Translational Regulation of the Vasopressin V1b Receptor Involves an Internal Ribosome Entry Site
Cristina Rabadan-Diehl,
Simona Volpi,
Maria Nikodemova and
Greti Aguilera
Section on Endocrine Physiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1862
Address all correspondence and requests for reprints to: Greti Aguilera, M.D., Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, 10 Center Drive, Mall Stop Code 1862, Bethesda, Maryland 20892-1862. E-mail: Greti_Aguilera{at}nih.gov.
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ABSTRACT
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Posttranscriptional mechanisms play an important role regulating pituitary levels of vasopressin V1b receptors (V1bR) during adaptation to stress. This study investigates the involvement of an internal ribosome entry site (IRES) in the 5'untranslated region (5'UTR) on V1bR translation. Transfection of bicistronic luciferase constructs into MCF-7 cells showed marked increases in translation of the second cistron after insertion of a 499-bp fragment of the V1bR 5'UTR in the intercistronic region, independently of cap-mediated translation, indicating the presence of IRES activity. IRES-mediated translation was potentiated by the protein kinase C activators, 12-O-tetradecanoylphorbol 13-acetate (PMA) and bryostatin 1, and appears to involve phosphorylation of amino terminus of eIF4G. In Chinese hamster ovary cells transfected with pV1bR-green fluorescent protein (pV1bR-GFP), PMA increased V1bR-GFP protein levels when cap-mediated translation was inhibited by rapamycin. The effect of PMA was due to increased translation because it persisted under transcriptional blockade by actinomycin D, and it was completely abolished by cycloheximide. In addition, PMA stimulated [35S]methionine incorporation into V1bR-GFP but not ß-actin in the absence of mRNA changes. The data show that regulation of IRES activity in the 5'UTR of the V1bR mRNA probably through phosphorylation of eIF4G may serve as a mechanism for rapid changes in V1bR translation to meet physiological demands.
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INTRODUCTION
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VASOPRESSIN (VP) SECRETED into the pituitary portal circulation from hypothalamic parvicellular neurons regulates pituitary ACTH secretion partly by potentiating the stimulatory effect of CRH (1, 2). VP binds to VP V1b receptors (V1bR) coupled to calcium-phospholipid-dependent mechanisms in the pituitary corticotroph (3, 4). Previous studies showing a good correlation between pituitary ACTH responses and the number of VP receptors in the pituitary suggest that regulation of V1bR may be a critical determinant of corticotroph responsiveness during adaptation of the hypothalamic-pituitary-adrenal (HPA) axis stress (5). The mechanisms controlling the number of V1bR in the pituitary are complex. Although transcriptional activation is likely to account for the increases in V1bR mRNA observed in most stress conditions, it is clear that mRNA levels are not the major determinant of receptor synthesis (6). Northern blot and in situ hybridization studies have shown that changes in the number of VP receptors in the pituitary is not always accompanied by parallel changes in V1bR mRNA. For example, both glucocorticoid administration and suppression of endogenous glucocorticoid by adrenalectomy lead to marked down-regulation of VP binding but V1b-R mRNA levels are normal or increased (6). Similarly, down-regulation of VP binding during osmotic stimulation is associated with normal V1bR mRNA levels and the increases in VP binding after the combined psychosensory and osmotic stimulus of ip hypertonic saline injection is associated with reduced mRNA levels (7). This lack of correlation between V1bR mRNA levels and VP binding suggests that the number of receptors is regulated at the posttranscriptional level.
The regulation of expression of several mammalian proteins occurs at the translational level. All mRNAs show a cap structure at the 5' end of the 5' untranslated region (5'UTR), which has a critical role in the initiation of translation and controls the rate of translation initiation (8, 9). Cap-dependent translation is mediated by the eukaryotic initiation factor 4F (eIF4F), which is a complex containing the scaffolding protein, eIF4G, a cap binding protein, eIF4E, and the RNA helicase, eIF4A (10). The translation initiation complex eIF4F unwinds secondary structure and scans the 5'UTR of the mRNA until reaching the first initiating methionine, AUG, before inducing ribosome binding and initiation of translation (10). High complexity of the 5'UTR can repress cap-dependent translation, and many mRNAs with structured 5'UTRs are poorly translated using this mechanism (10). Some mRNAs are translated by a cap-independent mechanism mediated by ribosome binding to an internal ribosome entry site (IRES). The latter seems to require secondary structures in the 5'UTR that allow binding of the ribosomes to the initiating AUG and permit translation independently of cap binding and scanning. Factors involved in the initiation of cap-mediated translation such as the scaffolding protein, eIF4G, are also required for IRES-dependent translation. Translational initiation by internal ribosome entry was first shown in viruses (11), but it has also been described for several mammalian mRNAs. These include growth factors such as the proto-oncogenes, c-myc, and c-sis, (12, 13), the transcription repressor nuclear factor-
B (14), hematopoiesis transcription factors, (15) vascular endothelial growth factor, (16) fibroblast growth factor (17), IGF-I and IGF-II receptors (18, 19), cardiac voltage-gated potassium channel Kv1, (20), and the ß subunit of mitochondrial H+-ATP synthase (21). The 5'UTR of the V1b receptor mRNA is unusually long (826 b) contains several small open reading frames (ORF) and has a nucleotide sequence predictive of a complex secondary structure. These characteristics of the 5'UTR led us to hypothesize that an IRES may be involved in the regulation of V1b receptor levels. In this study, we provide evidence that translation of the mRNA encoding the V1b receptor can be initiated by an internal initiation mechanism, and that IRES activity is under potential regulation by protein kinase C (PKC). These findings have important implications for understanding mechanisms by which receptors for regulatory neuropeptides can be rapidly regulated to meet the physiological demands during adaptation to stress.
