Endocrinology Vol. 142, No. 6 2631-2640
Copyright © 2001 by The Endocrine Society
Dexras1/AGS-1, a Steroid Hormone-Induced Guanosine Triphosphate-Binding Protein, Inhibits 3',5'-Cyclic Adenosine Monophosphate-Stimulated Secretion in AtT-20 Corticotroph Cells1
T. E. Graham,
T. A. Key,
K. Kilpatrick and
R. I. Dorin
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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Abstract
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Dexras1 is a novel GTP-binding protein that shares structural
similarity with the Ras family of small molecular weight GTPases
and is strongly and rapidly induced during treatment with
dexamethasone. The function of Dexras1 and its contribution to
glucocorticoid-dependent signaling in the corticotroph cell are
unknown. The present study was undertaken to examine the potential role
of Dexras1 in the regulation of peptide hormone secretion in the AtT-20
corticotroph cell line. To determine the effects of Dexras1 expressed
independently of glucocorticoid treatment, expression plasmids for
wild-type and constitutively active mutant Dexras1 proteins were
cotransfected with human GH (hGH), which provides an ectopic marker for
the stimulus-coupled secretory pathway. GTP binding properties and the
GTP to GDP ratio of wild-type and mutant Dexras1 proteins were examined
in transiently transfected AtT-20 and COS-7 cells. Stimulated and
constitutive components of secretion were assessed after 2-h
incubations with 5 mM 8-Br-cAMP or control. cAMP treatment
led to a 2-fold increase in hGH secretion relative to control.
Cotransfection of wild-type Dexras1 had no effect on cAMP-stimulated
hGH secretion, but a constitutively active mutant, Dexras[A178V],
attenuated stimulated secretion by 86% (P <
0.01). A double-mutant containing a deletion of the carboxyl terminus
isoprenylation site, Dexras[A178V/C277term], did not inhibit
cAMP-stimulated hGH secretion, indicating that the effect is
prenylation dependent. These findings suggest that activation of
Dexras1 has important functional consequences leading to inhibition of
stimulus-secretion coupling in corticotroph cells. Because Dexras1
messenger RNA is strongly and rapidly induced during glucocorticoid
treatment, these results raise the possibility that Dexras1 may
participate in the signal transduction pathways that govern the rapid
regulatory effects of glucocorticoids on peptide hormone secretion in
corticotroph cells.
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Introduction
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GLUCOCORTICOID-DEPENDENT INHIBITION of ACTH
synthesis and secretion in the corticotroph cell of the anterior
pituitary is a well-established and integral component of long-loop
negative feedback regulation of the hypothalamic-pituitary adrenal
axis. The inhibitory effects of glucocorticoids in primary
corticotrophs of the adenohypophysis and in mouse corticotroph AtT-20
cells are mediated through the classical type II glucocorticoid
receptor (GR) (1). The cellular effects of glucocorticoids
are pleiotropic, involving multiple intracellular targets and occurring
in distinct temporal domains (2, 3). Some of these
effects, such as inhibition of POMC gene transcription, appear to
involve direct effects of the ligand-bound GR at the gene promoter
(4). Transcriptional regulation may also be mediated
independently of direct DNA binding (5) through a
mechanism involving protein-protein interactions between GR and other
trans-activating proteins, such as c-Jun, c-Fos,
and NF-
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,
Gi
2, 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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Construction of Dexras1 [A178V] and [A178V/C277term]
mutants
Wild-type, mouse Dexras1 complementary DNA (cDNA) was kindly
provided by Dr. Robert Kemppainen (Auburn University College of
Veterinary Medicine, Auburn, AL) in the cloning plasmid pZL-1.
