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INTRODUCTION |
Rab proteins are a family of small GTP-binding proteins that are
important regulators of vesicle transport (1-3). They undergo nucleotide exchange to establish the active GTP-bound form and are
incorporated onto transport vesicles either during or after vesicle
formation. The GTP-bound form of Rab proteins recruit effectors, either
directly or indirectly, to target vesicles to the appropriate sites on
acceptor membranes. These effectors include motor proteins which link
the vesicles to the cytoskeleton (4, 5), docking complexes which are
recruited from the cytosol or tethering factors that mediate the
initial contact of membranes that are destined to fuse (6, 7).
To date, about 40 distinct Rab proteins have been identified and each
is believed to be specifically associated with a particular vesicle
transport pathway (2, 8). However, only a fraction of known Rab
proteins have their functions characterized in detail. Among these,
Rab8 (which we term Rab8a) has been shown to be a key regulator of
constitutive polarized vesicle transport to the dendrites in the
neurons or to the basolateral membrane in epithelial cells (9-11).
Using immunofluorescence and electron microscopy, Rab8a is localized to
the Golgi region, cytoplasmic vesicular structures, and the plasma
membrane of Madine-Darby canine kidney epithelial cells. When
the wild type and dominant active mutant forms of Rab8a are
overexpressed in baby hamster kidney fibroblast cells, a
dramatic change in cell morphology occurs. The cells form elongated
processes as a result of a reorganization of both their actin filaments
and microtubules. In this case, newly synthesized vesicular stomatitis
virus, a basolateral marker protein is preferentially delivered
into these cell outgrowths.
While the role of Rab8a in vesicle traffic has been elucidated, the
function of Rab8b, a closely related isoform, remains unclear. Rab8b
was first identified during a library screen to look for novel Rabs
involved in secretory granule fusion in mast cells and basophils (12).
Rab8b has an overall amino acid identity of 80% with Rab8a. While the
NH2 termini are highly conserved, the COOH-terminal domain
of Rab8b is substantially divergent from that of Rab8a. Transient
expression of the Myc-tagged Rab8b fusion protein in neuroblastoma and
basophilic leukemia cells shows staining of both the plasma membrane
and ill-defined vesicular structures. When overexpressed, Rab8b induces
striking plasma membrane outgrowth, similar to that observed for Rab8a.
Northern immunoblot analysis shows the highest expression level of
Rab8b in spleen and brain. In contrast, Rab8a has very low expression
levels in spleen and brain. From these data, we postulated that like
Rab8a, Rab8b may also play a similar role in the vesicle traffic from
the Golgi apparatus to the plasma membrane. Furthermore, because of the substantial COOH-terminal differences between Rab8b and Rab8a and their
different tissue expression patterns, we also suspected that Rab8b may
be involved in roles in secretion that are not shared or substituted by Rab8a.
Our study was aimed to further characterize Rab8b's function in the
secretory pathway. Here, we report the cloning and characterization of
a Rab8b interacting protein named TRIP8b and the function of Rab8b and
TRIP8b in the regulated secretion of adrenocorticotropic hormone
(ACTH)1 secretion in AtT20 cells.
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EXPERIMENTAL PROCEDURES |
Cloning of Rab8b--
Rat Rab8b cDNA was cloned from a rat
brain cDNA library by polymerase chain reaction screening using
primers specific to the 5' and 3' sequences of Rab8b (12). The forward
primer used was 5'-CGGGATCCATGGCGAAGACGTAC-3' and the reverse primer
was 5'-CGGGATCCTCAAAGCAGAGAACA-3'. For subsequent cloning, both primers
were designed to contain a BamHI restriction site. The
polymerase chain reaction products were cloned into pBluescript vector
(Stratagene) and colonies were screened with a
-32P-labeled primer (5'-GGTGGGCCAGTGAAAATAACAGAA-3')
corresponding to a portion of the C terminus of Rab8b. Positive
colonies were confirmed by DNA sequencing.
GAL4-based Yeast Two-hybrid Screening--
For two-hybrid
screens, Rab8b was cloned into the pAS2-1 vector at the
EcoRI and BamHI sites. The Rab3a construct used
was previously described (13). Rab8a (kindly provided by Johan Peranen) was cloned into pAS2-1 at the EcoRI and NcoI
sites, while Pex5 (kindly provided by Wolfgang Schliebs) was cloned
into the NcoI and XhoI sites. The expression of
the GAL4BD fusion proteins were confirmed by immunoblotting before
starting the library screen.
An oligo(dT) rat brain cDNA library was cloned into the pGAD424X
vector as described (14). Rab8b and the rat brain cDNA library were
co-transformed into the yeast strain Y190 according to manufacturer's
instructions (CLONTECH).
Cloning of TRIP8b--
To obtain the amino-terminal TRIP8b
sequence, a TRIP8b PstI fragment derived from the original
R38 clone that was isolated in the yeast two-hybrid system was used to
screen a rat brain cDNA phage library (Stratagene). Several clones
containing the NH2-terminal sequence were identified.
Enzyme digestion and cloning strategies were used to produce a
full-length TRIP8b clone whose sequence has been deposited to the
GenBankTM data bank (accession number AF324454).
DNA Constructs and Mutagenesis--
Rab8b mutants were created
using the Stratagene Quikchange Site-directed Mutagenesis kit. These
mutants include: Rab8bQ67L, Rab8bT22N, Rab8bC204S, and Rab8bN121I. All
the mutations were confirmed by DNA sequencing. These mutants were
either cloned into pAS2-1 (CLONTECH) for two-hybrid
studies, pXJ40-Myc or pXJ40-hemagglutinin (HA) (15) for
immunofluorescence staining and pXJ40-glutathione S-transferase (GST) (16) for in vitro translation
and binding studies.
