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Research Division, New Mexico Veterans Affairs Health Care System, and Department of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87108
Address all correspondence and requests for reprints to: Richard I. Dorin, M.D., Professor of Medicine, Departments of Medicine and Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Chief, Section of Endocrinology and Metabolism, New Mexico Veterans Affairs Health Care System, Medical Service (111), 1501 San Pedro Boulevard Southeast, Albuquerque, New Mexico 87108. E-mail: rdorin{at}salud.unm.edu
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Introduction |
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B (6, 7). Other effects, such as inhibition of
ACTH secretion, require new messenger RNA (mRNA) and protein synthesis
(8, 9), suggesting the participation of secondary
signaling proteins, the identity and mechanism of action of which are
unknown at present. In an effort to identify genes mediating the earliest effects of glucocorticoids, Kemppainen and Behrend (10) identified several mRNA transcripts that are rapidly up-regulated by glucocorticoid treatment in AtT-20 mouse corticotroph cells. One of these mRNAs, termed Dexras1, predicts a novel protein that has structural elements consistent with a GTP-binding protein and bears significant homology to members of the small molecular weight G protein (SMWG) family, such as Rap, R-Ras, and H-Ras. Dexamethasone treatment results in a 40-fold increase in Dexras1 mRNA within 30 min, with levels declining sharply after 2 h of treatment (10).
The role of SMWG proteins in a variety of cell regulatory processes is
well established. These include regulation of cell proliferation
(11, 12), gene transcription (13, 14), mRNA
stability and translation (15, 16, 17), cytoskeletal
organization (18, 19), peptide trafficking
(20, 21, 22), and secretion (23, 24). To our
knowledge, no functional role for Dexras1 in any of these areas has
been evaluated in mammalian systems. However, the human homolog of
Dexras1 recently has been identified by means of a genetic
complementation system in yeast cells on the basis of its ability to
activate signaling by the heterotrimeric G protein 
subunit,
Gi2, in a receptor-independent fashion
(25). This effect appears to involve a direct interaction
between Dexras1 and Gi (25) that causes enhanced guanyl
nucleotide exchange by Gi (26).
In view of its strong homology to other SMWG proteins and the potential
interaction of Dexras1 in signaling via heterotrimeric G proteins, we
have examined the effects of Dexras1 activation on peptide hormone
secretion in AtT-20 corticotroph cells. We focused on regulated or
stimulus-coupled secretion on account of previous reports demonstrating
that inhibition of stimulus-coupled ACTH secretion by glucocorticoids
requires newly synthesized protein (8, 9). Furthermore,
both glucocorticoids and agonists of Gi-coupled receptors, such as
somatostatin, inhibit stimulus-coupled ACTH secretion via regulation of
signaling targets that are downstream from or independent of adenylate
cyclase (27, 28, 29). Therefore, we hypothesized that
glucocorticoid- dependent inhibition of secretion from the
cAMP-stimulated pathway is regulated by Dexras1. This hypothesis
predicts that over-expression of a wild-type or constitutively active
Dexras1 protein in the absence of glucocorticoids will result in
inhibition of cAMP-stimulated peptide hormone secretion.
Our experimental approach involved expression and characterization of wild-type and mutant Dexras1 proteins in both AtT-20 and COS-7 cells. Stimulation-secretion coupling was evaluated in AtT-20 cells using a well-characterized technique of Moore and colleagues (30, 31) that employs transfected human GH (hGH) as an ectopic marker for the stimulus-coupled secretory pathway associated with dense core storage granules. This technique allowed us to selectively examine the effects of cotransfected Dexras1 species on spontaneous and stimulus-coupled peptide hormone secretion. Our results indicate that expression of a constitutively active Dexras1 mutant significantly attenuates cAMP-stimulated hGH secretion. These findings establish that over-expression of activated Dexras1 has biologically important effects in the regulation of peptide hormone secretion, and suggest that endogenous Dexras1 participates in specific aspects of glucocorticoid-dependent signal transduction.