Full-length wild-type and mutant forms of Dexras1 were amplified by PCR
from pZL-1 using a primer pair designed with EcoRI and
NotI overhangs (5'-ATAGAATTCGCAATGAAACTGGCCGCGATGATC-3',
sense; 5'-ATAGCGGCCGCCTCCTAACTGATGACACAGCGC-3', antisense) and
subcloned into the pGEX-6P1 plasmid (BD PharMingen,
San Diego, CA). PCR-based, site-directed mutagenesis was used to
introduce the [A178V] and [A178V/C277term] mutations into a
carboxyl terminus fragment of wild-type Dexras1. The primers used for
mutagenesis were 5'-GCCTACTTCGAGATCTCAGTCAAAAAGAACAGCAGCTTG-3',
sense, and 5'-ATAGCGGCCGCCTCCTAACTGATGACACAGCGC-3', antisense, for
[A178V](or 5'-ATAGCGGCCGCCTCCTAACTGATGACTCAGCGCTCCT-3', antisense,
for [A178V/C277term]). PCR products were digested with
BglII and NotI and subcloned into corresponding
unique sites of pGEX-P1 already containing wild-type cDNA. The mutants
were confirmed by automated dye-termination nucleotide sequencing.
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 142984 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 (0.050.1 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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Comparison of Dexras1 and related GTP-binding proteins
The full-length cDNA of Dexras1 predicts a 280-amino-acid protein
with a calculated molecular mass of 31,700 Da (10). The
structural organization of Dexras1 is shown in Fig. 1
, 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).
Sequence homology analysis indicates that Dexras1 is most closely
related to members of the Ras superfamily of SMWG proteins, with 55%
amino acid homology to Rap2B, 54% homology to R-Ras, and 50% homology
to the prototypical Ras protein, H-Ras. Recently, several nucleotide
sequences predicting proteins with high degrees of homology to mouse
Dexras1 have been reported, including human Dexras1 (GenBank Accession
No. AF069506; Ref. 25) and rat Dexras1 (GenBank Accession
No. AF239157), which share 98% homology with mouse Dexras1. Human
Dexras1 is located at chromosome 2q32. The most closely related
homologs to Dexras1 are human Dexras2 and rat Dexras2 (GenBank
Accession Nos. HS569D19 and AF134409) which share approximately 80%
homology with human and mouse Dexras1. Human Dexras2 is located at
chromosome 22q13.1, and was recently identified as tumor endothelial
marker-2, a potential regulator of tumor angiogenesis and
revascularization (41). Rat Dexras2 is also known as the
ras homolog enriched in striatum due to a particularly high level of
expression in that tissue (42). Drosophila
Dexras (GenBank Accession No. CAB43324) shares 68% homology with mouse
Dexras1. The alignment of mouse Dexras1 with these proteins and other
Ras family members, along with their key structural features, is shown
in Fig. 2
. Two additional homologs with
less overall similarity have been identified in
Caenorhabditis elegans (GenBank Accession
No. AAA68305) and Saccharomyces cerevisiae
(GenBank Accession No. CAA97166).
Induction of Dexras1 mRNA by dexamethasone in AtT-20 cells
To examine the time course of glucocorticoid-induced Dexras1 mRNA,
we performed RT-PCR on total RNA pools isolated from AtT-20 cells
treated with 100 nM dexamethasone (see Fig. 3
). 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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Figure 3. RT-PCR of full-length Dexras1 and verification of
time course of induction by 100 nM dexamethasone. During
induction with dexamethasone, Dexras1 mRNA, amplified by nonlinear
RT-PCR, peaks at approximately 90' and rapidly decays after 2 h.
The figure is representative of two replicate experiments.
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Detection of overexpressed Dexras1 and mutants by Western
blotting
We detected equal amounts of [A178V] and [A178V/C277term]
mutants overexpressed in COS-7 cells by Western blotting with an
antiepitope antibody, as shown in Fig. 4
.
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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Figure 4. Detection of transfected wild-type and
mutant forms of Dexras1 expressed in cell culture. Expression of
6xHis-tagged Dexras1 species in COS-7 cells was analyzed by
Ni-NTA-agarose precipitation and Western blotting with the anti-Xpress
epitope antibody. The single band (identified by an
arrow) has a calculated Mr of approximately
36.5 kDa. The figure is representative of two replicate experiments.