The full-length TRIP8b was cloned into pBluescript at the
EcoRI and XhoI sites. Digestion with
EcoRI, BglII, and XhoI produced a
570-base pair NH2-terminal fragment (TRIP8b-N) and a
1.3-kilobase fragment containing the tetratricopeptide repeat (TPR)
domain (TRIP8b-TPR). TRIP8b, TRIP8b-N, and TRIP8b-TPR were cloned
separately into pGEX4T-1 (Amersham Pharmacia Biotech) for GST fusion
protein production and pXJ40-Myc or pXJ40-HA vectors for transient
transfection and immunofluorescence staining. TRIP8b and TRIP8b-N were
also cloned into pXJ41-neomycin (neo) (16) for stable transfection of
AtT20 cells. For the two-hybrid experiments, TRIP8b-N and TRIP8b-TPR were cloned into pACT2 (CLONTECH).
To create the GST-SLL construct, two oligonucleotides were designed.
The upper primer was 5'-AATTCTCTCTGCTTTGAAAGCTT-3' and the lower primer
was 5'-TCGAAAGCTTTCAAAGCAGAGAG-3'. Each primer was phosphorylated by
the polynucleotide kinase in a 20-µl reaction at 37 °C for 30 min
according to the manufacturer's instructions (Roche Molecular
Biochemicals). To anneal the two primers, the reactions were mixed
together, incubated at 65 °C for 15 min, and slowly cooled to room
temperature. The annealed primers produced a double stranded fragment
with 5' and 3' overhangs which was then cloned into pGEX4T-1 at the
EcoRI and SalI sites. The GST-STOP construct was
created in the same manner using 5'-AATTCTGATGATGATGAAGATCT-3' as the
upper primer and 5'-TCGAAGATCTGCATCATCATCAG-3' as the lower primer.
Expression and Purification of GST Fusion Proteins and
Preparation of TRIP8b Sera--
For the bacterial expression of GST
fusion proteins, pGEX4T-1 constructs were transformed into BL21 cells
(Stratagene). Cultures were grown at 37 °C until an
A600 of 0.6 was reached and induced with 0.1 mM isopropyl-1-thio-
-D-galactopyranoside at
37 °C for 3 h before being harvested. The cells were
resuspended in phosphate-buffered saline (PBS) containing 2 mM phenylmethylsulfonyl fluoride (PMSF) and subjected to
sonication. The supernatant was obtained by centrifugation at
12,000 × g in an SS34 rotor (Beckman) for 20 min and
loaded onto a column containing glutathione-Sepharose (Amersham
Pharmacia Biotech). The column was washed 5 times with PBS containing
0.5 M NaCl and 0.1% Triton X-100, 3 times with PBS
containing 0.1% Triton X-100, 2 times with 50 mM Tris-HCl,
pH 8.0, 50 mM KCl, 20 mM MgCl2, and
5 mM ATP, and then 2 times with PBS containing 0.1% Triton
X-100 before the GST fusion protein was eluted with 10 mM
reduced glutathione (Sigma) in 50 mM Tris, pH 8.0, 0.1% Triton X-100 and 5% glycerol. To obtain antibodies against
TRIP8b, serum was obtained from a rabbit injected with purified
GST-TRIP8b-N and affinity purified on a GST-TRIP8b-N column.
Tissue Culture--
Human embryonic kidney (HEK) 293T cells were
grown in Dulbecco's modified Eagle's medium with 1000 mg/liter
glucose and 10% fetal bovine serum. AtT20 cells were grown in
Dulbecco's modified Eagle's medium with 4500 mg/liter glucose and
10% fetal bovine serum while stably transfected AtT20 cells were grown
in the same media plus 250 µg/ml G418 (Life Technologies, Inc.). HEK
293T cells or AtT20 cells were maintained at 37 °C in a 5 or 15%
CO2 incubator, respectively.
In Vitro Translation and Binding Assay--
TRIP8b was cloned
into pBluescript vector and in vitro translated in the
presence of [35S]methionine using the T7 TNT kit from
Promega. For studies with bacterially expressed proteins, 25 µg of
GST or GST fusion proteins (GST-SLL, GST-STOP, GST-Rab8b,
GST-Rab8b
SLL, and GST-Rac2) were bound onto glutathione-Sepharose
beads. For the cellular expressed proteins, cell extracts from HEK 293T
cells transiently transfected with pXJ-GST constructs containing Rab8b,
Rab8b
SLL, or Rab8bC204S were bound onto glutathione-Sepharose beads.
The beads were washed 5 times with PBS containing 0.1% Triton X-100
and incubated with 400 µl of 1 × GTP/GDP exchange buffer (50 mM KCl, 25 mM Hepes-OH, pH 7.3, 2.5 mM EDTA, pH 8.0, and 0.05% Triton X-100) including 1 mM GTP, GTP
S, or GDP, or 10 mM EDTA in a
1.5-ml Eppendorf tube at room temperature for 4 min. The beads were
spun down at 2000 rpm for 30 s, the supernatants were discarded
and the beads were stored on ice. 50 µl of 35S-labeled
TRIP8b was diluted with 800 µl of binding/wash buffer (PBS with 25 mM Hepes-OH, pH 7.3, 5 mM MgCl2,
and 0.05% Triton X-100) including 25 µg of GST protein. The 850-µl
mixture was then incubated with the GST fusion proteins bound on beads
at 4 °C for 15 min. The beads were spun down and the supernatants were discarded. The beads were washed 4 times with 800 µl of ice-cold binding/wash buffer. At the end, proteins were extracted from the beads
with 2 × SDS loading buffer, run onto a SDS-PAGE gel, and the gel
was dried under vacuum and exposed to x-ray film.