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RESULTS
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The 5'UTR of the V1bR Contains an IRES
Because in vivo studies have suggested that V1bR levels are regulated at the translational level we sought the possibility that translation of the rat V1bR mRNA can be initiated by an IRES. For this purpose, we used a bicistronic vector in which a first cistron is translated by a cap-dependent scanning mechanism and a second cistron would require the presence of an internal ribosome entry for efficient translation. To determine whether the V1bR contained an IRES, we subcloned the 5'UTR of the receptor mRNA into the bicistronic vector, pSL2 (22), which contains two cistrons, renilla luciferase (r-Luc) under cap-mediated translation, and firefly luciferase (f-Luc) translated at low levels due to ribosomal rescanning. A stable stem loop structure between the two cistrons minimizes ribosomal reinitiation for f-Luc, but this second cistron will be efficiently translated if an IRES element is placed upstream of its initiating codon. These plasmids were transfected into MCF-7 breast cancer cells, which express the V1bR endogenously, and tested for f-Luc and r-Luc activities. IRES activity was calculated as a ratio of f-Luc and r-Luc activities and normalized by the luciferase activities ratio of basic pSL2. Introduction of 499 bp of the V1bR 5'UTR (pV1bSL2) into the intercistronic region of the pSL2 vector increased the ratio f-Luc/r-Luc by 10- to 13-fold when transfected into MCF-7 cells (Fig. 1
). The increase in f-Luc/r-Luc suggests the presence of an IRES element in the 5'UTR of the V1bR. Similar results were obtained after transfection of the bicistronic constructs into Chinese hamster ovary (CHO) cells (data not shown). A construct containing the same fragment but in the antisense orientation did not show any significant increase in the f-Luc/r-Luc when compared with the pSL2 vector (Fig. 1
), suggesting that IRES activity is either dependent of the sequence alignment and/or sequence orientation. Two different deletion constructs, pV1bSL2-d, containing 217 bp, and pV1bSL2-d2 with 47 bp, of the 5'UTR showed a decrease of IRES activity (Fig. 1
) when compared with the full construct (pV1bSL2), suggesting that the region upstream of bp 217 is required for full IRES activity. To investigate whether the increase f-Luc activity could be the result of changes in transcription, f-Luc mRNA levels were measured by semiquantitative RT-PCR after transfection of cells with the bicistronic vector without (pSL2) and with the V1bR 5'UTR (pV1bSL2). Levels of f-Luc mRNA were similar in cells transfected with pV1bSL2 when compared with pSL2. The ratio f-Luc/cyclophillin was identical for pV1bSL2 and pSL2 (0.22 and 0.22, respectively), indicating that the increase in activity was due to posttranscriptional mechanisms, probably translation.

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Fig. 1. The V1bR 5'UTR Contains an IRES Element
A, Schematic representation and nomenclature of bicistronic constructs containing different lengths of the V1b 5'UTR. B, Relative IRES activity of different lengths of the V1bR 5'UTR inserted in the intercistronic region of the basic bicistronic vector, pSL2, after transfection into MCF-7 cells. IRES activity corresponds to the ratio between f-Luc and r-Luc in the bicistronic vector containing the V1bR 5'UTR fragment relative to the f-Luc/r-Luc ratio of pSL2 considered as 1. In a typical experiment, for pSL2, f-Luc and r-Luc values were 1,071 and 267,033, respectively, and for pV1bSL2, 12,566, and 203,735 for f-Luc and r-Luc, respectively. *, P < 0.001 vs. SL2.
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To further determine that the increase in f-Luc activity is due to increase in translation, we performed in vitro translation using equal amounts of in vitro transcribed mRNA from the bicistronic vectors, pSL2, and pV1bSL2, in the presence of 35S methionine. The autoradiogram revealed two major bands of 36 and 61 kDa, which are consistent with the molecular size of r-Luc and f-Luc. As shown in Fig. 2
, the intensity of the f-Luc band obtained from pV1bSL2 was significantly higher (100.6 ± 12.7 arbitrary units) than that obtained from the bicistronic vector lacking the V1bR 5'UTR (46.8 ± 7.2).

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Fig. 2. The V1b R 5'UTR Increases Translation of the f-Luc Independently from Transcriptional Mechanisms
A, In vitro translation of equal amounts of mRNA from the bicistronic construct without (SL2) or with (V1bSL2) the V1b-R 5'UTR. The bands showed by the arrow correspond to 35S-labeled proteins molecular size expected for f-Luc and r-Luc. B, The bar graph shows the mean and SE of the transmittance values of the in vitro translated bands in three different experiments. AU, Arbitrary units.
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To confirm that the increases in f-Luc mRNA translation by the V1bR 5'UTR were independent of the cap structure, MCF-7 cells were cotransfected with the bicistronic vector and an expression vector encoding for Lb-protease, which cleaves the amino terminus of the translational factor eIF4G (Fig. 3A
). This procedure has been shown to blunt cap-mediated translation by preventing eIF4G to interact with the cap-binding protein, eIF4E, in the eIF4F translation initiation complex, whereas the remaining two thirds of the protein preserve its capacity of activating IRES mediated translation. As shown in Fig. 3B
, Western blot analysis using an antibody against the carboxy terminus of eIF4G showed that transfection of Lb-protease in MCF-7 cells effectively cleaved eIF4G. Cotransfection of the bicistronic constructs and the Lb protease plasmid increased the ratio f-Luc/r-Luc, due to the predominant decrease in r-Luc activity (Fig. 3C
). IRES activity expressed as the f-Luc/r-Luc ratio in pV1bSL2 over as the f-Luc/r-Luc ratio in pSL2, showed a small decrease in presence of Lb-protease, but it was still significantly higher than the basic bicistronic vector, pSL2 (Fig. 3D
).

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Fig. 3. Blockage of cap-Mediated Translation by Lb Protease Cleavage of eIF4G Does Not Affect IRES Activity
A, Diagram showing the site of cleavage of eIF4G by Lb protease. B, Representative Western blot for eIF4G using an antibody against the carboxy terminus of the protein. C, Effect of cotransfection of an expression vector for Lb-protease (+Lb) or the empty vector with V1bSL2 in MCF-7 cells on the f-Luc/r-Luc ratios, and D, IRES activity in the presence and absence of Lb-protease, expressed as fold increase over SL2. Bars represent the mean and SE of the values obtained in five different experiments.