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In vitro GTP binding activity of Dexras1 and mutants
In an effort to elucidate the function of Dexras1, we selectively
introduced an Ala
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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Figure 5. In vitro GTP binding
characteristics of wild-type and mutant forms of Dexras1. Four
replicate experiments using 6xHis-tagged Dexras1 species purified by
Ni-NTA-agarose precipitation from AtT-20 cells are depicted by this
data set (n = 4) ± SEM. Due to variation in
conditions between experiments, binding is normalized to wild-type
Dexras1.
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In vivo [32P]orthophosphate labeling of Dexras1
and mutants
To further characterize the guanyl nucleotide binding status of
Dexras1 and DR[A178V] mutants, we performed in vivo
orthophosphate labeling experiments. For these studies, Dexras1 or
mutants were transiently expressed in COS-7 cells, and intracellular
guanyl nucleotide pools were radiolabeled with
[32P]orthophosphate in vivo. Guanyl
nucleotide-bound Dexras1 was then affinity purified from cell lysates
with Ni-NTA-agarose, and bound guanyl nucleotides analyzed by TLC, as
shown in Fig. 6
, 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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|
Figure 6. In vivo guanyl nucleotide binding
state (GTP to GDP ratio) of wild-type and mutant forms of Dexras1. The
A V mutation at position 178 of Dexras1 is predicted to cause
increased binding of GTP due to enhanced nucleotide exchange and
normally higher intracellular levels of GTP. In COS-7 cells labeled
in vivo with [32P]orthophosphate,
Dexras[A178V], and Dexras[A178V/C277term] demonstrate greater
guanyl nucleotide exchange, evident by increased incorporation of
32P-labeled nucleotides (1.8 x 106 counts
vs. 1.2 x 106 counts;
P < 0.01) and an increased ratio of GTP to GDP, or
%GTP (16.9 ± 0.5% vs. 7.9 ± 1.3%;
P < 0.01), compared with wild-type Dexras1. The
figure is representative of two replicate experiments (n = 2) from
which average percent GTP ± SEM was derived.
|
|
Constitutively active Dexras1 inhibits cAMP-dependent
secretion
The effects of expression of wild-type and constitutively active
Dexras1 species on spontaneous and stimulated secretion of coexpressed
hGH are shown in Table 1
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.
Glucocorticoids have been reported to affect levels of hGH mRNA in
somatotroph cells through a mechanism that is poorly understood.
Because we hypothesize that Dexras1 may mediate some biological effects
of glucocorticoids, we also sought to examine how the expression of
Dexras species affects the synthesis of hGH expressed ectopically in
corticotroph cells. hGH was coexpressed with Dexras species or control
plasmid, cells were lysed with 1% Triton X-100, and detergent-soluble
hGH was measured. As shown in Table 1
, 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).
 |
Discussion
|
|---|
The Ras superfamily can be divided into groupings of proteins
(subfamilies) related by similarity in structure and function
(49). Overall homology of greater than 60% is typical
within the same subfamily, and homology of 3550% is typical between
members of different subgroups (50). The recent
identification of Dexras1 and related proteins distinguishes a new
family of Ras-related G proteins. As a group, they have molecular
masses ranging from 30,200 to 33,400 Da, which is significantly larger
than other Ras family members that have typical weights of 20,000 to
24,000 Da. Their increased molecular mass can be accounted for by the
presence of an extended carboxyl terminus variable region. The group is
also defined by unusually high net isoelectric points (ranging from 8.2
to 9.2), with a predicted excess of positively charged surface residues
at physiological pH, based on Emini surface probability analysis and
the regional distribution of pKa (51, 52).
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 1216% 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.
 |
Footnotes
|
|---|
1 This work was generously supported by the New Mexico Veterans
Affairs Health Care System (Albuquerque, New Mexico) and by Grant
2726264 of the Howard Hughes Medical Institute. 
Received September 19, 2000.
 |
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