Co-immunoprecipitation of Rab8b and TRIP-8b--
4.5 µg of
total DNA (1.5 µg of pXJ40-HA-TRIP8b and 3 µg of pXJ40-Myc-Rab8b),
20 µl of Superfectin (Qiagen), and 300 µl of serum-free Dulbecco's
modified Eagle's medium were incubated at room temperature for 15 min
before another 2.5 ml of growth media was added. The total transfection
mixture was added to a 10-cm tissue culture plate containing HEK 293T
cells and incubated at 37 °C for 3 h. The media was removed and
the cells were grown overnight in 10 ml of fresh growth media. The next
day, the cells were briefly and gently washed with PBS before cellular
proteins were extracted at 4 °C for 3 h with 10 mM
Hepes-OH, pH 7.3, 150 mM NaCl, 0.1% Nonidet P-40, 2 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol, and 1 mM PMSF. The extract
was centrifuged at 14,000 × g for 15 min at 4 °C
and the supernatant was incubated with 20 µl of protein A beads
(Roche Molecular Biochemicals) at 4 °C for 1 h, spun down, and
the beads discarded. The supernatant was then incubated with either 1 µg of anti-HA polyclonal antibody (Y-11, Santa Cruz Biotechnology) or
1 µg of rabbit IgG (Sigma) at 4 °C for 2 h. Then 20 µl of
protein A beads were added and incubated with the supernatant for 30 min. The beads were recovered by brief centrifugation and washed 5 times with 10 mM Hepes-OH, pH 7.3, 150 mM NaCl,
and 0.05% Nonidet P-40. The amount of immunoprecipitated TRIP8b was
determined using an anti-HA monoclonal antibody (F-7, Santa Cruz
Biotechnology) and the amount of Rab8b that co-immunoprecipitated with
TRIP8b was observed with an anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology).
Immunofluorescence Staining of AtT20 Cells--
AtT20 cells were
plated onto 2-well chamber slides (Nunc) and transiently transfected
the next day with Superfectin. The day after the transfection, the
cells were washed 2 times with PBS and fixed with 3.7% formaldehyde in
PBS at room temperature for 10 min or with
20 °C acetone at room
temperature for 2 min. Then the cells were permeabilized with 0.1%
Triton X-100 in PBS for 5 min and blocked with 1% bovine serum albumin
in PBS for 30 min. Subsequently, the cells were incubated with the
primary antibody in blocking buffer for 1 h at room temperature,
washed three times with blocking buffer, and incubated with a
fluorescent secondary antibody for 30 min. The rabbit polyclonal TRIP8b
antibody was used at a dilution of 1:100 and the monoclonal anti-Myc
antibody was used at 1:500. The anti-rabbit Cy3-conjugated antibody
(Sigma) was used at 1:200 and the fluorescein isothiocyanate-conjugated anti-mouse antibody was used at 1:100 (Sigma).
Subcellular Analysis of AtT20 Cells--
A 10-cm plate of
confluent AtT20 cells was scraped in PBS, centrifuged at 2,000 rpm for
5 min, and the cell pellet resuspended in ice-cold homogenization
buffer containing 250 mM sucrose, 10 mM
Hepes-OH, pH 7.3, 2 mM EGTA, 1 mM EDTA, and 1 mM PMSF and lysed by sonication. An aliquot of the
homogenized fraction was taken for Western immunoblot analysis and the
remainder centrifuged at 200,000 × g in a TLA 100.2 rotor (Beckman) at 4 °C for 1 h. The pellet was solublized with
20 µl of 2 × SDS gel loading buffer. Proteins in the
supernatant were precipitated with 10% trichloroacetic acid and
solublized with 20 µl of 2 × SDS gel loading buffer. The
homogenate, pellet, and supernatant fractions were loaded onto a 10%
SDS-PAGE gel, transferred to a nitrocellulose membrane, incubated with
anti-TRIP8b sera or anti-Rab8 monoclonal antibody (Transduction
Laboratories), and detected using goat anti-mouse or anti-rabbit
horseradish peroxidase-conjugated secondary antibody and Pierce
Supersignal. Note, the Rab8 antibody reacts against both Rab8a and
Rab8b, while reverse transcriptase-polymerase chain reaction of poly(A)
RNA revealed that similar amounts of Rab8a and Rab8b messenger RNA were
present in AtT20 cells (data not shown).
For the extraction studies, AtT20 membrane pellets were resuspended in
0.1 mM Na2CO3, pH 11, or 1% Triton
X-100 as described (17). 40 µg of membrane fraction was incubated on
ice for 1 h with 0.1 M Na2CO3,
pH 11, or 1% Triton X-100 in 20 mM Tris-HCl, pH 7.5, 1 mM EGTA, and 1 mM EDTA. Following the
incubation, the fractions were centrifuged at 200,000 × g for 30 min. The supernatant fractions were precipitated
with 10% trichloroacetic acid and the resulting precipitates were
solubilized with 2 × SDS loading buffer. While the pellet
fractions were resuspended with 2 × SDS loading buffer. Both
fractions were subjected to Western immunoblot analysis as described above.
Solubilization of TRIP8b with GDP Dissociation Inhibitor
(GDI)--
GST-GDI1 protein was expressed and purified as described
above except for the absence of detergent in the wash and elution buffers. Instead, the wash buffers were 25 mM Tris-HCl, pH
7.5, 10% glycerol, and 1 mM dithiothreitol with 500 or 150 mM NaCl. The protein was eluted with the 150 mM
NaCl wash buffer containing 7.5 mM reduced glutathione.
An AtT20 membrane fraction was prepared as described (17) and the
membrane pellet resuspended in 10 mM Tris-HCl, pH 7.5, and
300 mM sucrose by sonication. 25 µg of resuspended
membranes were preincubated with 1 mM GDP for 20 min at
37 °C and then incubated with 400 nM GST-GDI1 in the
presence of 1 mM GDP and analyzed as described above.