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The V1bR IRES Element Is Regulated by PKC Activators
Because increased expression of V1bRs during stress is associated with augmented expression and secretion of VP by the parvocellular neuron (2, 5), it is possible that PKC activation by VP stimulates synthesis of its own receptor through IRES activation. To test this hypothesis, MCF-7 cells transfected with pSL2 or pV1bSL2 were incubated with the PKC activators, 12-O-tetradecanoylphorbol 13-acetate (PMA) (1 µM) or bryostatin 1 (10 nM). PMA increased both luciferase activities but the effect was higher on f-Luc than on r-Luc resulting in higher f-Luc/r-Luc ratios. There was a time-dependent increase in IRES activity, with significant increases to 152.7 ± 6 and 189.3 ± 14.1% at 4 and 6 h, respectively (Fig. 4A
), over basal levels. As shown in the dose response in Fig. 4B
, 10 nM PMA was sufficient for significant stimulation of IRES activity (144.3 ± 5.7% P < 0.001 after 4 h PMA). Similar effects were observed with 10 nM of the PKC activator, bryostatin 1, though the potentiation of IRES activity was not as marked as that observed with PMA (124 ± 3.3%, P < 0.001, data not shown). The stimulatory effect of PKC activation of f-Luc/r-Luc ratios was not affected by preincubation of the cells with the cap-mediated translation inhibitor rapamycin (not shown). To rule out the possibility that PMA may increase luciferase activity through changes in mRNA levels, the amount of f-Luc mRNA was measured by quantitative RT-PCR in MCF-7 cells transfected with pV1bSL2 with or without PMA treatment. As shown in Fig. 5
, incubation for 4 h with PMA had no significant effect on f-Luc mRNA levels, indicating that the increase in f-Luc activity was not due to higher mRNA levels.

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Fig. 4. PMA Stimulates V1bR IRES Activity
A, Time course of the effect of PMA (1 µM) on V1b-R IRES activity after transfection of SL2 or V1bSL2 into MCF-7 cells. Data are expressed as the mean and SE of the increases in IRES activity after PMA over the basal (100%) in each of three experiments. *, P < 0.001 vs. basal, Sheffés test. In a representative experiment, IRES activity ratio between f-Luc/r-Luc for V1bSL2 over f-Luc/r-Luc for SL2 increased from 9.1 to 10.5, 11.5, 13.3, and 16.5, after 1, 2, 4, and 6 h PMA, respectively. In the same experiment, f-Luc values for V1bSL2 increased from 11,185 to 31,536 and 44,154 after 4 and 6 h of PMA incubation, respectively, whereas r-Luc values changed from 135,893 to 240,498 and 21,618 after the same times. B, Dose response of the effect of PMA on V1b-R IRES activity after transfection of MCF-7 cells with the bicistronic constructs. Cells were incubated with PMA (10 nM to 1 µM) for 4 h. Data are expressed as the mean and SE of the increases in IRES activity after PMA over the basal (100%) in each of three experiments. *, P < 0.001 vs. basal. In a representative experiment, IRES activity values increased from a basal value of 9.713.8, 14, and 15.4 with 10, 100, and 1000 nM PMA, respectively.
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Fig. 5. PMA Does Not Increase f-Luc mRNA Levels in Transfected Cells
A, Representative RT-PCR for f-Luc mRNA and ß actin in MCF-7 cells transfected with the bicistronic vector, V1bSL2, in basal conditions or after 4 h incubation with 1 µM PMA. B, Bars represent the mean and SE of f-Luc mRNA after correction for RNA loading assessed by the levels of ß-actin mRNA in two experiments. Both f-Luc and ß-actin were measured using 25 PCR cycles, after determining the linear range by subjecting aliquots of cDNA from 1530 PCR cycles using specific primers for f-Luc and for ß-actin (inset). AU, Arbitrary units.
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To confirm the involvement of PKC on the effect of PMA and bryostatin 1, MCF-7 cells transfected with the bicistronic vectors 24 h earlier, were preincubated for 30 min with the PKC inhibitor, bis-indolyl maleimide I (BIM), before addition of the stimulants. Preincubation with 100 nM BIM, abolished the stimulatory effect of bryostatin 1, and reduced the effect of PMA by 50% from 155.3 ± 5.4 for PMA to 127.4 ± 7.5 for PMA plus BIM, P < 0.001. Higher concentrations of BIM (1 µM) also abolished the potentiating effect of PMA, while having no effect on basal IRES activity (Fig. 6A
).

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Fig. 6. Stimulation of the V1bR IRES by PMA Is Mediated by PKC and PI-3 Kinase Pathways
Effect of the PKC inhibitor BIM (100 nM and 1 µM) (A) or of the PI-3 kinase inhibitor, LY294002, (5 µM) (B) on PMA- or bryostatin 1-stimulated V1bR IRES activity. Transfected cells were incubated with PMA (1 µM) or bryostatin I (10 nM) with of without the inhibitors for 4 h. The data are expressed as the percent of change of IRES activity (calculated as the ratio f-Luc/r-Luc for V1bSL2 over f-Luc/r-Luc for SL2) under treatment conditions relative to basal IRES activity. Bars represent the mean and SE of the values obtained in four different experiments. *, P < 0.001 vs. basal. #, P < 0.001 vs. PMA stimulated. In a representative experiment, IRES activity increased from 14.4 to 23.0 with PMA, and to 17.4 with bryostatin. The effects of the inhibitors on IRES activity were 14.5 for 100 nM BIM, 15.0 for 1 µM BIM and 13.9 for LY294002. The effect of PKC activators in the presence of inhibitors was 18.3 (PMA+100 nM BIM), 15.7 (PMA + 1 µM BIM), 14.7 (bryostatin + 100 nM BIM), 18.2 (PMA + LY294002) and 14.2 (bryostatin + LY294002). nd, Not determined.
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It has been shown that phosphatidylinositol 3-kinase (PI-3 kinase)-dependent phosphorylation of eIF-4G contributes to the stimulation of translation by serum in JB6 cells. To determine whether such a mechanism is implicated in potentiating V1bR IRES activity by PKC stimulators, MCF-7 cells were incubated with PKC activators in the presence and absence of the PI-3 kinase inhibitor, LY 294002. As shown in Fig. 6B
, LY 294002 blunted the increases of IRES activity in response to bryostatin 1, but it only decreased the effect of PMA by 43% (155.3 ± 5.4% without and 131.6 ± 4.6% with the inhibitor; P < 0.001). The inhibitory effects of 100 nM BIM on PMA-potentiated IRES activity were not additive with the effect of LY294002 (150.4 ± 5.4% without and 128.5 ± 7.5% with both inhibitors; P < 0.001) suggesting that PKC and PI-3 kinase are sequential steps of a common pathway.
