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CELLULAR AND MOLECULAR
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas
Received November 10, 2006; accepted January 23, 2007.
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Abstract |
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2-fold) profoundly affect 
2-adrenergic receptor (AR) function and preferentially regulate neuronal 2A- and 2B-AR signaling. In the present study, we provide evidence that epinephrine (EPI)-induced up-regulation of GRK3 protein expression in two neuronal cell lines, BE(2)-C cells (endogenously express 2A- and 
2-AR) and BN17 cells [endogenously express 2B (NG108) and transfected to express 2-AR] is due in part to increased GRK3 gene expression. In both cell lines, the increase in GRK3 transcription occurred via an extracellular signal-regulated kinase (ERK) 1/2-dependent mechanism because the increase in GRK3 mRNA is eliminated in the presence of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene)]. EPI-induced GRK3 mRNA up-regulation also is prevented in the presence of propranolol or phentolamine. Moreover, GRK3 mRNA did not increase in response to EPI treatment in NG108 cells (endogenously express 2B-AR with no 2-AR). Both these results suggest that simultaneous activation of 2- and 2-AR by EPI is required for the ERK1/2-dependent increase in GRK3 mRNA. The EPI-induced increase in GRK3 mRNA was unaffected in the presence of the protein kinase C inhibitor, chelerythrine chloride. Finally, EPI treatment resulted in increased nuclear translocation and accumulation of the transcription factors, Sp-1 and Ap-2, in BE(2)-C cells. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up-regulation of GRK3 mRNA expression, possibly via activation of Sp-1 and Ap-2 transcription factors in neuronal cells.
; Penela et al., 2006). GRK3, a member of the GRK2/3 subfamily of the GRK group of kinases, is modestly expressed in most cell types compared with GRK2. Hence, it has not been extensively studied. Traditionally, the genes for GRK2/3 were considered to be part of a family of housekeeping genes, without selective regulation. However, recent observations from our laboratory and others suggest otherwise.
Several previous studies have examined the regulation of GRK2 expression utilizing multiple approaches. For example, GRK2 mRNA levels were reported to be regulated in a variety of disease states and during differentiation (De Blasi et al., 1995; Penela et al., 2000, 2006; Sefton et al., 2000). An association between modest changes in GRK2 expression and impaired GPCR signaling is also observed in various pathophysiological situations, including experimental models of hypertension (Gros et al., 1997); heart failure (Ungerer et al., 1993); cardiac hypertrophy, myocardial ischemia, and infarction (Choi et al., 1997; Dorn et al., 2000; Yu et al., 2000); rheumatoid arthritis (Lombardi et al., 2001); ventricular volume overload disease (Dzimiri et al., 2004); and cystic fibrosis lung (Mak et al., 2002). Almost universally, these studies have reported increase in GRK2 expression with no changes in GRK3 expression. In an attempt to define the mechanisms responsible for changes in GRK2 expression, the promoter for the GRK2 gene has been identified. GRK2 promoter-luciferase reporter constructs have demonstrated that phorbol esters, 1-AR agonists, and serum stimulation (Ramos-Ruiz et al., 2000; Theilade et al., 2005) increase GRK2 expression at the transcriptional level. However, there are relatively few reports of the selective increase in GRK3 expression (Dautzenberg and Hauger, 2001; Dautzenberg et al., 2002), and the GRK3 gene promoter has not been characterized but is predicted to lie in the genomic region immediately 5' to exon 1 (Barrett et al., 2003).
Recent observations suggest that the regulation of GRK3 expression also warrants greater attention. For example, using a neuronal cell model, we observed that the stress hormone/neurotransmitter, EPI, selectively up-regulates GRK3 expression and that this increase renders the 2A/B-AR more sensitive to agonist-stimulated desensitization and down-regulation (Bawa et al., 2003; Desai et al., 2005). No changes in GRK2 expression were observed. Other reports suggest that 
-opioid agonists and corticotropin-releasing factor (CRF) selectively regulate GRK3 expression and that signaling through both the CRF1 and -opioid receptor is preferentially regulated by GRK3 over GRK2 (Dautzenberg and Hauger, 2001; Dautzenberg et al., 2002; McLaughlin et al., 2004). Therefore, selective changes in GRK3 expression without changes in GRK2 transcription have been reported and have been implicated in selective alterations in GPCR signaling.
Given our observations of the selective up-regulation of GRK3 expression by EPI, but not NE, in two different neuronal cell lines, the present study was undertaken to begin to identify the mechanisms responsible for these changes in GRK3 expression. Our results reveal a transcriptional regulation of GRK3 expression that requires the simultaneous activation of both 2- and 2-adrenoceptors and the participation of the extracellular signal-regulated kinases (ERKs) 1/2.
