Correspondence to Tomoshige Kino: kinot{at}mail.nih.gov
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Introduction |
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The actions of glucocorticoids are mediated by a ubiquitous intracellular receptor protein, the glucocorticoid receptor (GR), which functions as a hormone-activated transcription factor of glucocorticoid target genes (Kino et al., 2003a). The GR consists of three domains: the NH2-terminal or "immunogenic" domain, the central, DNA-binding domain (DBD), and the COOH-terminal, ligand-binding domain. The functions of the latter two domains have been studied extensively, whereas those of the immunogenic domain are less well known (Kino et al., 2003a). In the unliganded state, GR is located primarily in the cytoplasm, as part of hetero-oligomeric complexes containing heat shock proteins 90, 70, and 50, and, possibly, other proteins. After binding to its agonist ligand, the GR undergoes conformational changes, dissociates from the heat shock proteins, homodimerizes, and translocates into the nucleus through the nuclear pore via an active process (Kino et al., 2003a). There, the ligand-activated GR directly interacts with DNA sequences, the glucocorticoid response elements (GREs), in the promoter regions of target genes, or with other transcription factors via proteinprotein interactions, indirectly influencing the activity of the latter on their target genes (Kino and Chrousos, 2002; Kino et al., 2003a). The GRE-bound GR stimulates the transcription rate of responsive genes by facilitating the formation of a transcription initiation complex, including the RNA polymerase II and its ancillary components via its activation function (AF)-1 and AF-2 domains (Kino et al., 2003a). The former is localized in the immunogenic domain, whereas the latter spans the entire ligand-binding domain.
Because glucocorticoids have a broad array of life-sustaining functions and play an important role in therapeutic interventions, changes of tissue sensitivity to glucocorticoids may be associated with and influence the course and therapy of many pathological states (Kino and Chrousos, 2002; Kino et al., 2003a). Such changes may present on either side of an optimal range, respectively, as glucocorticoid resistance or hypersensitivity, and may be generalized and/or tissue specific. Several autoimmune/inflammatory/allergic states, such as rheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerative colitis and asthma, are often associated with resistance of the inflamed tissues to glucocorticoids (Chrousos, 1995; Kino and Chrousos, 2002). On the other hand, glucocorticoid hypersensitivity has been suggested in visceral obesityrelated insulin resistance associated with components of the dysmetabolic syndrome, and in the acquired immunodeficiency syndrome caused by human immunodeficiency virus type 1 infection (Chrousos, 2000; Kino et al., 2003b). These changes in tissues' sensitivity to glucocorticoids associated with such pathological conditions may be possible by local modifications of GR functions, where altered concentrations/production of hormones, neurotransmitters, cytokines, growth factors and autacoids may play important roles (Kino and Chrousos, 2002; Kino et al., 2003a). The biological activities of such extracellular molecules are transduced into the intracellular compartment via their specific cell surface receptors (Cotecchia et al., 2004; Radeff-Huang et al., 2004). Binding of the compounds to their receptors activates signal-transducing molecules located on the cytoplasmic side of the plasma membrane, which subsequently communicate with downstream effector molecules, finally exerting a variety of biological effects on their target cells.
The heterotrimeric guanine nucleotide-binding proteins (G proteins) are downstream signal transducers for the G proteincoupled receptors (GPCRs), which form a large family consisting of >1,000 members (Cabrera-Vera et al., 2003). There are three subunits, G, Gß, and G
, each of which is also composed of many isoforms (Hamm, 1998; Cabrera-Vera et al., 2003). Among them, Gß contains a portion characterized as a seven timesrepeated blade-like structure, called a WD repeat (Clapham and Neer, 1997; Smith et al., 1999). Crystallographic analyses revealed that all WD-repeats of Gß are made up of four twisted ß strands and are arranged in a ring, thus, forming a propeller-like structure (Clapham and Neer, 1997). Gß is associated with G
through its NH2-terminal
helical portion and the fifth blade of the WD repeats.
The heterotrimeric complex consisting of G, Gß, and G
is attached to the cytoplasmic surface of the plasma membrane through the prenylated G
(Clapham and Neer, 1997). This heterotrimeric complex is inactive in the GDP-bound state. Once ligands bind to their GPCRs, GDP on G
is catalyzed to GTP, leading to dissociation of the GTP-bound G
from the Gß/G
heterodimer (Hamm, 1998). Liberated GTP-G
and the Gß/G
complex then interact with their downstream effector molecules and exert their biological actions (Clapham and Neer, 1997). The Gß/G
complex binds to and modulates the activity of diverse molecules, such as several forms of potassium and calcium ion channels, the enzymes phospholipase A2 and Cß, several adenylyl cyclases and the plasma membrane Ca2+ ATPase pump (Clapham and Neer, 1997).