Stable Transfection of AtT20 Cells--
AtT20 cells were plated
out onto a 6-well plate with a density of 4 × 105
cells/ml, 2 ml/well. The next day, cells were transfected with pXJ41-neo-Myc-Rab8b, TRIP8b, or TRIP8b-N. Briefly, 2 µg of DNA, 5 µl of Superfectin, and 100 µl of serum-free media were mixed and
incubated at room temperature for 20 to 30 min before another 600 µl
of growth media was added. Then the total 700-µl mixture was added
drop by drop onto each well of cells. Cells were incubated for 4 to
5 h, changed to new growth media, and grown at 37 °C overnight
in a 15% CO2 incubator. The next day, each well was trypsinized and the cells plated out onto a 150-mm plate, and after
24 h the media was changed to a media containing 250 µg/ml G418.
Cells were kept growing until colonies formed. Several colonies were
isolated and continuously grown in 96-, 24-, and 6-well plates. Positive clones were identified by protein expression using Western immunoblot analysis and immunofluorescence.
ACTH Release Assay--
The experiment was performed as
described with modifications (18). Cells were grown to 50-75%
confluence in 12-well plates for 3 days before the assay. The cells
were washed three times with serum-free media and then incubated in 150 mM NaCl, 20 mM Hepes-OH, pH 7.3, 0.7 mM CaCl2, 5 mM KCl, 1 mM MgCl2, and 0.1 mg/ml bovine serum albumin,
with or without 100 mM 8-Br-cAMP at 37 °C for 1 h.
The media fraction from each well was collected and PMSF was
immediately added. The cells were washed with ice-cold PBS and the wash
combined with the media fraction. The media fraction was precipitated
with 4 × volume
20 °C acetone overnight. The cells were
extracted with 50 mM Hepes-OH, pH 7.3, 250 mM
NaCl, 0.1% Nonidet P-40, and 1 mM PMSF for 20 min at
4 °C and centrifuged at 14,000 × g for 20 min. The
supernatant was transferred to a fresh tube and 15-20% of the
supernatant was precipitated with 4 × volume of
20 °C
acetone overnight. For both the media and cell fractions, precipitated
proteins were resuspended in 2 × SDS gel loading buffer,
electrophoresed onto a 12% SDS-PAGE gel, transferred to
nitrocellulose, and subjected to Western immunoblot analysis. For
immunodetection of ACTH products, the polyclonal antibody against ACTH
(RGG8502, Peninsula Laboratories) was used at 1:1000 to detect both the
30-kDa ACTH biosynthetic intermediate (ABI) and the 13-kDa mature form
of ACTH. Densitometric quantification of the autoradiographs was
performed with a Bio-Rad/GS700 imaging densitometer. The percentage of
ABI and ACTH secreted was calculated by dividing the media value by the
sum of the media and cellular values. All experiments were performed in triplicate.
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RESULTS |
Identification of a Rab8b-interacting Clone in the Yeast Two-hybrid
System--
Over 2 × 106 clones from a rat brain
cDNA library were screened using the GAL4-based yeast two-hybrid
system (14) with either Rab8b or the GTPase-defective mutant
(Rab8b-Q67L) to identify Rab8b interacting proteins. A single clone was
observed to interact with both proteins. A full-length clone (R38) was
obtained by screening a rat brain cDNA library and predicted to
encode a hydrophilic 66-kDa protein. BLASTP analysis of the R38 protein
sequence against GenBankTM reveals that the R38 had 44%
identity with the peroxisomal targeting signal 1 receptor (also
referred to as PEX5), a protein involved in the transport of
peroxisomal enzymes. Analysis of the R38 protein sequence revealed that
the most significant homology (66% identity) between the two proteins
occurs at the COOH-terminal half of R38 where six TPR motifs are
located to form a TPR domain (Fig. 1). TPR motifs are 34-amino acid repeats that are observed in a variety of
proteins and are implicated in protein-protein interaction (19-21). In
the case of PEX5, its TPR domain consists of seven TPR motifs and is
involved in the binding of peroxisomal enzymes containing the
tripeptide motif SKL at their carboxyl terminus (22-25).

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Fig. 1.
Schematic of TRIP8b amino acid sequence.
TRIP8b is 602 amino acid (aa) residues in length and
contains 6 TPR motifs within its COOH-terminal half, as shown in
shaded boxes. TPR motifs are 34 amino acid residues in
length and are involved in protein-protein interactions (see text for
details).
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We further analyzed the interaction of R38 with several other mutant
forms of Rab8b with properties described for a number of Rab proteins,
including Rab8a (26) (Table I). In
addition to Rab8b and Rab8b-Q67L, R38 was able to interact with
Rab8b-T22N (a form of Rab8b which is predominantly in the GDP-bound
state) and Rab8b-C204S (a mutant which abolishes the terminal cysteine required for isoprenylation) constructs. No binding to R38 was observed
with the dominant negative nucleotide-free mutant, Rab8b-N121I. Finally, we examined the interaction of two other Rab proteins, Rab8a
and Rab3a, with R38. While Rab8a has over 80% homology with Rab8b, no
binding of R38 was observed with Rab8a as well as Rab3a (Table I).
These results suggested that the binding of Rab8b to R38 was specific,
independent of the nucleotide state and prenylation status of Rab8b. We
have therefore termed R38 as TRIP8b, for TPR-containing Rab8b interacting protein.
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Table I
Interaction of Rab8b with TRIP8b in the yeast two-hybrid system
Bait and prey constructs were co-transformed into yeast Y190. The
strength of the interaction between bait and prey constructs was
determined by the -galactosidase activity of the transformants.