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Materials and Methods |
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Amplification and cloning of Dexras1 full-length cDNA by
RT-PCR
Using the nucleotide sequence reported by Kemppainen and Behrend
(10), oligonucleotide primers (above) were designed for
the amplification of full-length Dexras1 by RT-PCR. Total RNA was
prepared by the method of Chomczynski and Sacchi (32), and
reverse-transcribed using oligo-dT primer and avian myeloblastosis
virus reverse transcriptase (Life Technologies, Inc., Bethesda, MD). cDNA was amplified with primers that
correspond to open-reading frame nucleotides 142–984 of murine Dexras1
cDNA (GenBank Accession No. NM009026), which yielded the expected bands
of approximately 840 bp. PCR products were separated by agarose gel
electrophoresis and visualized by ethidium bromide staining. PCR
products were also subcloned into the pCR2000 vector
(Invitrogen, Carlsbad, CA) and sequenced using
T3 and M13 reverse primers.
Expression of Dexras1 in AtT-20 and COS-7 cells
Full-length cDNAs were cut from pGEX-6P1 and ligated into the
polycloning site of the cytomegalovirus promoter-driven
expression plasmid, pcDNA3.1/His-C (Invitrogen).
Fragments and plasmid were matched so that the Dexras1 coding sequence
remained in frame with the N terminus 6xHis and anti-Xpress epitope
tags provided by the plasmid. Plasmids were confirmed by sequencing, as
above, and purified by CsCl2 gradient. For
expression of protein, cells grown to 70% confluency were transfected
by the CaCl2 method (0.05–0.1 µg plasmid DNA
per square centimeter of monolayer culture surface) (33).
Cells were harvested at 24 or 48 h post transfection.
Western blotting of Dexras1
Wild-type Dexras1, Dexras[A178V], and Dexras[A178V/C277term]
mutant proteins were expressed as 6xHis fusion proteins by transfection
in COS-7 cells (100-mm plates), as above. pcDNA3.1/His empty vector was
transfected as the control. At 24 h posttransfection, cell layers
were washed with ice-cold PBS and harvested in 1 ml each lysis buffer
(50 mM Tris-HCl, 140 mM NaCl, 5 mM
MgCl2, 2% Triton X-100, 0.2% SDS, 1% sodium
deoxycholic acid, 1 mM phenylmethylsufonylfluoride, and 10
µg/mL aprotinin and leupeptin). Lysates were clarified by
centrifuging 10 min at 12,000 x g, and incubated for
2 h at 4 C with 25 µl nickel-nitrilotriacetic (Ni-NTA)-agarose
(QIAGEN, Valencia, CA) for affinity purification of the
6xHis-tagged Dexras1 proteins. Agarose beads were washed 6 times by
low-speed centrifugation/resuspension in 1.5 ml ice-cold lysis buffer.
Proteins were eluted from the beads by boiling 10 min in SDS-PAGE
sample buffer. Proteins were separated by 12% PAGE, transferred to
nitrocellulose, and detected by Western blotting with the anti-Xpress
monoclonal Ab (0.2 µg/ml; Invitrogen) directed to
epitope-tagged Dexras1. Detection was performed with the Western Breeze
Chemiluminescence kit (Invitrogen), with
visualization on Kodak-Eastman Scientific Imaging Systems (Rochester, NY) film by
autoluminography.
[3H]GTP binding to Dexras1
Wild-type Dexras1, Dexras[A178V], and Dexras[A178V/C277term]
mutant proteins were expressed as 6xHis fusion proteins in AtT-20 or
COS-7 cells and affinity purified as above, but without boiling in
Laemmli buffer. Washed Ni-NTA-agarose beads were rinsed in 1.5 ml
ice-cold binding buffer, and then resuspended in 40 µl ice-cold
binding buffer [25 mM Tris-HCl (pH 7.8), 100
mM NaCl, 1 mM EDTA, 0.5 mM
DTT, 1 µCi/ml GTP] (Amersham Pharmacia Biotech,
Piscataway, NJ; 10 Ci/mmol specific activity). The beads were then
transferred to a thermal mixer where binding was performed for 20 min
at 30 C. Binding was terminated by transferring the reactions to an ice
bath and adding 1.5 ml ice-cold wash buffer [25 mM Tris-Cl
(pH 7.8), 100 mM NaCl, 20 mM
MgCl2, 0.5 mM dithiothreitol]. Beads
were washed 6 more times in wash buffer, then resuspended in
scintillation cocktail for quantification of retained (Dexras1-bound)
[3H]GTP. Ni-NTA-agarose precipitates from
pcDNA3.1/His (empty vector)-transfected cells were used as blank
controls. Two replicate experiments were performed with triplicate data
points for each condition. Because counts varied between each
experiment, data are expressed as fold-binding relative to wild-type
Dexras1.