The receptor for activated C-kinase 1 (Rack1), also a WD-repeat protein that harbors 42% amino acid similarity to Gß with many conserved amino acids, plays regulatory roles in cell development and growth, as well as in the immune response and brain function (McCahill et al., 2002). Rack1 exerts its biological activities by binding to numerous partner molecules, including PKCb, phosphodiesterase 4-D5, Src family kinases, the Interleukin-3 and Interleukin-5, and granulocyte macrophage-colony stimulating factor receptors, and by regulating their activities as a scaffold molecule providing steric hindrance (McCahill et al., 2002). For example, Rack1 acts as a negative scaffold on the Src family kinases by binding to the phosphotyrosine-binding pocket of their Src homology-2 domains and by inhibiting their further association with downstream molecules (Chang et al., 2001; McCahill et al., 2002).
To look for partner molecules that may interact with GR and explain the local modification of GR activity in several physiological or pathological conditions, we performed a yeast two-hybrid screening assay using fragments of the GR immunogenic domain. We found that the WD repeat proteins Gß2 and Rack1 are specific interactors of GR, negatively regulating its transcriptional activity. Because Gß proteins play key roles in many signal transduction cascades, GR-associated Gß might play a role in the development of target tissue resistance or hypersensitivity to glucocorticoids in states in which the G protein system is affected and the sensitivity to glucocorticoids is altered.
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Results |
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The Gß/G heterodimer, but not G
i, is associated with GR in vivo
To test if Gß interacts with GR in vivo, we performed a regular coimmunoprecipitation assay using specific antibodies to Gß, G, G
i, and GR in HeLa cells, which express all these molecules endogenously. Both Gß and G
, but not G
i, were coprecipitated with GR in the absence of dexamethasone, and addition of dexamethasone increased the precipitation of these molecules with GR (Fig. 2 G, top three gels). This effect of dexamethasone was not through induction of any of these three proteins, because their expression levels did not change throughout the experiment (Fig. 2 G, bottom four gels). These results indicate that the Gß/G
heterodimer, but not G
i, is a component of the complex formed with GR both in the absence and presence of glucocorticoids. Gß2 mutants, G10K and Gß2(34340), both of which are defective in the association with G
(Garritsen et al., 1993), lost the wild-type Gß suppressive effect on GR-induced transactivation in HCT116 cells (unpublished data), further confirming that Gß2 acts as a heterodimer with G
to suppress the transcriptional activity of the GR. They also suggest that the overexpressed Gß1 and Gß2 acted on GR by forming complexes with endogenous G
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Somatostatin suppresses GR-induced transcriptional activity by activating Gß/G in rat GH3 cells
Somatostatin binds its receptor and activates the Gß/G dimer by causing dissociation of the complex from GTP-bound G
i (Law et al., 1991; Brown and Schonbrunn, 1993). Thus, we tested the effect of somatostatin on GR-induced transactivation in rat pituitary GH3 cells, which express endogenous somatostatin receptor and GR (Yatani et al., 1987; Levitan et al., 1991). These cells also express the Kv1.5 potassium channel, whose expression levels are positively regulated by glucocorticoids possibly though GREs located in its promoter region (Levitan et al., 1991). 24-h incubation of cells with 106 M of dexamethasone stimulated mRNA expression of the Kv1.5 potassium channel by eightfold and somatostatin suppressed this dexamethasone effect in a dose-dependent fashion (Fig. 3 A). Transfection of Gß1 and Gß2 siRNAs, which strongly suppressed the mRNA expression of these subunits (Fig. 3, B and C), enhanced dexamethasone-induced induction of Kv1.5 potassium channel mRNA and attenuated the suppressive effect of somatostatin (Fig. 3 A). These results indicate that somatostatin suppressed GR-induced transcriptional activity of the endogenous Kv1.5 potassium channel gene by activating the Gß/G
complex.
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To further examine the subcellular localization of Gß and G, we constructed plasmids expressing EGFP-fused Gß2 or G
2 and examined their localization in the HCT116 cells (Fig. 4, C and D). Consistent with our results obtained with indirect immunofluorescence staining and Western blots, EGFP-Gß2 was mainly distributed in the cytoplasm, with a relatively small fraction in the nucleus and a very small fraction at the plasma membrane (Fig. 4 C, left and Fig. 4 D). EGFP-G
2 was also detected in the cytoplasm, as well as at the plasma membrane, whereas a small fraction was detected in the nucleus (Fig. 4 C, right, and Fig. 4 D). The limited localization of EGFP-Gß2 at the plasma membrane was not caused by a small number of binding sites at the plasma membrane, as coexpression of G
2 and the human somatostatin receptor type 2 (SSTR2) that provided binding sites for EGFP-Gß2 at the plasma membrane, did not change the characteristic subcellular localization of EGFP-Gß2 (unpublished data).
