist was strongly enhanced
(~50%) by both constructs (Fig. 2A).

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Fig. 2.
Overexpression of full-length or N-terminal
Rim1 enhances secretion in intact (A) and
permeabilized (B) chromaffin cells. Chromaffin
cells were cotransfected with plasmids encoding hGH and full-length
Rim1 (Rim(1-1553)), N-terminal Rim1 (Rim(1-399)) or pCMV.neo. Four to
5 days after transfection, cells were incubated in physiological salt
solution for 2 min ± 20 µM DMPP (A) or
were permeabilized with 20 µM digitonin in KGEP buffer
without Ca2+ and with 2 mM MgATP for 4 min
followed by incubation with or without 30 µM
Ca2+ for 2 min (B). There were four
wells/group.
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The enhancement of secretion from intact cells could result from
altered Ca2+ signaling upon nicotinic receptor activation
or from a direct effect on the intracellular pathway triggered by
Ca2+. To investigate the direct effects of the proteins on
the secretory pathway, secretion from digitonin-permeabilized cells was
examined. Secretion stimulated by 30 µM Ca2+
was strongly enhanced by both the N-terminal construct and full-length Rim1 (Fig. 2B), indicating that the proteins act downstream
of the Ca2+ signal.
Effects of Rim1 on the Ca2+ Sensitivity of Secretion
and on ATP-dependent and ATP-independent
Secretion--
During the first few minutes of a constant
Ca2+ stimulus in permeabilized cells, several steps in the
secretory pathway are evident (31, 34, 35). One step does not require
the continuing presence of MgATP and has been termed "primed"
secretion. Primed secretion probably reflects the prior effects of ATP
in cells before permeabilization. It decays rapidly after
permeabilization in the absence of MgATP. Another step requires the
continuing presence of MgATP, which maintains the primed state. Once
priming has occurred, Ca2+ can trigger secretion. The steps
are summarized as follows.
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(Eq. 1)
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To determine whether Rim(1-399) alters the sensitivity of the
secretory pathway to Ca2+, the effects of Rim(1-399) on
secretion stimulated by different Ca2+ concentrations were
investigated (Fig. 3A). Rim1
enhanced secretion at 1, 3, and 30 µM Ca2+ by
64, 62, and 61%, respectively. The almost identical enhancements of
secretion at the different Ca2+ concentrations indicate
that Rim(1-399) does not alter the sensitivity of the secretory
pathway to Ca2+.

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Fig. 3.
Effects of Rim(1-399) on the
Ca2+ sensitivity of secretion (A) and on
ATP-dependent and -independent secretion in permeabilized
cells (B). Chromaffin cells were cotransfected
with plasmids encoding hGH and Rim(1-399) or pCMV.neo as in Fig. 2.
A, cells were permeabilized with 20 µM
digitonin in Ca2+-free KGEP containing 2 mM
MgATP for 4 min followed by incubation with the indicated
Ca2+ concentrations for 4 min in the continuing presence of
MgATP. B, cells were permeabilized with 20 µM
digitonin in Ca2+-free KGEP with or without 2 mM MgATP for 4 min followed by incubation with or without
30 µM Ca2+ in the continuing presence or
absence of 2 mM MgATP for 2 min. ATP-dependent
secretion was calculated as the difference of secretion in the presence
and absence of ATP. *, p < 0.001 versus
pCMV.neo.
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The effects of Rim(1-399) on ATP-dependent and
-independent secretion from permeabilized cells were determined. Cells
transfected with or without Rim(1-399) were permeabilized for 4 min in
the presence or absence of 2 mM MgATP and then were
stimulated with 30 µM Ca2+ for 2 min in the
continuing presence or absence of MgATP. The expression of Rim(1-399)
more strongly enhanced secretion in the presence of MgATP (Fig.