[32P]Orthophosphate loading and analysis of
Dexras-bound guanyl nucleotides
Wild-type Dexras1, Dexras[A178V], and Dexras[A178V/C277term]
were expressed as 6xHis fusion proteins by transfection in COS-7 cells
(100 mm plates). At 18 h posttransfection, cell layers were washed
with phosphate-free medium, and incubated 2 h in phosphate-free
media. [32P]Orthophosphate (10 mCi/ml,
HCl-free, Amersham Pharmacia Biotech) was then added to a
final specific activity of 0.2 mCi/ml, and the cells were incubated for
an additional 4 h. Cell layers were washed twice with ice-cold PBS
and harvested in 1 ml lysis buffer supplemented with 20 mM
MgCl2 to stabilize nucleotide binding. Lysates
were incubated 15 min on ice and insolubilites were removed by
centrifugation at 10,000 x g for 10 min. Supernatants
were incubated for 5 min at 4 C with 300 µl prewashed and 1%
BSA-blocked Nordit-A charcoal (as a 50% slurry; J. T. Baker,
Phillipsburg, NJ) to remove unbound nucleotides. Charcoal was removed
by centrifugation at 10,000 x g for 2 min, and
6xHis-Dexras1 proteins were affinity purified from the supernatants
with Ni-NTA-agarose as described above, except that the lysis buffer
used for washes contained 20 mM
MgCl2. After the final wash, Dexras-bound
nucleotides were eluted from Ni-bound 6xHis-Dexras1 by incubating the
beads for 20 min at 68 C in 25 µl elution buffer containing 20
mM Tris (pH 8.0), 20 mM
EDTA, 20 mM DTT, 2% SDS, 10
mM GTP, 10 mM GDP. Beads
were centrifuged at 12,000 x g for 1 min, and 5 µl
supernatant was loaded on fluorescent polyeohyleneimine-cellulose TLC
plates (J. T. Baker) that were prewashed with a 1:1
MeOH-H2O solution. Separation of guanyl
nucleotides was performed using stepped concentrations of
NH4 formate, pH 3.4 (0.75, 1.5, and 3.0
M concentrations), as described by Graham
et al. (34). Mobility of GTP and GDP was
determined by migration of afluorescent pools of genuine,
nonradioactive nucleotides (Sigma, St. Louis, MO).
Radioactive nucleotides were visualized by phosphor-imaging, and
quantitated by volume integration (area x total counts per unit
area) using ImageQuant software (Molecular Dynamics, Inc.,
San Diego, CA). Background radioactivity comigrating with GTP and GDP
pools, which represents nonspecific binding of nucleotide and
protein-nucleotide complexes to the Ni-NTA-agarose beads, was
quantified using identical lysates from pcDNA3.1/His (empty
plasmid)-transfected cells. The percent of GTP (%GTP; also referred to
as the GTP to GDP ratio) was calculated according to the following
equation: %GTP = (GTP -
BGGTP)/(GTP - BGGTP)
+ 1.5 x (GDP - BGGDP). The BG terms
represent the background radioactivity from nonspecific nucleotide
binding. The multiplicative factor of 1.5 corrects for the difference
in [32P]PO4 content
between GTP and GDP.
Effects of wild-type and mutant Dexras1 on secretion of
cotransfected hGH
AtT-20 cells in 12 well plates were cotransfected with pTK-GH,
an expression plasmid for hGH under control of the constitutive
thymidine kinase promoter (Nichols Institute Diagnostics,
San Juan Capistrano, CA), and pcDNA3.1/His plasmid containing wild-type
Dexras1, Dexras[A178V], or Dexras [A178V/C277term]. Empty
pcDNA3.1/His plasmid was used as a negative control. At 48 h post
transfection, cell layers were washed 3 times with 37 C secretion
medium (MEM + 1% FBS + 10 mM NaHEPES; pH 7.35), and then
incubated for 2 h with secretion medium containing 5
mM 8-Br-cAMP (stimulated and spontaneous secretion) at 37 C
in a 5% CO2 environment, or for 2 h with
secretion medium lacking 8-Br-cAMP (spontaneous secretion). Secretion
was stopped by addition of a 2-fold volume of ice-cold PBS and transfer
of wells to ice slurry. Secreted hGH was diluted 1:100 in secretion
medium and quantitated by means of enzyme-linked immunosorbent assay
(Roche Molecular Biochemicals, Indianapolis,
IN).