Gß2 and GR comigrate either to the nucleus or to the plasma membrane in response to dexamethasone or somatostatin, respectively
Because the above results suggest that Gß2 and GR might communicate in the cytoplasm as well as in the nucleus, we examined colocalization of these two molecules by overexpressing EGFP-Gß2 and DsRed2-GR in HCT116 cells (Fig. 4, E and F). EGFP-Gß2 was localized mainly in the cytoplasm in addition to the nucleus, whereas DsRed2-GR was exclusively distributed in the former subcellular fraction in the absence of glucocorticoids. 30-min incubation of the transfected cells with 106 M of dexamethasone induced nuclear translocation of both DsRed2-GR and EGFP-Gß2 (Fig. 4, E and F).
Because very little Gß2 was found at the plasma membrane and EGFP-Gß2 comigrated from the cytoplasm into the nucleus with the GR in response to dexamethasone, we stimulated a GPCR to test the hypothesis that this treatment induces the plasma membrane localization of EGFP-Gß in HCT116 cells. We coexpressed EGFP-Gß2 and DsRed2-GR with G2 and SSTR2, added 100 nM of somatostatin in the medium and examined the localization of EGFP-Gß2 under the microscope. 30 min after addition of somatostatin, EGFP-Gß2 accumulated at the plasma membrane (Fig. 4 G). EGFP-Gß2 was not seen at the plasma membrane after 3 h, possibly because of internalization of the receptor complex into the cell (unpublished data). As expected, a small amount of DsRed2-GR also accumulated at the plasma membrane with EGFP-Gß2. These results indicate that EGFP-Gß2 migrated from the cytoplasm to the plasma membrane in response to stimulation of a G proteincoupled, cell surface receptor. They also indicate that some fractions of Gß and GR are associated with each other in the cytoplasm and can translocate to either the nucleus or the plasma membrane following the activation of the GR or the cell surface GPCR.
Forced cytoplasmic localization of Gß2 attenuates its suppressive effect on GR-induced transcriptional activity
To further examine the mechanism of Gß2-induced suppression of GR transactivation, we constructed plasmids expressing a nuclear export signal (NES)- or NLS-fused Gß2 and EGFP-Gß2. The NES sequence used was that of the human protein kinase inhibitor (Henderson and Eleftheriou, 2000), whereas the NLS sequence was from the SV40 large T antigen (Rihs et al., 1991). NES-fused EGFP-Gß2 was localized exclusively in the cytoplasm, whereas NLS-fused EGFP-Gß2 was located entirely in the nucleus (Fig. 5, A and B). As expected, NES-Gß2 lost its suppressive effect on GR-induced transactivation, whereas NLS-Gß2 demonstrated a stronger suppressive effect than the wild-type Gß2 (Fig. 5 C). Wild-type, and NES- and NLS-fused Gß2 were similarly expressed in HCT116 cells (Fig. 5 D). These results indicate that increased nuclear localization of Gß2 correlates with its suppressive effect on GR-induced transcriptional activity.
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We further examined the effect of Gß2 on the transcriptional activity of the two GR mutants GR(77-261) and (1-515) in HCT116 cells, which are respectively devoid of the AF-1 and AF-2 domains (Fig. 6 D). Gß2 suppressed the transcriptional activity of GR(
77-261) similarly to its effect on the wild-type GR, whereas it did not affect the transcriptional activity of GR(1-515), indicating that Gß2 acts on GR by suppressing AF-2 but not AF-1.
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Discussion |
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Components of the G protein system are presumably strictly located at the inner surface of the plasma membrane attached to it through the prenylated G (Clapham and Neer, 1997). However, there are previously reported exceptions (Kageyama et al., 1999; Jin et al., 2000; Zhang et al., 2001; Chen et al., 2004); for instance, one of the Gßs, Gß5 was reported in the nucleus as well as in the cytoplasm and the membrane fraction of the cell, and this protein translocated into the nucleus in response to surface stimuli in neuronal cells (Zhang et al., 2001). Similarly, Gß3 was found in the cytoplasmic pool and moved to the plasma membrane in response to stimulation of the ß1 adrenergic receptor in rat cardiac cells (Kageyama et al., 1999). Furthermore, endogenous Gß replaced with the GFP-fusion form in yeast cells was localized in the cytoplasm, in addition to the plasma membrane, and changed localization in response to a chemo-attractant (Jin et al., 2000). A distribution of Gß1/G
2 similar to the one we found in our study was also reported recently (Chen et al., 2004). In addition to these previous reports, our results indicate that the localization of Gß is more diffuse than previously thought, being distributed in the cytoplasm and the nucleus, as well as at the cytoplasmic surface of the plasma membrane. EGFP-Gß2 comigrated with DsRed2-GR into the nucleus in response to dexamethasone, whereas it comigrated to the plasma membrane with the GR after addition of somatostatin. Together, these results indicate that Gß can migrate between subcellular compartments, such as between the cytoplasm and the nucleus or the plasma membrane, depending on the type of stimuli and the responsive signaling molecules activating the cell. Thus, it appears that cytoplasmic Gß may function as a reservoir pool, which supplies G proteins into various subcellular compartments according to the needs of the cell (Fig. 7).