3B). In three experiments, Rim(1-399) enhanced
ATP-dependent secretion (the difference in secretion in the
presence and absence of MgATP) and ATP-independent secretion by 71 ± 3 and 38 ± 5%, respectively (p < 0.005). The
protocol used in the above experiments gives excellent estimates of
ATP-dependent secretion, but because of the run-down of
already primed secretion in permeabilized cells (31, 34), the amount of
ATP-independent secretion was small. An alternative protocol in which
Ca2+ is added together with digitonin (in the absence of
ATP) greatly increases ATP-independent secretion. Using this protocol,
Rim(1-399) enhanced secretion by 28%. Taken together, these results
indicate that Rim1 preferentially enhances ATP-dependent secretion.
Minimal Region Required to Enhance Secretion in Chromaffin
Cells--
To determine the minimal region necessary for the
enhancement of secretion, N- and C-terminal deletion mutants were
constructed from Rim(1-399). Stimulated secretion from control cells
(15-20% of the total expressed hGH) was normalized to 100%. The
results of a series of experiments are summarized in Fig.
4. The enhancement of secretion was
maintained upon deletion of the N-terminal 50 amino acids (Fig.
4A). Further deletions from the N terminus caused loss of
activity, with Rim(71-399) having no effect on secretion.

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Fig. 4.
Effects on secretion of various N-terminal
and C-terminal deletions of Rim1. Chromaffin cells were
cotransfected with plasmids encoding hGH and either pCMV.neo or Rim
constructs. Four to 5 days later, cells were incubated in physiological
salt solution for 2 min ± 20 µM DMPP. Data are
presented as percent enhancement of DMPP-induced hGH secretion relative
to hGH secretion from cells transfected with control plasmid pCMV.neo.
The numbers of experiments performed are indicated in parentheses above
the error bars. Constructs labeled with an asterisk are
without the deletion in splice site #1. Constructs labeled with a
number sign (#) have residues 83-105 deleted in splice site #1. All
other Rim constructs have residues 56-105 deleted in splice site
#1.
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Constructs Rim(11-399)* and Rim(28-399)* contained the region in
splice site #1. Rim(1-214)#, a different splice variant, had a
23-amino acid deletion in splice site #1. Experiments with these
plasmids showed that the spliced regions do not contribute to the
enhancement of secretion.
C-terminal deletions revealed that activity was maintained in
constructs as short as Rim(1-190) (Fig. 4B). This construct does not contain the SGAWFF motif (residues 198-203) that in
Rabphilin3 plays a critical role in binding to Rab3a-GTP. In addition,
the enhancement of secretion does not require the high charged region (Rim(226-399)). Rim(1-190) contains the 4 pairs of cysteines that constitute a zinc finger domain. This domain is homologous to those in Rabphilin3, EEA1, and HRS-2. Deletion of even one pair of
cysteines (Rim(1-180)) resulted in complete loss of activity. The
results indicate that the zinc finger domain is important for the
enhancement of secretion by Rim1.
The above results lead to the prediction that a peptide containing
residues 51-190 should still enhance secretion. This was indeed the
case. Construct Rim(51-190) enhanced hGH secretion in chromaffin cells
as effectively as Rim(1-399), whereas Rim(71-190) was without effect
(Fig. 5). Because there was a 50-amino
acid deletion in splice site #1, Rim(51-190) actually encodes 90 amino acids. Thus, the minimal enhancing domain of Rim1 lacks the highly charged region, the SGAWFF motif and the N-terminal 50 amino acids. The
integrity of the entire zinc finger is important for the enhancement of
secretion.

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Fig. 5.
The minimal domain necessary to enhance
secretion in chromaffin cells includes the zinc finger. Chromaffin
cells were cotransfected with plasmids encoding hGH and either
Rim(51-190), Rim(71-190), Rim(1-399), or the parent plasmid
pCMV.neo. Secretion experiments in intact cells (A) and in
permeabilized cells (B) were performed as described in Fig.
2.
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Identification of Rab3a Binding Domain in Rim1--
N-terminal
deletion mutants of Rim1 were put into pGEX.2T vectors and expressed as
GST-Rim1 fusion proteins. The expressed proteins were bound to
glutathione-Sepharose beads, which were incubated in the presence of
either 1 mM GTP
S or GDP with lysates from human
embryonic kidney 293 cells transiently expressing HA-Rab3a. The
proteins on the beads were subjected to 10% SDS-polyacrylamide gel
electrophoresis, and the bound Rab3a was detected with anti-HA antibody.