The potentiating effect of PMA on IRES activity was abolished when cells were cotransfected with the Lb-protease expression vector (Fig. 7A
), suggesting that, although the truncated protein is sufficient to sustain IRES activity, regulation of IRES activity by PKC requires the integrity of eIF4G. Western blot analysis after 4 h incubation with PMA revealed no significant changes in eIF4G protein (Fig. 7B
), suggesting that the stimulatory effect of PKC on IRES activity involves phosphorylation rather than synthesis of eIF4G.

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Fig. 7. The Potentiating Effect of PKC Activators on IRES Activity Requires an Intact eIF4G
A, V1b-R IRES activity after 4-h stimulation with 1 µM PMA in MCF-7 cells cotransfected with the bicistronic constructs and an expression vector containing the Lb protease cDNA. IRES activity was calculated as the ratio f-Luc/r-Luc for V1bSL2 over f-Luc/r-Luc for SL2. Basal activity (100%) corresponds to the IRES activity in the absence of PMA. Bars represent the mean and SE of the values obtained in three different experiments. *, P < 0.001 vs. basal. In a representative experiment, PMA increased IRES activity from 9.213.1 in -Lb controls but had no significant effect in the presence of Lb-protease (+Lb) (6.4 basal and 5.4 after PMA). B, Western Blot for eIF4G using an antibody against the carboxy terminus of the protein on cell extracts untreated and treated for 4 h with PMA.
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PKC Activators Increase V1bR Levels
To determine whether PKC-stimulation of IRES-mediated translation is involved in regulating V1bR protein synthesis in cells, we examined the effects of PMA on V1bR protein levels by Western blot in cells transfected with a vector expressing V1bR-green fluorescence protein fusion protein (pV1bR-GFP), using a GFP antibody. Because initial experiments showed high levels of nonspecific background with the GFP antibodies in MCF-7 untransfected cells but not in CHO, experiments were conducted in CHO cells, 24-h after transfection with the pV1b-GFP construct. Western blot analysis of transfected cells revealed two major bands of 75 and 61 kDa, which were absent in untransfected cells. The high molecular size band represents a fully glycosylated mature protein because this band disappeared after N-deglycosylation, when incubations were performed either in the presence of 5% 2-mercaptoethanol (lane 2) or 0.1% sodium dodecyl sulfate (SDS) (lane 3) (Fig. 8
). In the pooled results from six experiments, incubation with PMA for 6 h caused a 2-fold increase in the intensity of the 61 kDa V1bR-GFP band (196.4 ± 17.4%, P < 0.001), whereas a smaller increase was observed for the 75 kDa, highly glycosylated band (134.6 ± 5.7%, P < 0.001) (Figs. 9
, 10A
, and 11
). Incubation of CHO cells transfected with pV1bR-GFP with PMA in the presence of the PKC inhibitor, BIM, abolished the stimulatory effect of PMA on V1bR levels (Fig. 9
, A and B). The increase in protein production was not altered in the presence of the inhibitor of cap-mediated translation, rapamycin (25 nM), 30 min before and during stimulation with PMA, suggesting that the effect was cap-independent (data not shown).

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Fig. 8. Western Blot Analysis of V1bR-GFP Protein in Lysates from CHO Cells Transfected with pV1bR-GFP
Aliquots of cell protein were incubated for 24 h at 37 C incubated without (lane 1) with 1 mU of N-deglycosilase A in the presence of 5% 2-mercaptoethanol (lane 2) or 0.1% SDS (lane 3). Western blot with GFP antibody reveals two major bands of 75 and 61 kDa. The high molecular size band was no longer apparent after N-deglycosilase treatment.
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Fig. 9. PKC Stimulation by PMA Increases V1bR Protein Expression in Transfected Cells
A, Representative Western blot of the effect of 4 h incubation with 1 µM PMA in the absence or in the presence of 1 µM of the PKC inhibitor, BIM, on V1b-GFP protein levels in CHO cells transfected with an expression vector for a V1bR-GFP fusion protein. B, Bars represent the mean and SE of the percent of change in V1bR-GFP protein levels in six separated experiments with PMA and two experiments in the presence of BIM. *, P < 0.001 vs. basal without PMA or BIM. The average integrated densities for the 61- and 75-kDa bands in basal conditions were 46 ± 3 and 105 ± 11, respectively.
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Fig. 10. PMA Increases V1bR-GFP Protein in the Absence of Transcription
CHO cells transfected with pV1bR-GFP were incubated with PMA in the presence and in the absence of 2 µg/ml actinomycin D (Act D) to inhibit transcription. A, Representative Western blot showing changes in the two major bands corresponding to V1bR-GFP protein after 4 h incubation with PMA. B, Bars represent the mean and SE of the values obtained in two experiments, expressed as percent of change from basal values in the absence of Act D or PMA. C, Representative PCR analysis for V1bR-GFP and cyclophillin mRNA levels using 17 and 27 PCR cycles, respectively. D, Bars represent the mean and SE of V1bR-GFP mRNA values obtained in two experiments after correction for cyclophillin. E, Effect of the number of PCR cycles on generation of cyclophillin and V1bR-GFP bands. The number of cycles used for measurements in experimental samples are indicated by the arrows. MK, DNA markers; AcD, actinomycin D; Cycloph, cyclophillin; UT, untransfected. *, P < 0.002 vs. basal without AcD or PMA; #, P < 0.01 vs. AcD alone.
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Fig. 11. PMA Fails to Increase V1bR-GFP Protein under Inhibition of Translation
Western blot analysis of V1bR-GFP protein in CHO cells transfected with pV1bR-GFP after 4 h incubation with PMA in the presence and in the absence of cycloheximide. Bars represent the mean and SE of the results of two separate experiments. *, P < 0.001 vs. basal without cycloheximide or PMA; #, P < 0.005 lower than basal; , P < 0.02 vs. basal by t test.
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The stimulatory effect of PMA on V1bR-GFP was also observed in the presence of actinomycin D (2 µg/ml), 30 min before and during the incubation with PMA, indicating that the effect is posttranscriptional (Fig. 10A
). In three experiments, PMA increased the V1bR-GFP 61-kDa band by 234 ± 24%, P < 0.002. Actinomycin D alone caused a slight and not significant increase in the 61-kDa band, which showed a further increase to 210.2 ± 17.3%, P < 0.01 after stimulation of the cells with PMA. In these experiments, the changes in the 75-kDa highly glycosylated band were not statistically significant. In addition, quantitative RT-PCR analysis revealed no significant differences in V1bR-GFPmRNA levels between control and PMA treated cells for 4 h. This indicates that the increase in protein levels is independent of changes in mRNA (Fig. 10B
).