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In our experiments, 24-h incubation of cells with somatostatin suppressed GR-induced transcriptional activity by stimulating the Gß/G heterodimer, possibly by increasing the fraction that can comigrate into the nucleus with the GR in response to dexamethasone. The underlying mechanism of this action is not understood, but it is possible that free Gß/G
heterodimers produced after stimulation with somatostatin might have a greater chance to be associated with the GR, comigrate with it into the nucleus and finally suppress GR-induced transactivation. Somatostatin has anti-inflammatory activity, and glucocorticoids increase local production of somatostatin in inflamed tissues, a phenomenon that may explain part of their anti-inflammatory actions (Karalis et al., 1994, 1995). Our results of the suppressive effect of somatostatin on GR-induced transactivation complete a closed regulatory circuit between glucocorticoids and somatostatin, with the ligand-activated GR stimulating somatostatin and the GR-associated somatostatin-stimulated Gß/G
complex suppressing the latter's effect.
Gß physically interacted with the GR as a heterodimer with G in the absence of dexamethasone, and addition of dexamethasone further increased the association of the Gß/G
complex with the GR. The ligand-free GR forms a multi-protein complex with several heat shock proteins. In this complex, GR is also associated with other molecules, such as protein 14-3-3, Raf-1, and p53, which modulate GR-induced transcriptional activity (Kino and Chrousos, 2002). The Gß/G
complex and Rack1 should be added to these GR-associated molecules, which suppress the transcriptional activity of the GR. Because Rack1 plays an important role in the regulation of signaling events induced by PKC, cAMP, and several growth factors and cytokines (McCahill et al., 2002), it is possible that these signaling cascades also regulate GR function through Rack1, as in the case of Gß, where the upstream stimulus somatostatin suppresses GR-induced transcriptional activity.
Gß together with G was attracted to GREs through the GR, inhibiting AF-2. Gß did not have any intrinsic transrepressive activity, and, hence, it is unlikely that it functions as an active repressor by attracting inhibitory molecules/complexes, such as, for instance, corepressors with histone deacetylase activity (Jones and Shi, 2003). Rather, Gß/G
may act as a negative scaffold, similarly to Rack1, by binding to a critical portion of the GR and/or other transcriptional intermediate molecules that are attracted to the AF-2, blocking full activation of AF-2 and hence transcription stimulation by the ligand-bound GR.
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Materials and methods |
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Yeast two-hybrid screening and assay
The yeast two-hybrid screening was performed using GR(263-419) as a bait in a human Jurkat cell cDNA library with the LexA system (CLONTECH Laboratories, Inc.). For a yeast two-hybrid assay, yeast strain EGY48 (CLONTECH Laboratories, Inc.) was transformed with pOP8-LacZ, pLexA-GR (263419), and indicated pB42AD-Gß1, -Gß2, and -Rack1 plasmids and ß-galactosidase activity was measured in the cell suspension as previously described (Kino and Chrousos, 2003). The ß-galactosidase activity was normalized for OD value at 600 nm. Fold induction was calculated by the ratio of adjusted ß-galactosidase values of cells transformed with pLexA-derived bait plasmids versus pLexA in the presence of the same prey plasmid.
Cell cultures, transient transfections, and reporter assays
Human colon carcinoma HCT116, African green monkey kidney COS7, uterine cervical carcinoma HeLa, and rat pituitary GH3 cells were purchased from the American Type Culture Collection and maintained in MacCoy's 5A or DME media supplemented with 10% FBS, 50 U of penicillin, and 50 µg/ml of streptomycin. Rat hepatoma HTC cells were provided by J.I. Webster (National Institute of Mental Health, Bethesda, MD) and were cultured in DME with the same supplements. HCT116 and COS7 cells do not contain functional GR, whereas HeLa, HTC, and GH3 cells express the fully active GR (Levitan et al., 1991; Thompson et al., 1966; Kino et al., 2003c; Kino and Chrousos, 2003; Webster et al., 2003; De Martino et al., 2004).