We confirmed that N-terminal Rim1 (Rim(1-399)) binds Rab3a in a
GTP-dependent manner (Fig.
6A). Rim1 constructs lacking
the N-terminal 19 amino acids still bound Rab3a-GTP, whereas further deletion (28 amino acids) totally prevented Rab3a-binding (Fig. 6A).

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Fig. 6.
Binding of various deletion mutants of Rim1
to GTP S- or GDP-bound Rab3a and Rab5.
GST-Rim1 constructs was expressed in E. coli and immobilized
on glutathione-Sepharose beads. HA-Rab3a or HA-Rab5 were expressed in
human embryonic kidney 293 cells and solubilized with Triton X-100.
Cell lysates were incubated with beads in the presence of 1 mM GTP S or GDP as described under "Materials and
Methods." Bound Rab was detected by 10% SDS-polyacrylamide gel
electrophoresis and immunoblotting for HA. A, binding of
HA-Rab3a to N-terminal deletions of Rim(1-399). B, binding
of HA-Rab3a to C-terminal deletions of Rim(1-399). C,
binding of HA-Rab5 to Rim(1-50) and Rim(1-399). Constructs labeled
with an asterisk do not have the splice deletion in splice site #1.
Constructs labeled with a number sign (#) have residues 83-105 deleted
in splice site #1. Other Rim constructs have residues 56-105 deleted
in splice site #1.
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Rim(28-399) does not contain the first splice region, whereas
Rim(28-399)* retains all the amino acids in splice site #1. The latter
construct also lacked the ability to bind Rab3a-GTP, indicating that
the splice insert does not restore specific Rab3a-GTP binding.
The SGAWFF motif (residues 198-203) in Rim1 is immediately C-terminal
to the zinc finger. The SGAWFF motif in Rabphilin3 has a similar
location and is required for the GTP-dependent binding to
Rab3a. Surprisingly, two C-terminal deletions, Rim(1-200) and Rim(1-190) bound Rab3a-GTP as strongly as Rim(1-399) (Fig.
6B), indicating that the SGAWFF motif is not required for
binding. Rim(1-190) contains the 4 pairs of conserved cysteines in the zinc finger. Removal of one pair (Rim(1-180)), two pairs
(Rim(1-160)), three pairs (Rim(1-150)), and four pairs (Rim(1-50))
of cysteines did not prevent binding to Rab3a. No binding was detected
for two shorter constructs, Rim(1-39) and Rim(1-28). We conclude that the minimal domain in Rim1 responsible for GTP-dependent
binding to Rab3a lies within residues 19-50. The zinc finger motif is not needed for binding to Rab3a-GTP.
It was demonstrated with the yeast two-hybrid system that Rim(1-345)
and Rim(11-399) bind Rab3a and Rab3c but not other Rab family members
(18). The present data indicate that a small region, less than 50 amino
acids, is responsible for GTP-dependent binding of Rim1 to
Rab3a. Although this domain is much smaller than those previously
examined, it nevertheless shows specificity for Rab3a. Rim(1-50),
although able to bind Rab3a-GTP, is unable to bind Rab5-GTP (Fig.
6C), a GTPase necessary for early endosome trafficking.
The abilities of different Rim1 N-terminal constructs to bind to
Rab3a-GTP and enhance secretion are compared in Table I.
Immunocytochemical Localization of Rim1 Constructs--
Chromaffin
cells were cotransfected with plasmids encoding various HA-tagged Rim1
constructs and ANP-GFP to label chromaffin granules (Fig.
7). Cells were fixed and permeabilized,
and double immunofluorescence was used to visualize the HA-Rim1
constructs. Full-length Rim1 (Rim(1-1553) (Fig. 7, A and
B) was diffusely distributed in the cytoplasm, without
apparent plasma membrane or chromaffin granule localization. There was
also full-length Rim1 in the nucleus. Rim(1-190) had a patchy
cytoplasmic distribution that partially overlapped (data not shown)
with the highly punctate ANP-GFP-containing chromaffin granules (Fig.