To rule out the possibility that the increase in V1bR-GFP protein after PMA incubation was due to decreased protein degradation, levels of V1bR-GFP protein were determined 6 h after incubation with PMA in the presence of the protein synthesis inhibitor cycloheximide. PMA alone increased the intensity of both V1bR-GFP bands, by 166.3 ± 3.3%, P < 0.001, and 131.8 ± 6.7%, P < 0.02, for the 61 kDa and 75 kDa proteins, respectively (Fig. 11
). This effect was no longer present in the cells incubated with cycloheximide. After 6 h incubation with cycloheximide alone (50 µg/ml), the amount of 61-kDa protein decreased by 72.5 ± 8.2, P < 0.01, due to inhibition in translation and ongoing protein degradation. The 75-kDa protein band showed a small decrease but this change was statistically significant from basal only by t test (Fig. 11
). These results indicate that PMA-induced increase in V1bR-GFP protein requires protein synthesis and that is it unlikely to result from increase protein stability.
To confirm that PMA increases V1bR-GFP protein synthesis, we examined the effect of PMA on [35S]methionine incorporation into protein in CHO cells transfected with the fusion protein construct. As shown in Fig. 12
, PAGE analysis of the radiolabeled proteins after immunoprecipitation with GFP antibody revealed two radiolabeled bands of 61 and 75 kDa, corresponding to the glycosylated and nonglycosylated forms seen by Western blot. PMA increased the incorporation of [35S]methionine into both bands but consistent with the Western blot the changes were more marked for the lower glycosylated band. Compared with the respective basal, PMA increased [35S]methionine incorporation by about 3-fold for the 61-kDa band and by 2-fold for the 75-kDa band. The increase in [35S]methionine by PMA was not accompanied by increases in mRNA, measured by semiquantitative RT-PCR.

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Fig. 12. Effect of PMA on the Incorporation of [35S]Methionine into V1bR-GFP and ß-Actin Protein
CHO cells transfected with pV1bR-GFP were incubated for 4 h with PMA or vehicle in the presence of [35S]methionine, and radiolabeled V1bR-GFP immunoprecipitated with a GFP antibody before PAGE and autoradiography. A, Representative image showing two radiolabeled V1bR-GFP bands of 61 and 75 kDa obtained by Phosphoimager. B, Mean and SE of the values obtained by phosphoimager in two experiments.
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DISCUSSION
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Adaptation of the organism to a changing environment requires rapid regulation of the hormones and receptors controlling ACTH secretion. An important factor contributing to this regulation is the hypothalamic peptide, VP, and evidence suggests that control of the number of pituitary VP receptors plays a significant role in this process. Previous studies in our laboratory showing a lack of correlation between VP binding and V1bR mRNA had suggested that pituitary content of VP receptors is mainly controlled at the posttranscriptional levels (6, 7). The V1bR 5'UTR is long, and displays a high guanosine-cytosine content and several small open reading frames upstream of the initiating methionine. These features are likely to reduce the ability of ribosomes to linearly scan the V1b 5'UTR mRNA according to the conventional scanning model. In fact, recent evidence indicates that mutation of the upstream ORFs of the V1bR increases the translational efficiency of the receptor (Ref.23 ; and Rabadan-Diehl, C., and G. Aguilera, manuscript in preparation). Such an inhibitory effect of upstream ORFs in the 5'UTR has been previously described for other G protein-coupled receptors such as the adrenergic (24), type 1 angiotensin II (25), and CRH (26) receptors. Whereas upstream ORFs may account for poor translational activity and the low levels of V1bRs in the pituitary in basal conditions, the present study provides evidence for a mechanism for positive regulation of V1b-R translation.
The data demonstrate that translation of the V1bR mRNA can be initiated by internal ribosome entry, and that this mechanism is a potential site of translational regulation by ligands coupled to PKC, such as VP itself. The presence of an IRES in the 5'UTR of the V1bR mRNA was clearly shown using the bicistronic vector strategy. In this system, cloning of the V1bR 5'UTR in the intercistronic region of the bicistronic vector increased the translation efficiency of the second cistron assessed either by reporter gene activity after transfection into cells, or the amount of protein produced in an in vitro translation system. This effect was preserved after inhibition of cap-dependent translation by Lb-protease cleavage of eIF4G. Lb-protease is involved in differential regulation of cap- and IRES-mediated translation in viruses (27), and it is believed to specifically cleave the amino terminus end of the translation factor, eIF4G. Whereas an intact eIFG4 is required for cap-mediated translation, the two thirds carboxy terminus portion of the molecule is sufficient to support IRES initiated translation. Although it is not possible to rule out other effects of Lb-protease, potentiation of translation of the second cistron, f-Luc, after total cleavage of eIF4G indicates that translation mediated by the 5'UTR of the V1bR is cap independent and supports the presence of an IRES. The in vitro translation data as well as the mRNA levels observed by RT-PCR confirm that the effects of the V1bR 5'UTR are due to translation and not to increases in f-Luc mRNA levels. Although the present experiments did not determine the precise elements constituting the IRES in the V1bR 5'UTR, the fact that the antisense sequence lacked any activity indicates that sequence alignment is important for the activity. The reduced activity of the deletion constructs compared with the full sequence suggests that the region upstream of bp 217 is required for full IRES activity.