Rab8b mutants were prepared as described under "Experimental
Procedures." TRIP8b-N represents a construct that encodes the
NH2-terminal 190 amino acids of TRIP8b, while the
TRIP8b-TPR construct encodes the COOH-terminal of TRIP8b containing the
TPR domain.
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In addition to the interaction of TRIP8b with Rab8b mutants, the
two-hybrid system was also employed to characterize the interaction of
Rab8b with different regions of TRIP8b, the amino-terminal half
(TRIP8b-N) and the COOH-terminal half (TRIP8b-TPR) that contains the
TPR domain. As displayed in Table I, Rab8b interacts only with TRIP8b
constructs containing the TPR domain (TRIP8b and TRIP8b-TPR). This
result is similar to that observed for PEX5, which only requires its
TPR domain to bind to enzymes that have the tripeptide motif SKL as
their COOH-terminal residues (22). Because of these results and the
homology between TRIP8b and PEX5, the interaction between Rab8b, which
has the tripeptide motif SLL at its COOH terminus, and PEX5 was also
examined. However, no interaction was observed (Table I), indicating
that the SLL motif of Rab8b cannot be recognized by the TPR domain of
PEX5.
Interaction of Rab8b and TRIP8b Using in Vitro Translation and
Co-immunoprecipitation--
To further characterize the interaction of
Rab8b and TRIP8b, TRIP8b was in vitro translated and the
binding of TRIP8b to bacterially expressed GST fusion proteins was
examined (Fig. 2A). Similar to
what was observed in the yeast two-hybrid studies, TRIP8b was able to
bind to GST-Rab8b. Because PEX5 only requires the COOH-terminal SKL
motif to bind proteins, we also examined the interaction of TRIP8b with
GST containing SLL as the last three COOH-terminal amino acids (as is
the case for Rab8b) and Rac2, another small GTP-binding protein with
the last three COOH-terminal amino acids being SLL. No interaction was
observed with these proteins suggesting that the mechanism of
interaction between TRIP8b with Rab8b is not similar to that observed
for PEX5 with SKL-containing peroxisomal enzymes. We also examined the
interaction of GST fusion proteins expressed in mammalian cell
cultures, which allows prenylation of Rab proteins. As shown in Fig.
2B, binding of TRIP8b to Rab8b bound with either GTP
S or
GDP was observed, consistent with the results from the bacterial and
yeast binding studies. In the presence of 10 mM EDTA, which
chelates the free magnesium and reduces the amount of guanine
nucleotide binding to Rab8b, significantly less binding of TRIP8b to
Rab8b was observed. Reduced binding was also observed for Rab8b with
the COOH-terminal SLL residues deleted (Rab8b
SLL) as well as the
prenylation defective Rab8b-C204S construct. Finally, the interaction
of Rab8b and TRIP8b was confirmed in HEK 293T cells co-transfected with
Myc-tagged Rab8b and HA-tagged TRIP8b. As shown in Fig.
3, Myc-tagged Rab8b was recovered in the
immunoprecipitate with anti-HA polyclonal antibody.

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Fig. 2.
In vitro interaction of Rab8b with
TRIP8b. A, TRIP8b was in vitro
translated in the presence of [35S]methionine and
incubated with bacterially expressed GST-Rab8b, GST, GST-SLL, or
GST-Rac2 bound to glutathione-Sepharose beads (as described under
"Experimental Procedures"). The amount of bound
[35S]TRIP8b was determined by autoradiography,
quantitated using a densitometer and the values represented as a
bar graph. B, TRIP8b binds to GST-Rab8b expressed
in mammalian cells in the presence of GTP S and GDP but with
significantly reduced binding in the presence of 10 mM
EDTA. TRIP8b also bound Rab8b mutants SLL and C204S that were
expressed in mammalian cells but bound to the two mutants with lesser
affinity compared with GST-Rab8b.
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Fig. 3.
Co-immunoprecipitation of Rab8b and
TRIP8b. HEK 293T cells were co-transfected with HA-TRIP8b and
Myc-Rab8b. Cell extracts (lane 1) were incubated with rabbit
IgG (lane 2) or anti-HA polyclonal antibodies (lane
3). The immunoprecipitates were electrophoresed onto a 12%
SDS-PAGE gel, transferred to nitrocellulose, and subjected to Western
immunoblot analysis. The upper (A) and
lower (B) blots were probed with monoclonal
antibodies against HA or Myc.
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Expression of TRIP8b in Brain and AtT20 Cells--
Western
immunoblot analysis using an antibody generated against a GST fusion
protein of TRIP8b revealed that the TRIP8b was present only in brain
(Fig. 4). The expression of TRIP8b was
also examined in several cell lines and detected in the AtT20 cells, a
neuroendocrine cell line but not in kidney fibroblasts (COS-7) or PC12
cells that are derived from a pheochromocytoma.

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Fig. 4.
Western immunoblot reveals that TRIP8b is
specifically expressed in brain and AtT20 cells. 40 µg (tissue)
or 25 µg (cell lines) of postnuclear supernatant were loaded per lane
and the immunoblot was probed with a rabbit polyclonal antibody raised
against TRIP8b.
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TRIP8b Is Associated with a Membrane Fraction via Its Interaction
with Rab8b--
Immunofluorescence staining of endogenous TRIP8b in
the AtT20 cells revealed punctate structures distributed over the
entire cell body as well as a concentration of TRIP8b at the
perinuclear and cell tips (Fig.
5A). These results suggest
that TRIP8b can interact with membrane structures. Additionally, this
staining pattern is similar to that observed for Rab8b (12) as well as other proteins involved in vesicle traffic to the plasma membrane such
as Rab3a (13).

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Fig. 5.