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Materials and Methods |
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2-AR (250–300 fmol/mg protein). BE(2)-C cells were generously provided by Dr. Robert Ross (Fordham University, Bronx, NY). Reagents used in immunofluorescence were purchased from the source indicated: anti-rabbit Alexa-488 (no. A11034
[GenBank]
; Invitrogen), VECTASHIELD mounting medium with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA), paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), Mowiol 4–88 reagent (EMD Biosciences, San Diego, CA), poly-D-lysine (Sigma-Aldrich), L-lysine hydrochloride (JT Baker, Phillipsburg, NJ), sodium periodate (Fisher Scientific, Fair Lawn, NJ), and goat serum (Sigma-Aldrich). Coverslips used to grow cells were from VWR Scientific (West Chester, PA), and the Superfrost microscope slides used to mount the cover slips were purchased from Fisher Scientific (Pittsburgh, PA).
Cell Culture. The neuroblastoma/glioma hybrid NG108 cells were maintained in a humidified atmosphere (5% CO2:95% air) in DMEM supplemented with 10% heat-inactivated fetal calf serum, penicillin, streptomycin, and HAT supplement. BN17 cells were maintained similarly except that the media contained G418 (0.4 mg/ml) to retain selection pressure. BE(2)-C cells were maintained in a humidified atmosphere (6% CO2:94% air) in a 1:1 mixture of Eagle's minimal essential medium with nonessential amino acids and Ham's F12 medium containing 10% fetal bovine serum, 100 U/ml penicillin G, and 0.1 mg/ml streptomycin sulfate. All cells were grown until 70 to 80% confluence before EPI treatment. 2-AR levels were expressed at comparable levels in BN17 as well as BE(2)-C cells (40–60 fmol/mg). 2-AR levels were very different, 250 fmol/mg in BN17 versus 20 fmol/mg in BE(2)-C cells. BE(2)-C cells endogenously express 2-AR, whereas BN17 are NG108 cells transfected to express 2-AR.
Pretreatment. BN17, NG108 and BE(2)-C cells were pretreated with vehicle (medium containing 0.1 mM ascorbate and 1 µM sodium metabisulphite) or vehicle containing 0.3 µM EPI for 5, 15, 30, or 60 min for RT-PCR experiments.
MEK1/2 Inhibitor, Chelerythrine Chloride, Propranolol, and Phentolamine Treatment. Cells, when 70% confluent, were treated in serum-free media for 2 h in the presence of the MEK1/2-inhibitor, U0126 (10 µM), chelerythrine chloride (CC) (10 µM), propranolol (1 µM), or phentolamine (10 µM) at 37°C within an incubator in the appropriate environment. After pretreatment, the cells were treated with vehicle/EPI for the desired time points in the continued presence of the pretreatment agent.
Western Blot Analysis. Cells were washed once with 1x PBS buffer, pH 7.4, lysed immediately in 100 to 200 µl of hypotonic lysis buffer (50 mM Tris-HCl, pH 7.4, 4 mM EDTA, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin) followed by five to six passes through a 23-gauge needle and subsequently centrifuged at 1000 rpm for 10 min to remove cellular debris and nuclei (Salim et al., 2003). The lysates thus obtained were checked for their protein concentration using Pierce's protein detection kit (Smith et al., 1985). The cell lysates were diluted with 4x Laemmli buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1 mg/ml bromphenol blue), resolved on SDS-PAGE (10% gel), and transferred to polyvinylidene difluoride membrane (GE Healthcare, Piscataway, NJ). Levels of Sp-1 and Ap-2 were determined by immunoblotting using anti-Sp-1 and anti-Ap-2 antibody (1:250 and 1:200 dilution, respectively). Levels of phospho-ERK1/2 and total ERK1/2 were determined by using specific phospho-ERK1/2 and total ERK1/2 antibodies (1:1000 dilution). The immunoreactive bands were detected by a horseradish peroxidase-conjugated secondary antibody, and the blots were developed using chemiluminescence reagent made by adding p-coumaric acid and luminol (Sigma-Aldrich) in 1 M Tris-HCl and hydrogen peroxide solution. Chemiluminescence was detected by an Alpha Innotech (San Leandro, CA) imaging system, and densitometry was quantified using Fluorchem FC8800 software (Alpha Innotech).
Protein Estimation. Protein concentrations were determined by the Pierce protein detection kit (catalog no. 232009; Pierce) using BCA protein assay reagent A (catalog no. 23223) and reagent B (catalog no. 23224) (Smith et al., 1985).