Flow cytometric analysis of ß-galactosidase expression in AtT-20
cells transiently expressing wild-type and mutant Dexras1
AtT-20 cells were cotransfected with a pcDNA3.1/His expression
plasmid for ß-galactosidase (lacZ) in a ratio of 1:4 with
expression plasmids for wild-type or mutant species of 6xHis-tagged
Dexras1, using the technique described above. At 48 h
posttransfection, medium was removed and cells were incubated in fresh
medium containing 300 µM chloroquine for 1
h at 37 C in a 5% CO2 environment to inhibit
endogenous lysosomal ß-galactosidase activity. Cells were washed
twice with warm (37 C) medium and incubated for 30 min with fresh
medium containing 30 µM
C12-fluorescein-di-ß-D-galactopyranoside
(C12-FDG; Molecular Probes, Inc., Eugene, OR), a
nonfluorescent, cell-permeant ß-galactosidase substrate. Cells were
incubate at 37 C in a 5% CO2 environment.
Reactions were stopped by addition of room temperature enzyme-free cell
dissociation solution (Sigma) supplemented with 1
mM phenylethyl
ß-D-thiogalactopyranoside (Molecular Probes, Inc.), an inhibitor of ß-galactosidase. Enzymatic
cleavage of C12-FDG to the fluorescent product C12-fluorescein was
measured on an individual cell by cell basis using a FACSCalibur flow
cytometer (BD PharMingen) under excitation by a 488-nm
argon laser, as described by Plovins et al.
(35). C12-fluorescein emission proportional to
lacZ expression/ß-galactosidase activity was measured on
fluorescence channel one. Individual cells were identified by
characteristic forward- and side-scatter light diffraction
characteristics. AtT-20 cells cotransfected with the pcDNA3.1/His empty
vector were used to gate for baseline C12-fluorescein incorporation in
a linear, one-dimensional histogram mode. A total of 200,000 individual
cells were analyzed for each cotransfection condition, and two
replicate experiments were performed. Numbers reported for
lacZ+ cells reflect the number of
individual cell events with channel one fluorescence intensities
greater than the established gate for baseline C12-fluorescein
incorporation, thus providing a simultaneous indicator of transfected
cell number, lacZ gene expression, and accumulation of
cotransfected ß-galactosidase.
Statistical analysis
To determine statistical significance, paired ANOVA was
performed on secretion data sets and paired t test on the
other data sets, with pairing assigned on the basis of replicate
experiments. A P value of less than 0.05 defined significant
variation.
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Results |
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, and includes highly conserved GTP
binding pocket (
1-4) domains and an effector loop which, by
analogy to Ras, participates in protein-protein interactions with other
signaling molecules and is necessary for full biological activity
(36, 37, 38). A third structural feature of importance is the
CAAX box, a consensus site for isoprenylation, at the extreme carboxyl
terminus of Dexras1. Based on analogy to other Ras family members,
Dexras1 is predicted to undergo C15 (farnesyl) isoprenylation, a
posttranslational modification that regulates the subcellular
localization and function of other GTP-binding proteins (39, 40).
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. Two additional homologs with
less overall similarity have been identified in
Caenorhabditis elegans (GenBank Accession
No. AAA68305) and Saccharomyces cerevisiae
(GenBank Accession No. CAA97166).
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). Using primers selected to amplify the
entire 841-nucleotide open reading frame of Dexras1, we were able to
confirm the rapid induction and disappearance of Dexras1 mRNA within
4 h of glucocorticoid treatment. Direct nucleotide sequencing
confirmed that the PCR product was identical with the open reading
frame of the clone reported by Kemppainen and Behrend
(10). These data establish the specificity of RT-PCR
methods for analysis of Dexras1 and confirm the time course of
up-regulation of Dexras1 mRNA by glucocorticoids.