The role of IRESs as positive translational regulators has been suggested in other systems such as the proto-oncogene, c-myc, in which IRES-dependent translation is activated during apoptosis (12). Similarly, the vascular endothelial growth factor IRES is active during hypoxia when protein synthesis is inhibited (28), and the platelet-derived growth factor IRES is more active during cell differentiation, in which protein synthesis rates are also reduced (29). In general, a translational mechanism mediated by an IRES confers clear advantages allowing translation under conditions that are not favorable for cap-dependent translation. A similar situation could apply to the pituitary V1bR up-regulation during stress, a condition in which the high circulating levels of glucocorticoids would inhibit cap-mediated translation (30). The present demonstration that phorbol esters and bryostatin 1 potentiate IRES activity, an effect that is blunted by PKC inhibitors, supports a role of PKC as a positive regulator of the V1bR IRES element. The potential physiological importance of this finding is further emphasized by the ability of PKC to stimulate cap-independent translation of pV1bR-GFP in cells, without changes in mRNA, as shown by the data. In addition to the lack of change in mRNA, the data provide strong evidence that the increases in V1bR-GFP protein induced by PMA were posttranscriptional and most likely translational. Firstly, the effect was not suppressed by blockade of transcription by actinomycin D. Secondly, the increase in [35S]methionine incorporation into V1bR-GFP by PMA in the absence on mRNA changes must reflect newly synthesized protein and indicates that the effect must involve translation. Lastly, the fact that PMA did not increase V1bR-GFP protein under inhibition of translation by cycloheximide, indicates that the effect of PMA increasing V1bR-GFP levels required protein synthesis and that it is unlikely that involves increases in protein stability. It is noteworthy that the major changes in V1bR-GFP protein after activation of PKC were observed in the lower molecular size band. This probably reflects changes in newly synthesized protein, which has not undergone complete glycosylation by a 6-h incubation with PMA.
A possible ligand involved in the modulation of V1bR IRES activity by PKC is VP itself, because the increases in pituitary VP binding observed during stress are associated with augmented expression and secretion of the peptide by the parvocellular neuron. The pituitary effects of VP are mediated by PKC (31, 32); thus, it is possible that PKC activation by VP contributes to the increase in V1bRs by potentiating IRES-mediated translation.
The blunting effect of Lb protease on the potentiation of IRES activity by PMA indicates that eIF4G is involved in IRES regulation by PKC. It should be noted that whereas the proteolytic fragment of eIF4G lacking the amino terminus is sufficient for IRES activity, PKC regulation requires the integrity of the translational factor. This suggests either that phosphorylation of the N terminus induces conformational changes which favor binding of eIF4G to the IRES, or that the protein conformation of the intact protein favors phosphorylation of the carboxy terminus to increase its activity. The demonstration by peptide mapping that serum induced phosphorylation of serines 1108, 1148, and 1192 in the C terminus of the eIF4G requires the amino terminus, would favor the latter possibility (33).
The lack of change in eIF4G protein in the Western blot after 4 h treatment with PKC stimulators suggests that the increase in IRES activity at this early time point is due solely to protein activation, likely through phosphorylation. However, it is likely that after longer time exposure to PMA, increases in cellular content of the translation factor eIF4G contribute to the increases in IRES activity. In fact, recent experiments in our laboratory have shown a much higher stimulation of V1bR IRES activity after a 24-h incubation with PMA, associated with increases in eIF4G protein levels by Western blot analysis. Similar increases in eIF4G have been described after long-term exposure of T cells to PMA (34). The differential ability of bryostatin 1 and PMA to stimulate IRES activity, and their different sensitivity to BIM inhibition shown by the data, could relate to the reported ability of these two stimuli to activate different isoforms of PKC (35, 36). The inhibitory effect of the PI-3 kinase inhibitor, LY294002, on the potentiating effects of bryostatin 1 and PMA on IRES activity, suggests that in addition to PKC, PI-3 kinase is involved in regulating IRES activity. It has been shown that phorbol esters can activate PI-3 kinase independently from PKC in JB6 cells (37). However, in the present study, the complete lack of additivity between the inhibition of PMA-stimulated IRES activity by BIM and LY294002, suggests that PKC and PI-3 kinase are sequential steps of a common stimulatory pathway. The blunting and partial inhibitory effects of LY294002 on bryostatin 1 and PMA action, respectively, would indicate that the PKC isozyme activated by both agents can stimulate the PI-3 kinase pathway, whereas additional isozymes stimulated solely by PMA do not.
In summary, we have identified an internal ribosome entry site in the 5'UTR of the V1bR mRNA that is able to initiate cap independent translation. Potentiation of the V1bR IRES activity by PKC activators, such as PMA and bryostatin 1, suggests that the IRES element is a possible site of translational regulation. Positive regulation of the V1bR mRNA IRES is likely to involve PKC and PI-3 kinase-dependent phosphorylation of the translation factor, eIF4G. The data provide a mechanism of regulation of the V1bR under conditions in which rapid changes in receptor content are required to respond to physiological demands. Because of the critical role of V1bR in the long-term control of pituitary corticotroph function, these findings have important implications for understanding both of normal and abnormal adaptation of the HPA axis to stress.
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MATERIALS AND METHODS
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Reagents and Protocol of Cell Culture and Treatments
MCF-7 breast cancer cells were purchased from ATCC (Manassas, VA) and cultured at 37 C under 5% CO2 atmosphere in DMEM supplemented with 10% fetal bovine serum, glutamine, and penicillin/streptomicin. CHO cells were cultured under the same conditions in AMEM (MEM alpha medium) with the same supplement as the MCF-7 cells. Unless specified, cells were plated in 24-well plates at a density of 50,000 cells/well and cultured for 24 h before transfection. A total amount of 0.4 µg of DNA per well was transfected using Lipofectamine plus (Life Technologies, Inc., Rockville, MD) according to the manufacturer instructions. In cotransfection experiments, a final concentration of 0.4 µg per well was maintained by adding 0.2 µg of each DNA construct. Twenty-four hours after transfection, cells were lysed for luciferase measurements. Where indicated, cells were changed to serum-free medium containing 0.1% BSA and incubated with the phorbol ester, PMA (Sigma, St. Louis, MO), or the PKC activator, bryostatin 1 (Biomol, Plymouth Meeting, PA), in the presence or absence of 100 nM or 1 µM of the PKC inhibitor, BIM (Calbiochem, La Jolla, CA), 5 µM of the PI-3 kinase inhibitor, LY294002 (Calbiochem) or 25 nM of the cap-mediated translation inhibitor Rapamycin (Calbiochem). When necessary, actinomycin D (2 µg/ml; Invitrogen, Carlsbad, CA) or cycloheximide (50 µg/ml, Sigma) were used 30 min before and during incubation to block transcription or translation, respectively. Cells were preincubated with the inhibitors LY294002, BIM, Rapamycin, actinomycin D, or cycloheximide for 30 min at 37 C before the addition of the PKC activators.