HCT116 cells were transfected as previously described (Kino et al., 2003c). For the experiments using pMMTV-Luc or pGAL4-E1B-Luc as reporter plasmids, different amounts of Gß1-, Gß2-, G2-, Rack1-, or GAL4-fused molecule-expressing plasmids were cotransfected with 0.5 µg/well of pRShGR
(for pMMTV-Luc), 1.5 µg/well of pMMTV-Luc or pGAL4-E1B-Luc and 0.5 µg/well of pSV40-ß-Gal. Empty vectors were used to maintain the same amounts of transfected DNA. 106 M of dexamethasone was added to 24 h after transfection. The cells were harvested after an additional 24 h and luciferase and ß-galactosidase assays were performed as previously described (Kino et al., 1999).
Introduction of Gß1 and Gß2 siRNAs into HTC and GH3 cells, the TAT assay, and the RT-PCR
The rat Gß1 and Gß2 siRNAs (5'-AAGCUCUGGGAUGUCCGAGAAdTdT-3' and 5'-AACAUCUGCUCCAUCUAUAGUdTdT-3', respectively), which respectively target nucleotides 625645 and 355375 of their coding regions, were produced by QIAGEN. The negative control siRNA (5'-UUCUCCGAACGUGUCACGUdTdT-3') was also purchased from QIAGEN.
HTC and GH3 cells were transfected with siRNAs by using the Nucleofector System (Amaxa GmbH). In brief, 106 of HTC or GH3 cells were resuspended in solution R and T (Amaxa GmbH), respectively, and were mixed with 5 µg of indicated siRNAs. The electricity was applied with the Nucleofector Device (Amaxa GmbH) by using the protocol T-27 and T-20 for HTC and GH3 cells, respectively. We achieved nearly 80% transfection efficiencies in both cell lines with these combinations of solutions and protocols. 24 h after plating these cells in the 24-well plates, the cells were stimulated with 106 M of dexamethasone and/or different concentrations (0100 nM) of somatostatin. After additional 24 h of incubation, cell lysates for the TAT assay and Western blots, and total RNA for the RT-PCR were harvested. TAT assays were performed as previously reported (Thompson et al., 1966; Webster et al., 2003).
The reverse transcription reaction was performed as previously described (De Martino et al., 2004). To detect mRNA levels of rat Gß1, Gß2, Kv1.5 potassium channel and control rat acidic ribosomal phosphoprotein P0 (RPLP0), primer pairs (Gß1: forward: 5'-CAGCAGACAACCACGTTTAC-3', reverse: 5'-CAGCCTGCATGTAGCATC-3'; Gß2: forward: 5'-CTCATCATTTGGGACAGCTAC-3', reverse: 5'-GTGATGATT-TGGTTGTCGTC-3'; Kv1.5 potassium channel: forward: 5'-CAACCTAGAAGGCTATCT-3, reverse: 5'-GTCGAAGAAGTATTCATTTC-3; RPLP0: forward: 5'-GACATGCTGCTGGCCAATAAG-3'; reverse: 5'-CAACATGTTCAGCAGTGTG-3') were used. The RT-PCR reaction, consisting of heat activation of the Taq polymerase (10 min at 95°C) and the subsequent 60 PCR cycles (denaturing: 15 s at 95°C; annealing/extension: 1 min at 60°C) was performed in quadruplicate using the SYBR green PCR Master Mix (Applied Biosystems) in an ABI PRIZM 7900 SDS lightcycler (Applied Biosystems). Obtained CT (threshold cycle) values of Gß1, Gß2 and the Kv1.5 potassium channel were normalized for those of RPLP0 and their relative mRNA expressions were demonstrated as fold induction of the baseline. The dissociation curves of used primer pairs showed a single peak and samples after PCR reactions had a single expected DNA band in an agarose gel analysis (unpublished data).
Confocal microscopy analyses
HCT116 were cultured on poly-L-lysinecoated cover slides (for fixed cells) or in Delta-T Culture Dishes (Bioptechs Inc.; for live cells) and transfected and/or treated with the indicated plasmids/compounds. They were fixed with 4% PFA, and were subsequently mounted on glass slides with the Vectashield with DAPI (Vector Laboratories, Inc.), or were directly examined under the microscope. Emitted signals were recorded with the LSM510 meta/axiovert 200M microscope (Carl Zeiss MicroImaging, Inc.) stand at 19 ± 1.0°C for fixed samples and at 37 ± 0.5°C for samples cultured in Delta-T Culture Dishes at the NICHD Microscopy and Imaging Core (National Institute of Child Health and Human Development) with the assistance of V. Schram. The plan-apochromat 63x oil (1.4 NA, DIC, working distance = 0.17 mm; Carl Zeiss MicroImaging, Inc.) objective lens (Carl Zeiss MicroImaging, Inc.) was used with the lens immersion medium (Immersol 518FF, n = 1.518: Carl Zeiss MicroImaging, Inc.) for image acquisition. Confocal images were built point by point by collecting the intensities from the photo-multiplier tube using LSM 5 software version 3.2 (Carl Zeiss MicroImaging Inc.). Endogenous Gß and G were visualized with anti-Gß and -G
2 antibodies (Santa Cruz Biotechnology, Inc.) followed by the FITC-labeled antirabbit IgG antibody (Santa Cruz Biotechnology, Inc.). To demonstrate the specificity of anti-Gß and -G
2 antibodies, blocking peptides (Santa Cruz Biotechnology, Inc.) were co-administered with them.