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.
This finding confirms the stability of the mutants relative to
wild-type Dexras1. The apparent Mr of Dexras1 is
approximately 36.5 kDa, consistent with the migration characteristics
reported by Cismowski et al. (26). The
increased Mr of Dexras1 above the expected 31.7
kDa may reflect altered migration due to its unusually high isoelectric
point (discussed below).
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Val mutation in codon 178,
based on structural analogy to a known activating mutation in H-Ras
(H-Ras[A146V]; Ref. 43). This mutation in the 4
region of Dexras1 is predicted to interrupt the guanyl
nucleotide-binding pocket, resulting in decreased affinity of the
mutant for both GTP and GDP, as well as an increase in the exchange
rate of GDP for GTP (43, 44). Guanyl nucleotide exchange,
particularly the release of GDP, is the rate-determining step in the
physiological activation of most G proteins (45). Due to
the overall higher intracellular levels of GTP relative to GDP,
increased nucleotide exchange results in increased occupancy in the
GTP-bound state. The H-Ras[A146V] mutant possesses normal GTPase
activity and transforms NIH-3T3 cells with an efficiency comparable to
other constitutively active Ras mutants (43). Thus, the
Dexras[A178V] mutant is also predicted to have an overall higher
ratio of bound GTP to GDP in vivo and behave functionally as
a constitutively active signal transducer, even in the absence of
upstream signals that normally lead to increased guanyl nucleotide
exchange activity under physiological conditions. We also created a
double mutant containing both the [A178V] mutation as well as a
premature translation termination codon that deletes the carboxyl
terminus CAAX box (Dexras[A178V/C277term]). This mutant was designed
to explore the potential role of prenylation on the functional
properties of Dexras1.
Binding of [3H]GTP was evaluated directly on
Ni-NTA-agarose beads using Dexras1 protein purified from transiently
transfected AtT-20 cells; two replicate experiments with duplicate data
points were performed. Total binding activity ranged from 12 x
103 to 40 x 103 cpm
above baseline. Results are described in terms of fold-binding relative
to wild-type, due to variation in baseline activity between
experiments. As shown in Fig. 5, compared
with wild-type Dexras1, GTP binding activity under saturating
conditions was significantly reduced in both Dexras[A178V] (38% of
wild-type) and the related mutant Dexras[A178V/C277term] that also
contains the carboxyl terminus CAAX box deletion (54% of wild-type).
Similar results were obtained from GTP binding experiments performed
using Dexras species purified from transfected COS-7 cells (data not
shown). The results indicate that, like H-Ras[A146V], Dexras[A178V]
has impaired steady-state binding of guanyl nucleotides relative to
wild-type.
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, which is representative
of two replicate experiments. Unlike in vitro GTP-binding
experiments, where mass action dictates the loading of G proteins with
radiolabeled guanyl nucleotides, in vivo labeling requires
an exchange of radiolabeled nucleotide for unlabeled nucleotide at
physiological concentrations. Therefore, due to the higher guanyl
nucleotide exchange rates associated with the [A178V] mutants, they
are predicted to copurify with a greater quantity of radiolabeled
guanyl nucleotides (total GTP and GDP). The absolute quantity of
copurifying radiolabeled guanyl nucleotides was increased in both
Dexras mutants (1.8 ± 0.2 x 106 and
1.8 ± 0.15 x 106 total counts for
[A178V] and [A178V/C277term], respectively) relative to wild-type
Dexras1 (1.2 ± 0.07 x 106 total
counts, P < 0.01). As shown in Fig. 6, the absolute
ratio of GTP to GDP (or %GTP) bound to [A178V] mutants was
approximately twice that of wild-type Dexras1 (16.9 ± 0.5% and
16.4 ± 0.6%, respectively for [A178V] and [A178V/C277term],
vs. 7.9 ± 1.3% for wild-type, P <
0.01). These findings confirm that the [Al78V] mutants possess an
enhanced rate of guanyl nucleotide exchange, and support the inference
that they behave in vivo as constitutively active forms of
Dexras1. The GTP to GDP ratio bound to wild-type Dexras1 (7.9%) is
comparable to the ratio bound to rat Dexras1 expressed in 293 cells
(6.2%) reported by Fang et al. (46) However,
our results differ from those reported by Cismowski et
al. (26) who observed preferential binding of GTP by
Dexras1 purified with similar techniques but expressed in yeast. This
difference suggests the possibility that Dexras1 expressed in a native
mammalian system may undergo different posttranslational modifications,
or may interact with proteins that stabilize a different guanyl
nucleotide binding conformation. A considerable amount of
32P-labeled material was retained at the
chromatographic origin, a common observation for this assay
(47). This material may represent coprecipitating
phosphorylated proteins, phosphorylated Dexras1, or a fraction of
noncovalently bound nucleotides that were not released during the
elution process.