7, C and D). Surprisingly, Rim(1-399) was
concentrated in the nucleus, with much smaller amounts in the cytosol
(Fig. 7, E and F). This nuclear localization was
probably caused by the highly charged region in the N terminus that was
exposed upon deletion of the C terminus of full-length Rim. Rim(1-190)
(without the highly charged region) did not localize to the nucleus
(Fig. 7, C and D), whereas the highly charged
region (Rim(226-399)), when expressed alone, was concentrated in the nucleus (Fig. 7, G and H). Rim(1-180), which did
not alter secretion, had a patchy, cytoplasmic distribution (Fig. 7,
I and J).

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Fig. 7.
Localization by confocal microscopy of
HA-tagged Rim constructs in chromaffin cells. Chromaffin cells
were cotransfected with plasmids encoding ANP-GFP, a chromaffin granule
marker, and HA-tagged Rim constructs. After 5 days, cells were fixed,
permeabilized, and immunocytochemistry performed to detect the
HA-tagged Rim. ANP-GFP was visualized in the fluorescein isothiocyanate
channel (B, D, F, H,
J); HA-tagged Rim was visualized in the lissamine-rhodamine
channel (A, C, E, G,
I).
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Full-length Rim1 was found both in the cytosol and nucleus. It may
shuttle between the nucleus and cytosol using nuclear import and export
pathways. Treatment with leptomycin B (200 nM), an inhibitor of the CRM1-dependent nuclear export pathway (36-38), did
not cause increased further accumulation of Rim(1-1553) in the nucleus
(data not shown). If Rim1 shuttles between the cytoplasm and nucleus, a
CRM1-mediated nuclear export pathway is not involved.
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DISCUSSION |
Rim1 is a large Rab3a-binding protein with multiple domains
including a N-terminal zinc finger domain, a central PDZ domain, potential phosphorylation sites, and two C2 domains at the C terminus (18). In this study, we investigated the structural basis for the
ability of Rim1 to bind Rab3a-GTP and to stimulate exocytosis from
chromaffin cells. We found that the abilities of Rim1 to enhance
secretion and to bind Rab3a-GTP reside on distinct and relatively small
domains that act independently. An analysis of the characteristics of
the enhancement of secretion indicates that Rim1 acts at a step after
Ca2+ entry in the cells to enhance the
ATP-dependent priming of secretion.
Rim1 and Rabphilin3 Possess Different Rab3a Binding
Domains--
The N termini of Rim1 and Rabphilin3 have similar
structures with homologous zinc finger regions and a distinctive SGAWFF motif (Fig. 1). They both preferentially bind the GTP-bound form of
Rab3a. Surprisingly, the region of Rim1 that binds Rab3a-GTP is
distinct from that in Rabphilin3. In Rabphilin3, an ~140-amino acid
is responsible for the binding of Rab3a-GTP (Fig. 1B) (22, 23, 28). The domain includes the entire zinc finger and flanking sequences. Ostermeier and Brunger explored the crystal structure of
Rab3a-GTP/Rabphilin3 and report two contact areas in the complex (39).
Although the zinc finger does not directly contact Rab3a, it serves as
a scaffold to hold the Rab3a-interacting domains in a binding
conformation. The SGAWFF motif that is C-terminal to the zinc finger
directly interacts with Rab3a.
Neither the zinc finger nor the SGAWFF motif of Rim1 is required for
binding to Rab3a-GTP. Either or both can be totally removed from
N-terminal constructs without altering the specific binding to
Rab3a-GTP. The minimal Rab3a binding domain in Rim1 was mapped to
residues 19-50, immediately N-terminal to the zinc finger. This domain
is predicted to be a coiled-coil domain (40). It has little homology to
the corresponding region in Rabphilin3. Thus, Rim1 and Rabphilin3
possess different Rab3a binding domains, suggesting that their
interaction sites on Rab3a are distinct.