Regular coimmunoprecipitation assay, subcellular fractionation, and Western blots
HeLa cells were treated with 106 M of dexamethasone or vehicle for 3 h. Cell lysis and coimmunoprecipitation were carried as previously described (Kino et al., 1999). Proteins were precipitated by anti-hGR antibody or control rabbit IgG (Santa Cruz Biotechnology, Inc.) and the proteinantibody complexes were collected with protein Agarose A/G PLUS (Santa Cruz Biotechnology, Inc.). After blotting on nitrocellulose membranes, G
i, Gß, and G
were detected by anti-G
i, -Gß, or -G
2 antibodies, respectively (Santa Cruz Biotechnology, Inc.). To evaluate endogenously expressed GR, G
i, Gß, and G
, 10% of cell lysates used in the coimmunoprecipitation reaction were run on SDS-PAGE gels.
To compare endogenous levels of Gß and G residing in cytoplasmic, nuclear or membrane fractions, HCT116 cells were harvested and homogenated in buffer containing 50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 0.25 M sucrose and 1 Tab/50 ml Complete Tablet, and were centrifuged at 500 g for 5 min to obtain the whole homogenate. All the procedures were performed at 4°C. The whole homogenates were then centrifuged at 2,000 g for 15 min to harvest the nuclear fraction (pellet) and the supernatant was further centrifuged at 105,000 g for 1 h to separate the cytoplasmic (supernatant) and membrane (pellet) fractions. The nuclear and membrane fractions were washed once with the same buffer. The samples (0.1 µg of protein) were run on SDS-PAGE gels, transferred to the nitrocellulose membranes, and Gß and G
were detected by reprobing the same membrane with anti-Gß or -G
2 antibodies (Santa Cruz Biotechnology, Inc.), respectively. The intracellular adhesion molecule 1,
-tubulin and Oct1 were also respectively detected as positive controls for the membrane, cytoplasmic and nuclear fractions by reprobing the membrane with their specific antibodies (Santa Cruz Biotechnology, Inc.). To evaluate the expression levels of wild-type and NES-/NLS-fused Gß2, plasmids expressing these molecules were transfected into HCT116 cells, and whole homogenates were prepared as described above. Expressed Gß-related proteins were then separated on a SDS-PAGE gel, blotted, and visualized with anti-His antibody (Santa Cruz Biotechnology, Inc.).
ChIP assay
ChIP assay was performed in COS7/MMTV cells, which have the genomically integrated MMTV-luciferase gene, (COS7/MMTV), using a ChIP kit (Upstate Biotechnology) with minor modifications. COS7/MMTV cells were transfected with control plasmid, pRShGR, or pRShGR
(
262-404) in the presence of pCDNA4His/MaxB-Gß2, and were exposed to either 106 M of dexamethasone or vehicle for 5 h. The cells were then fixed, DNA and bound proteins were cross-linked, and ChIP assays were performed by coprecipitating the DNAprotein complexes with anti-GR
and -Gß antibodies or rabbit control IgG (Santa Cruz Biotechnology, Inc.), as previously reported (De Martino et al., 2004). The promoter region 219 to 47 of the MMTV long terminal repeat was amplified from the prepared DNA samples using a primer pair: 5'-AACCTTGCGGTTCCCAG-3' and 5'-GCATTTACATAAGATTTGG-3'. Amplified products were then run on a 3% agarose gel and visualized DNA bands were photographed.
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Acknowledgments |
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Submitted: 24 September 2004
Accepted: 27 April 2005
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References |
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Brown, P.J., and A. Schonbrunn. 1993. Affinity purification of a somatostatin receptor-G-protein complex demonstrates specificity in receptor-G-protein coupling. J. Biol. Chem. 268:66686676.
Cabrera-Vera, T.M., J. Vanhauwe, T.O. Thomas, M. Medkova, A. Preininger, M.R. Mazzoni, and H.E. Hamm. 2003. Insights into G protein structure, function, and regulation. Endocr. Rev. 24:765781.