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and in Fig. 7, which represent a summary of four
replicate experiments. Wild-type Dexras1 had negligible effects on
stimulated secretion. However, transfection of the constitutively
active Dexras[A178V] mutant resulted in a profound inhibition of both
the spontaneous and stimulated portions of hGH secretion (53% and 86%
reductions, respectively). By contrast, the CAAX-box-deficient mutant,
Dexras[A178V/C277term], did not show inhibition of the stimulated
portion of secretion, suggesting a requirement for prenylation of
Dexras1 in this activity.
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, wild-type Dexras1 did not
appreciably alter the amount of detergent-soluble hGH. However, each
constitutively active mutant, Dexras[A178V] or
Dexras[A178V/C277term], caused a similar, approximately 50%
reduction in soluble hGH. Remarkably, expression of the CAAX-deficient
mutant Dexras[A178V/C277term] caused a similar reduction in soluble
hGH. This observation contrasts with the effects of
Dexras[A178V/C277term] on stimulus-coupled secretion, and suggests
that not all of the signaling activities of Dexras1 require
prenylation. This reduction in detergent-soluble hGH caused by Dexras[A178V] did not appear to be related to a generalized effect on cell turnover or viability, because expression of wild-type Dexras1 or mutant species did not alter the number or intensity of X-gal staining in cells cotransfected with a lacZ (ß-galactosidase) expression plasmid, nor reduce total protein (data not shown). To quantitatively test this observation, we performed flow cytometric analysis to determine expression of ß-galactosidase (lacZ) in individual cells cotransfected with wild-type and mutant forms of Dexras, using a fluorescent substrate of ß-galactosidase, C12-FDG, as described by Plovins et al. (35). This technique enabled us to simultaneously monitor the number of transfected cells surviving 48 h post transfection and the level of ß-galactosidase protein expression in 2 replicate experiments. Average transfection efficiencies (percentage of lacZ+ cells) on 2 different replicate transfection days were 7.5 ± 0.42% and 4.3 ± 0.19%. Total numbers of lacZ+ cells per 200,000 total cells analyzed on each day were 16,654 and 9,876 for control (empty vector) transfectants, 14,365 and 8,542 for wild-type Dexras1 transfectants, 13,850 and 8,324 for Dexras[A178V] transfectants, and 16,211 and 8,252 for Dexras[A178V/C277term] transfectants. The average percentage of lacZ+ cells relative to empty vector control cells at 48 h posttransfection was reduced by 14.6 ± 0.1% for wild-type Dexras1, 16.3 ± 0.4% for Dexras[A178V], and 12.6 ± 2.8% for Dexras[A178V/C277term]; P less than 0.01 for significant variation between control cells and each Dexras transfectant, and P nonsignificant for variation between the different Dexras transfectants. These data indicate that wild-type and mutant forms of Dexras exert a small, comparable inhibitory effect on expression of ß-galactosidase in transfected cells.
Although it is not clear whether variation in the quantity of
detergent-soluble hGH accurately reflects the pool of peptide hormone
available for secretion from the regulated pathway, we nevertheless
included an adjustment for this effect in our analysis of the data. We
calculated the combined effects in terms of a percentage of stimulated
secretion per total soluble hGH (see Fig. 7 and Table 1). As observed
for the absolute quantity of stimulated hGH secretion, wild-type
Dexras1 had negligible effects on the percentage of stimulated
secretion, whereas the constitutively active Dexras[A178V] mutant
caused a large reduction (75%, P < 0.01). Remarkably,
the CAAX-box-deficient mutant, Dexras[A178V/C277term], caused a
moderate increase in both percent-stimulated secretion and
percent-spontaneous secretion. Although this effect may reflect an
artifact of the percentage calculation, it may also indicate an ability
of Dexras[A178V/C277term] to antagonize the inhibitory effects of
endogenous, activated Dexras1, as described for other nonprenylated,
activated Ras family members (48).