Immunofluorescence and biochemical analysis
of TRIP8b reveal membrane association. A,
endogenous expression of TRIP8b in AtT20 was determined by indirect
immunofluorescence using a TRIP8b-specific antibody.
Arrowheads indicate the localization of TRIP8b at the cell
tips. B-D, all fractions were prepared as
described under "Experimental Procedures." B,
AtT20 cells were homogenized by sonication (Hom) and the
pellet (Pel) and supernatant (Sup) were separated
by centrifugation at 95,000 × g. C, a
membrane fraction was prepared by differential centrifugation of cell
lysates at 200,000 × g and incubated with buffer, 0.1 M Na2CO3, pH 11, or 1% Triton
X-100 for 30 min at 4 °C and then fractionated by centrifugation at
200,000 × g for 30 min. D, membrane
fractions were incubated with or without GDI1 for 20 min at 37 °C
and then fractionated by centrifugation as described above.
B-D, the fractions were subjected to electrophoresis on a
12% SDS-PAGE gel, transferred to nitrocellulose, and immunodetected
with antibodies against TRIP8b or Rab8.
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The distribution of Rab8b and TRIP8b in AtT20 cells was also examined
in subcellular fractions using Western immunoblot analysis. As shown in
Fig. 5B, both Rab8b and TRIP8b were detected in the supernatant and pellet fractions, indicating that both are present in
cytosolic and membrane compartments. While Rab8b contains a prenylation
site that enables it to interact with membranes, TRIP8b contains no
hydrophobic domains and may therefore interact with membranes through
another protein such as Rab8b or be peripherally associated. To further
investigate this interaction, the membrane fraction was incubated with
Na2CO3, pH 11, or Triton X-100 to determine the
nature of TRIP8b binding to membranes. As shown in Fig. 5C,
treatment with high pH caused TRIP8b to appear in the soluble fraction
suggesting that the membrane-bound TRIP8b is peripherally associated.
In addition, incubation in the presence of detergent also released
TRIP8b into the soluble fraction. One possibility is that the
association of TRIP8b with membranes is via its interaction with Rab8b.
To examine this, the membrane fraction was incubated with GDI1 that can
remove Rab proteins from membranes (27). As expected, some of the
membrane-bound Rab8b now appeared in the soluble fraction (Fig.
5D). More importantly, a fraction of TRIP8b also appeared in
the soluble fraction. These results indicate that the membrane
association of TRIP8b is through its interactions with Rab8b and that
removal of Rab8b from the membrane results in solubilization of TRIP8b.
TRIP8b and Myc-Rab8b Co-localize and Co-immunoprecipitate in the
AtT20 Cells--
Both the constitutive and regulated secretion of the
hormone ACTH have been well studied in AtT20 cells (28-30). To further examine the relationship between Rab8b and TRIP8b and their role in
membrane traffic to the plasma membrane, AtT20 cells stably expressing
vector only, Rab8b, TRIP8b, or a truncated TRIP8b containing the
amino-terminal 190 amino acids (TRIP8b-N) were established and examined
for their ability to release ACTH. AtT20 cells were stably transfected
with Rab8b, TRIP8b, or TRIP8b-N and their protein expression confirmed
by Western immunoblot analysis (data not shown). The expression of
Rab8b and TRIP8b in the AtT20 stable cells was also examined by
immunofluorescence staining (Fig. 6). As
previously observed, the overexpression of Rab8b resulted in an altered
morphology in the AtT20 cells, with a dramatic increase in the length
of the cell processes compared with the vector only cells (Fig.
6A). Costaining with antibodies against ACTH also reveals
that Rab8b has significant co-localization with ACTH, in particular at
the perinuclear region and cell tips where release of ACTH vesicles
occurs (Fig. 6A). While in the cells expressing TRIP8b,
co-localization was primarily observed at the cell tips.

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Fig. 6.
Immunofluorescence localization of Rab8b,
TRIP8b, and ACTH in AtT20 stable cells.
A, co-localization of Rab8b and ACTH in the AtT20
cells stably expressing Myc-Rab8b or Myc-TRIP8b. Pictures on the
left, cells stained with anti-Myc mouse monoclonal antibody
and fluorescein isothiocyanate-conjugated anti-mouse antibody;
middle, cells stained with anti-ACTH rabbit polyclonal
antibody and rhodamine-conjugated anti-rabbit antibody;
right, merge of the left and middle pictures.
Bar, 10 µM. B,
co-localization of Rab8b and TRIP8b in the Rab8b stable cells. Picture
on the left, cells stained with anti-TRIP8b rabbit
polyclonal antibody and fluorescein isothiocyanate-conjugated
anti-rabbit antibody; middle, cells stained with anti-Myc
mouse monoclonal antibody and rhodamine-conjugated anti-mouse antibody;
right, the merge of the former two pictures.
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To examine the co-localization of Rab8b and TRIP8b, Rab8b expressing
cells were stained for both Rab8b and endogenous TRIP8b. As shown in
Fig. 6B, there was significant overlap observed in the
staining patterns of Rab8b and TRIP8b suggesting that the two proteins
are present on similar membranes. However, while co-localization was
observed, this does not mean that the proteins are interacting. To
establish whether Rab8b and TRIP8b interact when expressed in cells, we
used antibodies against TRIP8b to co-immunoprecipitate Rab8b from the
Rab8b stable cells. As shown in Fig. 7,
TRIP8b was able to specifically co-immunoprecipitate with Rab8b. Thus,
immunofluorescence and co-immunoprecipitation data further confirm that
Rab8b and TRIP8b interact in cells.

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Fig. 7.