Chang, B.Y., M. Chiang, and C.A. Cartwright. 2001. The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1. J. Biol. Chem. 276:2034620356.
Chen, S., E.J. Dell, F. Lin, J. Sai, and H.E. Hamm. 2004. RACK1 regulates specific functions of Gbetagamma. J. Biol. Chem. 279:1786117868.
Chrousos, G.P. 1995. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N. Engl. J. Med. 332:13511362.
Chrousos, G.P. 2000. The role of stress and the hypothalamic-pituitary-adrenal axis in the pathogenesis of the metabolic syndrome: neuro-endocrine and target tissue-related causes. Int. J. Obes. Relat. Metab. Disord. 24:S50S55.[CrossRef][Medline]
Chrousos, G.P. 2004. The glucocorticoid receptor gene, longevity, and the complex disorders of Western societies. Am. J. Med. 117:204207.[CrossRef][Medline]
Clapham, D.E., and E.J. Neer. 1997. G protein beta gamma subunits. Annu. Rev. Pharmacol. Toxicol. 37:167203.[CrossRef][Medline]
Cotecchia, S., L. Stanasila, D. Diviani, K. Bjorklof, O. Rossier, and F. Fanelli. 2004. Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes. Biol. Cell. 96:327333.[CrossRef][Medline]
De Martino, M.U., N. Bhattachryya, S. Alesci, T. Ichijo, G.P. Chrousos, and T. Kino. 2004. The glucocorticoid receptor and the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II interact with and mutually affect each other's transcriptional activities: implications for intermediary metabolism. Mol. Endocrinol. 18:820833.
Falkenstein, E., H.C. Tillmann, M. Christ, M. Feuring, and M. Wehling. 2000. Multiple actions of steroid hormones -a focus on rapid, nongenomic effects. Pharmacol. Rev. 52:513556.
Garritsen, A., P.J. van Galen, and W.F. Simonds. 1993. The N-terminal coiled-coil domain of beta is essential for gamma association: a model for G-protein beta gamma subunit interaction. Proc. Natl. Acad. Sci. USA. 90:77067710.
Hamm, H.E. 1998. The many faces of G protein signaling. J. Biol. Chem. 273:669672.
Henderson, B.R., and A. Eleftheriou. 2000. A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. Exp. Cell Res. 256:213224.[CrossRef][Medline]
Jantzen, H.M., U. Strahle, B. Gloss, F. Stewart, W. Schmid, M. Boshart, R. Miksicek, and G. Schutz. 1987. Cooperativity of glucocorticoid response elements located far upstream of the tyrosine aminotransferase gene. Cell. 49:2938.[CrossRef][Medline]
Jin, T., N. Zhang, Y. Long, C.A. Parent, and P.N. Devreotes. 2000. Localization of the G protein betagamma complex in living cells during chemotaxis. Science. 287:10341036.
Jones, P.L., and Y.B. Shi. 2003. N-CoR-HDAC corepressor complexes: roles in transcriptional regulation by nuclear hormone receptors. Curr. Top. Microbiol. Immunol. 274:237268.[Medline]
Kageyama, K., T. Murakami, K. Iizuka, K. Sato, K. Ichihara, Y. Tokumitsu, A. Kitabatake, and H. Kawaguchi. 1999. Translocation of G-protein beta3 subunit from the cytosol pool to the membrane pool by beta1-adrenergic receptor stimulation in perfused rat hearts. Biochem. Pharmacol. 58:14971500.[CrossRef][Medline]
Karalis, K., G. Mastorakos, G.P. Chrousos, and G. Tolis. 1994. Somatostatin analogues suppress the inflammatory reaction in vivo. J. Clin. Invest. 93:20002006.[Medline]
Karalis, K., G. Mastorakos, H. Sano, R.L. Wilder, and G.P. Chrousos. 1995. Somatostatin may participate in the antiinflammatory actions of glucocorticoids. Endocrinology. 136:41334138.[Abstract]
Kino, T., and G.P. Chrousos. 2002. Tissue-specific glucocorticoid resistance-hypersensitivity syndromes: multifactorial states of clinical importance. J. Allergy Clin. Immunol. 109:609613.[CrossRef][Medline]
Kino, T., and G.P. Chrousos. 2003. Tumor necrosis factor alpha receptor- and Fas-associated FLASH inhibit transcriptional activity of the glucocorticoid receptor by binding to and interfering with its interaction with p160 type nuclear receptor coactivators. J. Biol. Chem. 278:30233029.
Kino, T., A. Gragerov, J.B. Kopp, R.H. Stauber, G.N. Pavlakis, and G.P. Chrousos. 1999. The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor. J. Exp. Med. 189:5162.