Full Length Rim1 and Its Zinc Finger Domain Enhance Secretion in
Chromaffin Cells--
A previous study had demonstrated that the
N-terminal domain of Rim1 enhances secretion when transiently expressed
in PC12 cells (18). In the present study we demonstrated that
full-length Rim1 and as well as truncation mutants without the two C2
domains and the PDZ domain stimulate secretion similarly in chromaffin cells. The ability of full-length Rim1 to stimulate secretion suggests
that the endogenous protein normally acts as a positive regulator of exocytosis.
The minimal N-terminal domain required for the enhancement of secretion
was located between residues 51 and 190 and did not require a splice
insert. It contains the 90 amino acids comprising the cysteine-rich,
zinc finger region. Importantly, the ability of transiently expressed
full-length and N-terminal constructs of Rim1 to stimulate secretion in
permeabilized cells indicates that the enhancing effects are a direct
effect of the proteins on the Ca2+ -dependent
secretory pathway and not on Ca 2+ entry.
The N-terminal Domain of Rim1 Stimulates Secretion by Enhancing
ATP-dependent Priming--
We had previously demonstrated
that ATP acts before the final Ca2+-triggering step to
prime the cells for exocytosis (31, 34). In the present study we found
that Rim1 increased the ability of ATP to prime secretion in
permeabilized cells without altering the sensitivity of the secretory
response to Ca2+ (Figs. 3 and 4). Rim1 also increased, but
to a lesser degree, secretion that did not require the continuing
presence of ATP (Fig. 4). This seemingly ATP-independent secretion
probably reflects the prior effects of ATP in the intact cell before
permeabilization (34). It is likely that the enhancement of
ATP-independent secretion reflects an increase in the degree of priming
in the intact cells.
ATP-dependent priming is a dynamic process (31, 34).
Rim(1-399) could be either increasing the rate of priming or
decreasing the rate of unpriming. If Rim(1-399) decreased the rate of
unpriming, then the primed state would be stabilized in the presence of
Rim(1-399), and the decline in the secretory response in the absence
of ATP would be slowed. We investigated the effects of Rim(1-399) on the stability of the primed state after permeabilization in the absence
of ATP. When Ca2+ was added after 4 min of
permeabilization, primed secretion decreased similarly in the presence
and absence of Rim(1-399) (75 and 73% declines, respectively, data
not shown). The data support the notion that Rim(1-399) increases the
rate of ATP-dependent priming rather than decreasing the
rate of unpriming.
Localization of Rim1 Constructs in Chromaffin
Cells--
Endogenous Rim1 is concentrated to presynaptic active zones
in neurons and presynaptic ribbons in ribbon synapses (18). Because of
the variety of possible splice variants, the actual protein that was
visualized in these experiments is unknown. The full-length Rim1
protein that we constructed had a diffuse, cytoplasmic distribution
with no evident plasma membrane association when transiently expressed
in chromaffin cells. The localization of endogenous Rim1 to the active
zone in neurons may require interaction with active zone-specific
proteins that are not present in the chromaffin cell plasma membrane.
The different subcellular distributions (diffuse, patchy, or nuclear)
of the various deletion mutations investigated in this study suggest
that multiple domains could determine localization. Despite differences
in subcellular distribution in chromaffin cells, all of the constructs
with an intact zinc finger domain similarly enhanced secretion.
Although Rim(1-399) was detected in the cytoplasm, its predominant
localization to the nucleus was unexpected and raised the possibility
that Rim1 enhances secretion because of an effect on gene expression.
However, an analysis of other Rim1 mutants revealed that nuclear
localization was unnecessary for the enhancement of secretion. Highly
charged Rim(226-399) localized entirely to the nucleus, indicating
that this sequence contains a nuclear localization domain. It had no
effect on secretion. Rim(1-190), a construct without the nuclear
localization domain, was entirely cytoplasmic and enhanced secretion.
Thus, it is likely that cytoplasmic rather than nuclear Rim1 enhances secretion.