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Our RT-PCR results confirm the rapid and transient induction of Dexras1 mRNA in corticotroph cells by glucocorticoids, achieving a maximum at 90 min after continual induction. A similar magnitude and time course of induction was previously reported by Kemppainen and Behrend (10), who also demonstrated induction of Dexras1 mRNA in brain, heart, liver, and kidney following ip injection of dexamethasone in mice. Interestingly, rat Dexras2 expressed in striatum is up-regulated by thyroid hormone (42). These observations suggest that Dexras1 and its homologs may be uniquely responsive to hormonal regulation. Dexras1 is predicted to have a relatively short half-life, which suggests that hormonal regulation of signaling by Dexras1 and its homologs could occur through dynamic changes in their gene expression (53). We thus suggest that this novel Ras subfamily encompassing Dexras1 and its homologs represents a unique family of hormone-responsive, basic G proteins.
By analogy to other, well-characterized G proteins, we anticipate that expression of many of the biological activities of Dexras1 occur in the GTP-bound state and are terminated by GTP hydrolysis, an enzymatic activity predicted to be intrinsic to Dexras1 itself. Because the signaling events leading to activation of wild-type Dexras1 are unknown, we sought to develop a constitutively active mutant, Dexras[A178V] that would promote signal transduction by Dexras1 independently of upstream activation. The mutant Dexras[A178V] was designed by analogy to a constitutively active mutant of H-Ras (H-Ras[A146V]) that possesses accelerated guanyl nucleotide exchange (43), and thus provided a means to identify the functional effects of activated Dexras1. Several lines of evidence support the conclusion that Dexras[A178V] is constitutively active. These include reduced guanyl nucleotide binding, an enhanced guanyl nucleotide exchange rate, and an increased ratio of bound GTP to GDP relative to wild-type Dexras1. The potent effect of Dexras[A178V], but not wild-type Dexras1, on a biological endpoint such as hGH secretion provides further evidence that this mutant confers signaling activities that are distinct from wild-type Dexras1. As with other constitutively active Ras family members (39, 40), inhibition of prenylation blocks this signaling activity.
We found that constitutively active Dexras1 regulates spontaneous and cAMP-stimulated secretion. Though the magnitude of Dexras1 effects were greater for cAMP-stimulated secretion, the independent inhibition of spontaneous hGH suggests that Dexras1 may be acting through effects on the dense core secretory pathway, which contributes to both net spontaneous as well as cAMP-stimulated secretion in AtT-20 cells (30, 31). Inhibition of spontaneous secretion by Dexras1 could conceivably be mediated at proximal points in the secretory pathway, such as trafficking of hGH into the dense core granules or development of the dense core storage granules themselves. This effect may reflect the same mechanism underlying the observed decrease in detergent-soluble hGH.
The basis for the decrease in detergent-soluble hGH is uncertain, but did not appear to be related to a generalized effect on protein synthesis or a decrease in cell number or viability. We speculate that the inhibitory effect on soluble hGH could reflect Dexras activities directed toward regulation of the thymidine kinase promoter driving the reporter plasmid, hGH mRNA stability, protein translation efficiency, or trafficking in the secretory pathway. Quantitative analysis of lacZ expression via a cotransfected reporter plasmid revealed a small but significant approximate 12–16% reduction in ß-galactosidase activity that was not selective for any particular form of Dexras tested. This effect is disproportionately small when compared with the more than 80% reduction in stimulated hGH secretion by Dexras[A178V], or the approximately 50% reduction in soluble hGH, and is compatible with the standard deviations calculated for the secretion experiments. The difference in magnitudes and lack of specificity in this case suggests a different mechanism.
Remarkably, inhibition of stimulated secretion by Dexras[A178V] is dependent on prenylation, whereas inhibition of soluble hGH accumulation is not. Signaling activity by other nonprenylated Ras family proteins has been reported, and observations such as these emphasize that prenylation regulates some but not all functions of Ras family proteins (48, 54, 55, 56, 57). We are currently studying the role of prenylation in determining the subcellular localization of Dexras1.