Co-immunoprecipitation of Rab8b and TRIP8b in
the Rab8b stable cells. Postnuclear extracts of AtT20 cells stably
expressing Rab8b were immunoprecipitated with either rabbit IgG or
anti-TRIP8b rabbit polyclonal antibody as described under
"Experimental Procedures." Precipitated proteins were subjected to
electrophoresis on a 12% SDS-PAGE gel and then probed with antibodies
against TRIP8b and anti-Myc monoclonal antibody to detect the
co-precipitation of Rab8b.
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Rab8b and TRIP8b Stimulate Secretion of ACTH from AtT20
Cells--
Post-translation processing of proopiomelanocortin leads to
a 30-kDa ABI product that is eventually cleaved to produce a 13-kDa mature ACTH product (31). Studies on the secretion of ABI and ACTH have
indicated that the ABI is constitutively secreted while the secretion
of ACTH is regulated (30). The release of ACTH and ABI from AtT20 cells
were measured in the presence or absence of the secretagogue 8-Br-cAMP
(Table II). In the vector only cells, no
increase in secretion of ABI was observed after incubation with
8-Br-cAMP when compared with unstimulated cells. In contrast, ACTH
product had ~10-fold increase in secretion after stimulation with
8-Br-cAMP. These results are consistent with other studies (13, 18, 28)
and indicate that the ABI is secreted via a constitutive-like process,
while ACTH is secreted in a regulated manner (28).
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Table II
Fold stimulation of ACTH release in AtT20 clones
Semiconfluent AtT20 cells grown in 12-well plates were washed with
media and incubated for 60 min in media in the presence or absence of 5 mM 8-Br-cAMP. The media and cells were collected
separately, the proteins precipitated, and the amount of ABI and ACTH
released determined as described under "Experimental Procedures."
The stable clones N4, F6, WT3, and WT6 represent high-expression single
clones, while Nm1, Nm2, Fm1, and Fm2 represent mixed clonal
populations. The fold stimulation value represents the amount of ABI or
ACTH released in the presence of 8-Br-cAMP over the amount released in
the absence of secretagogue. Values represent the average ± S.E.
of three independent experiments.
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As shown in Table II, all the stable cell lines displayed similar
results with regard to the secretion of ABI when compared with the
vector only cells. In fact, the fold stimulation of ABI was around 1 for all the stable cell lines, which demonstrated that overexpression
of Rab8b, full-length TRIP8b, and truncated TRIP8b did not affect the
secretion of ABI in AtT20 cells.
In contrast, the overexpression of Rab8b and TRIP8b had a significant
increase in the fold stimulation of secreted ACTH. As mentioned
earlier, the control cells had a 10-fold stimulation of ACTH release in
the presence of 8-Br-cAMP compared with untreated cells. The three
TRIP8b-N clones (N4, Nm1, and Nm2) gave similar results with fold
stimulations of ACTH release of 10, 13, and 11, respectively. This
suggests that the overexpression a truncated TRIP8b does not have a
significant effect on stimulated ACTH release. In contrast, the TRIP8b
clones F6, Fm1, and Fm2 showed a 17-, 19-, or 20-fold increase,
respectively, in the stimulated release of ACTH. Compared with the
10-fold increase shown by the vector-only cells, overexpression of the
TRIP8b had a significant stimulatory effect on ACTH secretion. In the
case of the Rab8b clones, similar results were observed. The two
clones, Rab8b-WT3 and Rab8b-WT6, had a 17- or 33-fold increase in the
secretion of ACTH when stimulated. In summary, these data demonstrate
that Rab8b and TRIP8b stimulate the regulated but not the constitutive
secretion of ACTH products in AtT20 cells. Furthermore, the stimulation
effect of TRIP8b was not due to its NH2-terminal domain
because TRIP8bN clones did not affect the secretion of mature ACTH.
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DISCUSSION |
We have identified a protein that interacts with Rab8b, termed
TRIP8b, that contains six TPR motifs within the COOH-terminal half of
the protein. Based on several in vitro and cell based approaches, the binding of Rab8b to TRIP8b appears to be independent of
the nucleotide bound to Rab8b, and does not require the prenylated form
of Rab8b (Table I, Figs. 2-4). The interaction with TRIP8b was
specific as no interaction was observed for several other small
GTP-binding proteins such as Rac2, Rab3a, or even the homolog Rab8a
(Table I). Recently, the sequence of several orthologs of TRIP8b have
been deposited in GenBankTM (accession numbers CAC01120,
NP_057643, NP_067458, and BAA92878) and based on their homology, termed
PEX5 related protein or peroxisomal targeting signal 1 receptor-like.
However, our data suggests that TRIP8b is not involved in peroxisomal
function. In contrast to PEX5, which binds to peroxisomal enzymes with
COOH-terminal residues of SKL (22-25), no interaction was observed
between TRIP8b and GST-SLL (Fig. 2) or GST-SKL (data not shown).
Furthermore, the interaction of TRIP8b with Rab8b can occur in the
absence of the last three COOH-terminal residues of Rab8b. In fact,
this is normally the case for mature prenylated Rab proteins, which undergo proteolytic cleavage of the last three residues after prenylation of the terminal cysteine residue (32, 33). Finally, immunostaining of AtT20 cells against the peroxisomal enzyme catalase revealed a different staining pattern when compared with TRIP8b (data
not shown).
Proteins containing TPR motifs are observed in a variety of species
from bacteria to human and are involved in several protein-protein interactions (19-21). Theses 34 amino acid repeats are frequently observed in tandem and observed to form a structural lattice consisting of two antiparallel
helices linked by a short loop (34-36). In fact, the interaction the small GTP-binding protein Rac2 to phox67 is
dependent on the TPR motifs of phox67. Recently, the crystal structure
of that interaction has been determined (36). The structural data
indicates that the residues of phox67 responsible for the interaction
occur between the loops that connect TPR1 with TPR2 and TPR2 with TPR3
as well as a
-hairpin insertion that occurs between TPR3 and TPR4.