Full-length Rim1 (Rim(1-1553)) was not concentrated in the nucleus.
Its cytoplasmic localization was not altered significantly by
leptomycin B, an inhibitor of CRM1-dependent nuclear
export. It is possible that deletion of the C terminus in Rim(1-399)
exposed a nuclear localization signal whose function is suppressed in the full-length protein.
Comparison of the Effects on Secretion of Rim1 and
Rabphilin3--
The domains required for the enhancement of secretion
by Rim1 and Rabphilin3 are strikingly different. In previous studies, we found that both C2 domains as well as the zinc finger were required
for the enhancement of secretion by Rabphilin3 (23, 27). In fact,
N-terminal Rabphilin3 constructs terminating after the zinc finger
inhibit rather than enhance secretion. The enhancement of secretion by
Rim1 requires only the zinc finger region and not the C-terminal C2 or
other identified domains. In addition, Rim1 (Figs. 2-5) but not
Rabphilin3 (data not shown) enhanced secretion from permeabilized
cells. These differences are likely to reflect different mechanisms
underlying the enhancements of secretion by these two Rab3a-binding proteins.
The enhancements of secretion by Rim1 and Rabphilin3 are similar in
that neither requires interaction with Rab3a. The main function of the
interaction of Rabphilin3 and Rab3a is to stabilize Rabphilin3 in
chromaffin cells (23). Rim1 constructs that were unable to bind Rab3a
were well expressed in chromaffin cells (e.g. Rim(51-399),
Rim(71-399)). There was no evidence that interaction with Rab3a
stabilized Rim1 in chromaffin cells.
Other Rim Family Members--
Rim1 belongs to a family of
proteins, each of which has many possible splice variants. Rim2 is
highly homologous to Rim1 (19). Although we did not investigate Rim2,
it is likely that its N terminus functions similarly to that of Rim1.
Amino acids 19-50 of Rim1 constitute the minimal sequence necessary to
bind Rab3a-GTP. Twenty-seven of the 32 amino acids are identical to
corresponding amino acids in Rim2 (residues 23-54). Similarly, Rim2
has a region that is 79% homologous to the smallest Rim1 construct
that enhanced secretion. This region contains a zinc finger whose
integrity is required for the enhancement of secretion. The zinc finger domains in Rim1 and Rim2 are 90% identical.
Nim2 and Nim3 have been also identified as part of the Rim family (19).
Both these smaller proteins contain a C2 domain and flanking domains
that are highly homologous to the C2B and adjacent regions in Rim1
(19). Neither contains zinc finger domains. Nim3 enhances elevated
K+-induced secretion in PC12 cells (19). It is possible
that C2B region as well as the zinc finger region in Rim1 can stimulate secretion. Thus, the protein may contain two regions that enhance the
secretory response.
Several Rim-binding proteins have been identified. Two of them bind
through SH3 domains to a specific region in the C terminus of Rim (19).
Their function in secretion has not been investigated. Another protein,
cAMP-GEFII, binds to a region C-terminal to the zinc finger domain and
N-terminal to the first C2 domain (C2A) in Rim2 (26). CAMP-GEFII
enhances secretion from PC12 and insulin-secreting cells through a
mechanism that requires both interaction with Rim2 and the direct
binding of cAMP. None of these Rim-binding proteins are likely to play
a role in the enhancement of secretion caused by the zinc finger of
Rim1, which does not contain the specific binding sites for these
various proteins.
In summary, our investigation together with other recent studies
highlights the importance of Rim as a regulator of exocytosis. Its many
distinct domains are likely to reflect a role of the protein in
integrating cellular signals, including those that modulate secretion.
Although chromaffin cells do not normally express Rim1, we suspect that
the activity of the transiently expressed protein in chromaffin cells
reflects aspects of the function of the endogenous protein. We have
identified the zinc finger domain as being sufficient for the
enhancement of secretion. The interaction with Rab3a-GTP, although not
necessary for the enhancement of secretion, is likely to serve another
function, perhaps allowing the proper transport of the protein in the neuron.