Although it is unclear where in the regulated secretory pathway coupling cAMP and peptide hormone secretion Dexras1 may exert its inhibitory effect, it is conceivable that it is activating or interfering with known targets of related proteins. Ras family proteins directly regulate effects on protein trafficking and stimulus-coupled secretion in AtT-20 cells (17, 23, 24), and Dexras1 may be affecting similar pathways. Structurally, Dexras1 is most closely related to Rap and R-Ras, whose role in secretion is less clearly established than for Rab family members.
An alternative mechanism is suggested by the recent observation that
human Dexras1 is capable of ligand-independent activation of Gi/o
family heterotrimeric G proteins in a yeast pheromone pathway reporter
system (25), with an analogous effect on activation of an
Elk-1 reporter plasmid in mammalian cells (26). As noted
by Cismowski et al. (26), the ability of a Ras
family protein to directly transactivate heterotrimeric G proteins
represents a novel paradigm for signal transduction. It raises the
possibility that the inhibition of secretion by Dexras[A178V]
observed in this study could be mediated by interactions with Gi/o
family members. This possibility is supported by the observation that
Gi-coupled receptors, such as somatostatin receptor, inhibit the
secretion of ACTH in AtT-20 cells (58). This effect
appears to be independent of adenylate cyclase regulation, and may
involve stimulation of inwardly rectifying potassium channels that
suppress voltage-dependent calcium influx (27, 28, 59, 60, 61, 62). Futhermore, Gi/o family subunits localize to the
Golgi apparatus, where they regulate Golgi structure and the production
of secretory granules (63, 64, 65). Because this pool of Gi
in the Golgi may represent a downstream signaling target of Dexras1, it
will be important in future studies to determine the specific
subcellular compartments in which Dexras1 interacts with Gi.
The discovery of Dexras1 by two independent, function-oriented cloning
methods (10, 25) further suggests that Dexras1 may
represent a nexus between Gi- and glucocorticoid-dependent
signaling pathways. The effects of glucocorticoids on several cell
types, including AtT-20 cells, are sensitive to pertussis toxin, an
inhibitor of most members of the Gi/o family (66, 67, 68, 69, 70, 71, 72, 73).
Large conductance calcium-activated potassium channels (BK-channels),
which have been specifically implicated in the glucocorticoid-dependent
inhibition of stimulated ACTH secretion (29, 74), are also
regulated by Gi (75). Taken together, these observations
raise the possibility that Dexras1 might link the signaling pathways of
glucocorticoid and Gi-coupled receptors, and thereby mediate the
glucocorticoid-dependent inhibition of ACTH secretion in AtT-20
cells.
The impressive induction of Dexras1 mRNA by glucocorticoids suggests that transcriptional regulation may be the principal mechanism by which glucocorticoids activate Dexras1 signaling. Nevertheless, over-expression of wild-type Dexras1 did not affect hGH secretion, and thus signaling events apart from increased expression of Dexras1 are required for biological activities leading to this effect. One question raised by this observation is whether glucocorticoids could lead to both induction and activation of Dexras1. It is certainly possible that dexamethasone treatment could activate endogenous Dexras1 through the coordinate expression or activation of a guanyl nucleotide exchange factor. Alternatively, glucocorticoids may act solely in a permissive fashion to induce Dexras1, with activation of Dexras1 mediated through independent signaling pathways.
Our studies do not directly address specific interactions between Dexras1 and other signaling pathways, but rather demonstrate significant effects of Dexras1 activation on a biological endpoint, stimulus-coupled peptide secretion. Based on this important effect, it will be important in future studies to determine the specific roles Dexras1 plays in mediating signal transduction by glucocorticoids and heterotrimeric G proteins.
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Received September 19, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, left y-axis) or as the percentage of
secreted hGH relative to total detergent-soluble hGH (
, right
y-axis). Data represent the mean of four replicate experiments
(n = 4) ± SEM. Coexpression of constitutively
active Dexras[A178V] resulted in a 86% reduction in stimulated hGH
secretion (P < 0.01,