Kino, T., M.U. De Martino, E. Charmandari, M. Mirani, and G.P. Chrousos. 2003a. Tissue glucocorticoid resistance/hypersensitivity syndromes. J. Steroid Biochem. Mol. Biol. 85:457467.[CrossRef][Medline]
Kino, T., M. Mirani, S. Alesci, and G.P. Chrousos. 2003b. AIDS-related lipodystrophy/insulin resistance syndrome. Horm. Metab. Res. 35:129136.[CrossRef][Medline]
Kino, T., E. Souvatzoglou, M.U. De Martino, M. Tsopanomihalu, Y. Wan, and G.P. Chrousos. 2003c. Protein 14-3-3sigma interacts with and favors cytoplasmic subcellular localization of the glucocorticoid receptor, acting as a negative regulator of the glucocorticoid signaling pathway. J. Biol. Chem. 278:2565125656.
Law, S.F., D. Manning, and T. Reisine. 1991. Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors. J. Biol. Chem. 266:1788517897.
Levitan, E.S., L.M. Hemmick, N.C. Birnberg, and L.K. Kaczmarek. 1991. Dexamethasone increases potassium channel messenger RNA and activity in clonal pituitary cells. Mol. Endocrinol. 5:19031908.[Abstract]
Liu, X., C.A. Wang, and Y.Z. Chen. 1995. Nongenomic effect of glucocorticoid on the release of arginine vasopressin from hypothalamic slices in rats. Neuroendocrinology. 62:628633.[Medline]
McCahill, A., J. Warwicker, G.B. Bolger, M.D. Houslay, and S.J. Yarwood. 2002. The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol. Pharmacol. 62:12611273.
Nagy, L., H.Y. Kao, D. Chakravarti, R.J. Lin, C.A. Hassig, D.E. Ayer, S.L. Schreiber, and R.M. Evans. 1997. Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell. 89:373380.[CrossRef][Medline]
Navarro, C.E., S.A. Saeed, C. Murdock, A.J. Martinez-Fuentes, K.K. Arora, L.Z. Krsmanovic, and K.J. Catt. 2003. Regulation of cyclic adenosine 3',5'-monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons. Mol. Endocrinol. 17:17921804.
Pazin, M.J., J.W. Hermann, and J.T. Kadonaga. 1998. Promoter structure and transcriptional activation with chromatin templates assembled in vitro. A single Gal4-VP16 dimer binds to chromatin or to DNA with comparable affinity. J. Biol. Chem. 273:3465334660.
Radeff-Huang, J., T.M. Seasholtz, R.G. Matteo, J.H. Brown, S. Cotecchia, L. Stanasila, D. Diviani, K. Bjorklof, O. Rossier, and F. Fanelli. 2004. G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. Structural determinants involved in the activation and regulation of G protein-coupled receptors: lessons from the alpha1-adrenegic receptor subtypes. J. Cell. Biochem. 92:949966.[CrossRef][Medline]
Rihs, H.P., D.A. Jans, H. Fan, and R. Peters. 1991. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO J. 10:633639.[Abstract]
Smith, T.F., C. Gaitatzes, K. Saxena, and E.J. Neer. 1999. The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci. 24:181185.[CrossRef][Medline]
Sze, P.Y., and Z. Iqbal. 1994. Regulation of calmodulin content in synaptic plasma membranes by glucocorticoids. Neurochem. Res. 19:14551461.[CrossRef][Medline]
Thompson, E.B., G.M. Tomkins, and J.F. Curran. 1966. Induction of tyrosine alpha-ketoglutarate transaminase by steroid hormones in a newly established tissue culture cell line. Proc. Natl. Acad. Sci. USA. 56:296303.
Webster, J.I., L.H. Tonelli, M. Moayeri, S.S. Simons Jr., S.H. Leppla, and E.M. Sternberg. 2003. Anthrax lethal factor represses glucocorticoid and progesterone receptor activity. Proc. Natl. Acad. Sci. USA. 100:57065711.
Yatani, A., J. Codina, R.D. Sekura, L. Birnbaumer, and A.M. Brown. 1987. Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes. Mol. Endocrinol. 1:283289.[Abstract]
Zhang, J.H., V.A. Barr, Y. Mo, A.M. Rojkova, S. Liu, and W.F. Simonds. 2001. Nuclear localization of G protein beta 5 and regulator of G protein signaling 7 in neurons and brain. J. Biol. Chem. 276:1028410289.
Zhu, B.G., D.H. Zhu, and Y.Z. Chen. 1998. Rapid enhancement of high affinity glutamate uptake by glucocorticoids in rat cerebral cortex synaptosomes and human neuroblastoma clone SK-N-SH: possible involvement of G-protein. Biochem. Biophys. Res. Commun. 247:261265.[CrossRef][Medline]