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Endocrinology Vol. 142, No. 6 2631-2640
Copyright © 2001 by The Endocrine Society

ARTICLES

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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-{kappa}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 {alpha} subunit, Gi{alpha}2, in a receptor-independent fashion (25). This effect appears to involve a direct interaction between Dexras1 and Gi{alpha} (25) that causes enhanced guanyl nucleotide exchange by Gi{alpha} (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{alpha}-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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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. 1Go, and includes highly conserved GTP binding pocket ({Sigma}1-{Sigma}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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  		Figure 1. Schematic representation of the primary structure of a hormone-responsive, basic GTP-binding protein. Dexras1 contains all four components of the guanyl nucleotide binding and hydrolysis pocket ({Sigma}1-{Sigma}4) arranged with an order and spacing similar to that of other G proteins (36 37 ); an effector loop region similar to that of Ras, Rap, and R-Ras family members; and a carboxyl terminus CAAX box site for prenylation. The residues spanning from the {Sigma}4 domain to the CAAX box comprise an extended carboxyl terminus variable domain that accounts for the greater molecular mass of hormone-responsive, basic GTP-binding proteins as compared with other Ras family proteins.

 
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. 2Go. 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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  		Figure 2. Alignment of Dexras1 with related hormone-responsive basic GTP-binding proteins (Dexras2, Drosophila Dexras) and representative Ras family members. MultAlign software (76 ) was used to align Dexras1 with its most closely related homologs. Regions defining the GTP-binding and hydrolysis domain ({Sigma}1-{Sigma}4) and the CAAX box site for isoprenylation are boxed and annotated with their identifying consensus sequences (36 37 ). Consensus abbreviations: B, basic residue; J, polar residue; O, hydrophobic residue; X, any residue. Asterisks denote locations of A -> V mutation at position 178 (*) and C -> termination mutation at position 277 (**).

 
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. 3Go). 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.

 
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. 4Go. 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.

 
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 {Sigma}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. 5Go, 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.

 
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. 6Go, 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. 6Go, 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 1Go and in Fig. 7Go, 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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  		Table 1. Analysis of spontaneous and stimulated peptide hormone secretion

 


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  		Figure 7. Effects of wild-type and mutant forms of Dexras1 on cAMP-stimulated secretion of cotransfected hGH. Control (pcDNA empty vector) or expression plasmids for wild-type or mutant Dexras1 proteins were cotransfected with expression plasmid for hGH. Secreted hGH was assayed following 2 h incubation with 5 mM 8-Br-cAMP or vehicle and expressed as the absolute quantity of cAMP-stimulated hGH secretion ({blacksquare}, 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, {blacksquare}), and a 75% reduction in percent-stimulated hGH secretion relative to total detergent-soluble hGH (P < 0.01, ). The CAAX-box-deficient mutant Dexras[A178V/C277term] did not significantly alter stimulated secretion (P = NS) but exerted a moderate positive effect on the percent of stimulated secretion (P = 0.04) and the percent of spontaneous secretion (P < 0.01).

 
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 1Go, 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. 7Go and Table 1Go). 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 35–50% 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 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{alpha}-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 {alpha} 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{alpha} 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{alpha}.

The discovery of Dexras1 by two independent, function-oriented cloning methods (10, 25) further suggests that Dexras1 may represent a nexus between Gi{alpha}- 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. Back

Received September 19, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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