Similarly, the interaction of Rab8b with TRIP8b requires the TPR
motifs. When TRIP8b was separated into two domains, only the
COOH-terminal domain containing the TPR domain was capable of
interacting with Rab8b. Based on the Rac2/phox67 model, it would be of
interest to further dissect the interaction of these TPR motifs with Rab8b.
In contrast to Rab8b, which is ubiquitously expressed (12), Western
immunoblot analysis of TRIP8b reveals it to be exclusively expressed in
brain. When we evaluated several cell lines for the expression of
TRIP8b, only the neuroendocrine cell line, AtT20, was found to contain
TRIP8b. Subcellular fractionation of these cells indicated that a
significant percentage of TRIP8b was membrane bound even though the
predicted protein sequence contains no hydrophobic domains. TRIP8b is
most likely peripherally associated with membranes, as it was capable
of being extracted by high pH from the membrane fraction. In fact,
further experiments with isolated membranes revealed that the binding
of TRIP8b to membranes occurs through its interaction with Rab8b. When
Rab8b was removed from the membrane using GDI1, a fraction of TRIP8b
was observed in the supernatant fraction. Thus, Rab8b acts as a
recruitment factor in this case, bringing TRIP8b to membranes.
The presence of TRIP8b in AtT20 cells allowed us to examine the effects
of overexpression of TRIP8b on both constitutive and regulated
secretion. The processing of the proopiomelanocortin leads to ABI that
is constitutively secreted and ACTH, which is packaged into dense core
granules and secreted in a regulated manner (28, 30, 31). When the
basal and stimulated release of ABI and ACTH were examined in several
stable constructs expressing Ra8b or TRIP8b, a noticeable pattern was
observed. Overexpression of either Rab8b or TRIP8b increased the fold
stimulation of ACTH while the release of ABI was constant. Thus, not
only is there no effect on the constitutive pathway in these stable
cell lines, but the release of ABI serves as an internal control for
the data obtained for stimulatory effects of Rab8b and TRIP8b on ACTH
release. As an additional control, either single (N4) or mixed (Nm1 and Nm2) clones of a truncated TRIP8b (TRIP8b-N) exhibited no stimulatory effects on ACTH secretion. It would have been of great interest to
examine the effects of a stable cell line expressing TRIP8b-TPR to see
if there was a negative or enhanced effect compared with the
full-length TRIP8b stables cells, however, repeated attempts to create
such a stable line were unsuccessful.
While there is significant homology between Rab8a and Rab8b, it appears
that functional differences are present. Rab8a was previously
identified as being involved in the transport of vesicles from the
trans-Golgi network to the basolateral membrane of polarized Madine-Darby canine kidney cells (9). Similarly in studies with
hippocampal neurons (10), Rab8a was demonstrated to be involved in
transport to the dendritic plasma membrane. These results suggest that
Rab8a is not involved in the regulated secretory pathway that involves
the apical or axonal surfaces. Furthermore, studies have demonstrated
that overexpression of Rab8a has no effect on the regulated secretion
of human growth hormone (37). In contrast, the overexpression of Rab8b
had a significant stimulatory effect on the regulated secretion of ACTH
that occurs at the tips of the cell processes (13, 38).
The majority of studies on Rab proteins and regulated secretion have
focused on the Rab3 family members. The Rab3 family consists of four
highly homologous isoforms associated with secretory granules and
synaptic vesicles. Overexpression of each of the four members of the
Rab3 family inhibited secretion (37). Moreover, knockout of Rab3A
increased the quantal release of synaptic vesicles (39). In contrast to
the inhibitory effect of Rab3 family on regulated secretion,
overexpression of Rab8b exerted a stimulatory effect and suggests that
several Rab may converge to regulate secretion.
While this is the first study to implicate Rab8b in regulated
secretion, a previous study indicated that the overexpression of Rab8b
in rat basophilic leukemia cells, a model system for mast cells,
resulted in significant plasma membrane extension (12). These results
are similar to the effect observed in the AtT20 stable cells lines
Rab8b-WT3 and Rab8b-WT6. One possibility is that the effects on cell
morphology observed by the overexpression of Rab8b are related to the
stimulatory effects on the regulated secretion of ACTH. In the AtT20
stable cells expressing TRIP8b, only a slight increase in plasma
membrane extensions was observed, however, the expression of TRIP8b was
several fold less than observed for the Rab8b stable cell lines (data
not shown). Since it is known that the dense core secretory granules
accumulate at the tips of these processes, the morphological changes
enabled by Rab8b expression may be the mechanism by which regulated
secretion is enhanced. In fact, similar results have been observed with other proteins involved in ACTH secretion. Kalirin is an
interacting partner of peptidylglycine
-amidating monooxygenase
(40), an enzyme essential for the proteolytic processing of ACTH in
AtT20 cells (41). In addition to peptidylglycine
-amidating
monooxygenase, kalirin also can interact with the small GTPase Rac1 and
serve as a guanine nucleotide exchange factor (42). The expression of
kalirin in AtT20 cells also affects the cytoskeleton and causes the
elongation of the cell processes, the same phenomena observed in the
Rab8b stables cells. It was also demonstrated that exogenous expression
of kalirin could restore the regulated ACTH secretion that is inhibited
by the overexpression of peptidylglycine
-amidating monooxygenase
(40). It is interesting to speculate that these two systems
(kalirin/peptidylglycine
-amidating monooxygenase/Rac1 and
TRIP8b/Rab8b) may utilize similar pathways to regulate the secretion of
ACTH. Further exploration of this system may unveil the communication
between Rab proteins on secretory vesicles and the Rho family proteins
that regulate the cytoskeletal network and lead to a greater
understanding of the regulation of the secretory pathway.