Dual Luciferase Assay
f-Luc and r-Luc were measured using the dual-luciferase reporter assay system (Promega, Madison, WI), which allows simultaneous measurement of both luciferase activities. After treatment and removal of incubation medium, cells were washed and lysed by addition of 100 µl of lysis buffer provided with the kit. Ten microliters of cell lysate were added to 100 µl of the luciferase assay reagent II and f-Luc activity measured for 10 sec. One hundred microliters of stop and Glo buffer, which contains the r-Luc substrate and stops the f-Luc reaction, were added to the same tube, and r-Luc activity was measured for 10 sec. Chemiluminescence signal was measured in a luminometer, Monolight (Analytical Luminescence Laboratory, Ann Arbor, MI). IRES activity of V1bRSL2 was calculated as the ratio of the values of f-Luc/r-Luc activities of pV1bSL2 over f-Luc/r-Luc activity values of the basic pSL2 construct.
Cloning of DNA Fragments
To construct the pV1bSL2 plasmid, 499 bp of the rat VP V1bR 5'UTR were amplified by PCR using the V1bR cDNA clone, rAP9-1 (3) as template, and a forward primer (39-59 bp) and reverse primer (537517 bp) containing an additional XhoI site on their 5' ends. The PCR products were XhoI digested, purified, and subcloned into the XhoI site of the basic bicistronic vector, pSL2, provided by Dr. Rudolf Werner, University of Miami (Miami, FL) (22). The 5'UTR deletion constructs, pV1bSL2-d and pV1bSL2-d2 were made by using the Exsite PCR-Based Site directed mutagenesis kit (Stratagene, La Jolla, CA) on the pV1bSL2 plasmid with forward primers directed to different regions of the 5'UTR (BIC-F2 5'-GGG TCA GGG ATG TTG GTC CT-3', for pV1bSL2-d, and BIC-F4 5'-ACC TTT CTC TCT CAT TCC AT-3' for pV1bSL22d), and a reverse primer (BIC-Rev 5' CTC GAG TAG AAT TCA CTA GA-3') directed to the bicistronic vector backbone. A plasmid containing foot and mouth disease viruses. Lb protease provided by Dr. Encarnacion Martinez-Salas (Madrid, Spain), in a pBluescript vector (37) was digested with PstI and XbaI and the Lb-protease cDNA fragment subcloned into the corresponding sites of the expression vector pCDNA 3.1/Zeo (+) (Invitrogen). The pV1b-GFP construct was made as follows. The V1bR cDNA was amplified from the original rap91 V1bR (3) by PCR using a Pfu Polymerase system (Promega) and specific primers directed toward position 35 bp of the V1bR 5'UTR (GFP-F 5'-AGA GCG GTG GGA GCA CAC GGC-3'), and the region of the V1bR before the stop codon (GFP-R 5'-GAA AGA TGC TGG TCT CCA TAG-3'). The PCR fragment was cloned in frame in the expression vector pcDNA 3.1/CT-GFP (Invitrogen) to create a fusion construct of the V1bR and a GFP protein in the carboxyl terminus. The accuracy of the fusion constructs was confirmed by enzyme digest and sequence analysis.
In Vitro Translation
One microgram of pSL2 and pV1bSL2 plasmids linearized with BamHI was in vitro transcribed using a Riboprobe in vitro transcription system (Promega) and a T7 RNA polymerase. Equal amounts of in vitro-transcribed mRNA from pSL2 and pV1bSL2 were in vitro translated in a Wheat Germ cell free System (Promega) in the presence of 35S methionine. Aliquots of one fifth of the in vitro translated product were centrifuged through a 3% sucrose cushion for 1 h at 4 C, the supernatant was acetone precipitated, resuspended in Laemmli sample buffer, separated in a 10% SDS-PAGE, and exposed to a BIOMAX film (Kodak, Rochester, NY) at room temperature from 1624 h. The length of film exposure was chosen to obtain transmittance values in the linear range determined by exposure of 14C standards. Light transmittance of the autoradiographic bands for f-Luc and r-Luc were quantitated using a computerized imaging system (Imaging Research, St. Catherine, Ontario, Canada), using the public domain NIH Image program (developed at the U.S. NIH and available on the Internet at http://rsb.info.nih.gov/nih-image).
Preparation of mRNA and Quantitative RT-PCR
For quantitative RT-PCR of f-Luc or V1bR-GFP mRNAs, cells were plated in 10-mm culture dishes and transfected with 400 ng of SL2, V1bRSL2 or V1bR-GFP and cultured for 24 h before mRNA isolation using RNAzolB (Tel-Test, Inc., Friendswood, TX) after the manufacturers protocol. RNA was subjected to digestion with 1 U deoxyribonuclease I, Amp Grade (Life Technologies, Inc.), to eliminate contaminant endogenous DNA, and quantified by UV spectrometry.
Aliquots of each total RNA preparation (1 µg) were used for RT-PCR and PCR amplification was performed using SUPERSCRIPT One-Step RT-PCR PlatinumTaq system (Life Technologies, Inc.), at 54 C for 30 min, followed by 3 min at 90 C. To quantitate V1bR-GFP mRNA the following primers were used: rat V1bR, forward, 5'-ggatgagaatgcccccaatgaaga-3'; reverse, 5'-gagagagagtggcccatacctaca-3', and rat cyclophilin primers as internal control, forward, 5'-tgggaaggtgaaagaagg-3'; reverse, 5'-gctagacttgaaggggaatg-3'. F-Luc mRNA levels for pSL2 and pV1bRLS2, were determined using the following f-Luc primers: forward, 5'-gctca-ctgagactacatcagc-3'; reverse, 5'-tccacaaacacaactcctcc-3'; human ß actin as internal control: forward, 5'-ccccaggcaccagggcgtgat-3'; reverse 5'-ggtcatcttctcgcggttggccttggggt-3'.
To determine the linear range in the PCR, aliquots of single-stranded cDNA for V1bR-GFP or pV1bRSL2 were subjected to different number of PCR cycles, 20, 25, 30, 35, and 40 cycles). For quantification of V1bR-GFP each cycle (23 cycles for cyclophilin and 27 for rat VP receptor) consisted of 40 sec at 94 C, 40 sec at 60 C, and 40 sec at 72 C, followed by a 10-min extension at 72 C. For pV1bRSL2 each cycle consisted of 40 sec at 94 C, 40 sec at 58 C and 40 sec at 72 C, followed by a 10-min extension at 72 C. Preliminary experiments demonstrated that 25 PCR cycles yielded amounts of product within the linear range for both for ß actin and luciferase. The PCR products were separated and visualized in a 2% Tris-acetate EDTA-agarose gel containing ethidium bromide and sized using PCR Markers (Promega). Images of the PCR generated bands were captured electronically and quantified using Kodak 1D Image Analysis Software.
Western Blot
For Western blot analysis of eIF4G, cells were plated in 24-well plates at a density of 50,000 cells/well and cultured for 24 h before transfection. A total amount of 400 ng of DNA (200 ng of each DNA construct) per well was transfected using Lipofectamine plus (Life Technologies, Inc.) according to the manufacturer instructions. Twenty-four hours after transfection, cells were washed with PBS and lysed with 100 µl of lysis buffer (Promega). Twenty micrograms of total cell protein lysate were loaded and separated by 6% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ), incubated with 5% blocking agent in 1x PBST (PBS plus 0.1% Tween-20) for 1 h and incubated with anti-eIF4G antibody at a 1:2000 dilution overnight. After washing in 1x PBST, membranes were incubated for 1 h with peroxidase-linked antirabbit IgG at a 1:200,000 dilution. Detection of immunoreactive eIF4G band was performed by using ECL Plus reagents (Amersham Pharmacia Biotech) and exposure to Hyper film (Amersham Pharmacia Biotech) for 1 min. Light transmittance quantitation was done as described for in vitro translation. For Western blot analysis of the V1bR-GFP fusion protein, five million CHO cells were plated in 75-cm2 flasks, and transfected 24 h later with 4 µg of DNA. Twenty-four hours after transfection, cells were incubated under the appropriate conditions, as described in results and figure legends, washed with PBS and lysed with a T-PER Tissue protein extraction reagent (Pierce, Rockford, IL) supplemented with protease inhibitors, and 410 µg of proteins were separated in an 8% SDS-PAGE. Western blot procedure was identical as for MCF-7 cells, but detection of the V1b-GFP fusion construct was done by incubating the polyvinylidene difluoride membranes with a 1:5,000 dilution of a GFP antibody (Invitrogen) overnight and 1:100,000 diluted antirabbit IgG as the second antibody.
N-Deglycosylation of V1bR-GFP Protein
CHO cells transfected with pV1b-GFP were lysated with RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, and 50 mM Tris) containing protease inhibitors cocktail (500 µM [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride], 150 nM aprotinin, 1 µE-64, 0.5 mM EDTA, 1 µM leupeptin). Aliquots of 8 µg of protein were incubated with 1 mU of N-deglycosilase A (Roche Diagnostics Corp., Indianapolis, IN), in the presence of 5% 2-mercaptoethanol or 0.1% SDS, at 37 C for 24 h. Bands containing V1b-GFP protein were identified by Western blot as described above.
Incorporation of [35S]Methionine into V1bR-GFP
CHO cells (1 x 106) plated in 10-cm plates were transfected with the expression vector for V1bR-GFP construct were incubated in methionine-free DMEM containing 0.1% BSA for 30 min, before addition of 170 µCi of 35S-methionine (Amersham) and 1 µM PMA and incubation for an additional 6 h. After discarding the medium and two washes in ice-cold DMEM, cells were harvested in 50 mM Tris-HCl (pH 7.4), containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and protease and phosphatase inhibitors, homogenized by 10 strokes in a Dounce homogenizer (Blaessing Glass, Rochester, NY), and centrifuged at 12,000 x g for 10 min at 4 C. Total protein concentration was determined using the bicinchoninic acid reagent (Pierce), before immunoprecipitation of the V1bR-GFP fusion protein.
A 150-µg aliquot of total protein was preincubated for 3 h at 4 C with 50 µl of protein G-agarose (Roche), the supernatant separated by centrifugation (12,000 x g for 10 min) and incubated overnight at 4 C with 20 µl of GFP antibody (Roche), before addition of 50 µl of protein G agarose and incubation for an additional 3 h at 4 C. Imunoprecipitated proteins were pelleted down at 12,000 x g for 20 sec and washed twice in lysis buffer for 10 min at 4 C. After a final wash in 50 mM Tris-HCl (pH 7.4), containing 250 mM NaCl, 0.1% Nonidet P-30, and 0.05% sodium deoxycholate, 30 µl, the pellet was dissolved in of 1x SDS loading buffer containing 1 µl of ß-mercaptoethanol, and loaded in a 10% SDS gel. 35S-labeled GFP V1bR bands were quantified using Phosphor Screen software Storm (Amersham Bioscience, Little Chalfont, UK).
Statistical Analysis
Data are expressed as the mean ± SE of the number of experiments indicated in Results or figure legends. The statistical significance of the differences between experimental groups was calculated by one-way ANOVA followed by Sheffés F least significant different procedure. P < 0.05 was considered to be statistically significant.
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ACKNOWLEDGMENTS
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We thank Dr. Rudolf Werner for providing the bicistronic pSL2 plasmid and for helpful discussions, Dr. Encarnación Martínez-Salas for the foot and mouth disease virus Lb protease, Dr. Amelia Nieto for the antibody against eIF4G, and Drs. Orna Elroy-Stein and John Cidlowski for helpful discussions.
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FOOTNOTES
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Present address for C.R.-D.: Division of Heart and Vascular Diseases, National Heart Lung and Blood Institute, NIH, Bethesda, Maryland.
Abbreviations: BIM, Bis-indolyl maleimide I; CHO, Chinese hamster ovary; eIF4F, eukaryotic initiation factor 4F; f-Luc, firefly luciferase; GFP, green fluorescent protein; HPA, hypothalamic-pituitary-adrenal; IRES, internal ribosome entry site; ORF, open reading frame; PI-3 kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PMA, 12-O-tetradecanoylphorbol 13-acetate; r-Luc, renilla luciferase; SDS, sodium dodecyl sulfate; 5'UTR, 5'untranslated region; V1bR, VP V1b receptor; VP, vasopressin.
Received for publication December 14, 2001.
Accepted for publication July 9, 2003.
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