Semiquantitative RT-PCR. Total RNA was isolated from NG108, BN17, or BE(2)-C cells after vehicle/EPI treatment at the indicated time intervals using the RNeasy RNA isolation kit per manufacturer's guidelines. The purity of the RNA was determined by the ratio of spectrophotometric readings at 260 and 280 nm. The RNA used for RT-PCR had a ratio of 1.5 to 2.0. RT-PCR was carried out in a programmable thermal controller (iCycler; Bio-Rad) with the following oligonucleotide primers: forward primer for detecting human GRK3 in BE(2)-C cells, 5'-AGAGCACAGCTTTTTCAAAGGTGTTG-3', which corresponds to position 1344 to 1369 of human GRK3 cDNA (Dautzenberg et al., 2002); reverse primer for detecting human GRK3 in BE(2)-C cells, 5'-GTCAGAAATGGGTTTCCCAGTTTCAG-3', which corresponds to position 1721 to 1696 of human GRK3 cDNA (Dautzenberg et al., 2002); forward primer for detecting human -actin in BE(2)-C cells, 5'-AAGATGACCCAGATCATGTTTGAGAC-3', which corresponds to position 393 to 418 of human -actin (Palchaudhuri et al., 1999); reverse primer for detecting human -actin in BE(2)-C cells, 5'-CTGCTTGCTGATCCACATCTGCTGG-3', which corresponds to position 1121 to 1096 of human -actin (Palchaudhuri et al., 1999); forward primer for detecting GRK2, 5'-GTTGCTGCAGAGGGATGTCAACCG-3', which corresponds to position 1388–1411 of human GRK2 cDNA (Dautzenberg et al., 2002); reverse primer for detecting GRK2, 5'-GTCAGAAAGGGGTTGCCCATCTTGG-3', which corresponds to position 1828–1804 of human GRK2 cDNA (Dautzenberg et al., 2002); forward primer for detecting GRK3 in BN17 cells, 5'-AATTGAGGCCAGGAAGAAGGCTA-3', which corresponds to position 1605–1627 of rat GRK3 cDNA (Arriza et al., 1992); reverse primer for detecting GRK3 in BN17 cells, 5'-TCAGAGGCCGCTGCTATTTCTGTGACA-3', which corresponds to position 2041–2067 of rat GRK3 cDNA (Arriza et al., 1992); forward primer for detecting GAPDH in BN17 cells, 5'-TACTCCTTGGAGGCCATGTA-3 (Andresen et al., 2004); and reverse primer for detecting GAPDH in BN17 cells, 5'-CGTGGAGTCTACTGGCGTCT-3' (Andresen et al., 2004).
Both cDNA synthesis and PCR were performed using superscript RT-PCR kit in a single tube with gene-specific primers and total RNA isolated from NG108, BN17, or BE(2)-C cells using the RNeasy minikit. One cycle of 50°C for 30 min and 94°C for 2 min was carried out for cDNA synthesis and predenaturation, followed by 40 cycles of 94°C for 15 s (denature), 55°C for 30 s (anneal), and 72°C for 10 min for final extension. PCR products were separated and visualized in an ethidium bromide-stained agarose gel (2%) by electrophoresis.
Immunofluorescence. The cells were processed for immunofluorescence as described previously (Desai et al., 2006). In brief, BE(2)-C cells were grown on poly-D-lysine-coated 20-x 20-mm glass cover slips to 40 to 70% confluence. The cells were then exposed to vehicle/EPI (0.3 µM) for 5 and 15 min. Next, the cells were washed with PBS containing 1.2% sucrose (PBSS) and fixed with 4% paraformaldehyde in PBSS at 4°C for 15 min. The following steps were carried out at room temperature. The fixed cells were incubated in 0.034% L-lysine, 0.05% Na-m-periodate for 20 min, washed, and permeabilized with 0.2% Triton X-100 for 10 min. After further wash, the cells were blocked with 10% normal goat serum for 15 min. Primary (anti-Sp-1 or anti-Ap-2 antibody) and secondary (anti-rabbit Alexa488 conjugated antibody) antibodies were diluted in PBSS with 0.2% goat serum and 0.05% Triton X-100. The cells were incubated with anti-Sp-1 or anti-Ap-2 antibody for 1 h at room temperature or overnight at 4°C, followed by Alexa 488-conjugated secondary antibody for 1 h in complete darkness. The cells were washed three times with PBSS before and after incubation with secondary antibody. The cover slips were then mounted on slides with a drop of a 1:1 mixture of Mowiol solution and VECTASHIELD mounting medium with DAPI. Fluorescence examination of at least six fields on the same slide was performed under an oil immersion objective (x60, 1.4 numerical aperture) using a filter selective for Alexa 488 or DAPI using an Olympus IX81 Fluorescence Deconvolution Microscope System (Olympus America, Center Valley, PA). DAPI staining enabled us to determine the area occupied by the nucleus in the BE(2)-C cells for us to monitor movement of Sp-1 and/or Ap-2 into the nucleus from the cytosol. At each time point, a representative group of cells was assessed for the extent of nuclear translocation. As a negative control, we stained the cells either with primary (anti-Sp-1/Ap-2) or secondary (anti-rabbit Alexa 488 conjugated) antibody alone to determine the specificity of the fluorescence signal. Images were optimized using AutoDeblur and Autovisualization deconvolution software (Media Cybernetics, Silver Spring, MD) and transferred to Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA) for the production of final figures.
Data Analysis. For comparison between groups, the values were expressed as mean ± S.E.M. Comparisons between groups were made either by Student's t test or one-way analysis of variance followed by Tukey's post hoc test where appropriate (GraphPad Software, Inc. San Diego, CA), and groups were considered significantly different if p < 0.05.
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Results |
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). In BE(2)-C cells that endogenously express 2A-AR and 2-AR, acute (5–60 min) exposure to 0.3 µM EPI produced an increase in GRK3 mRNA within 5 min; maximal expression was observed between 15 and 60 min (Fig. 1A). Preincubation of the cells with the MEK1/2 inhibitor, U0126 (10 µM; 2 h), before EPI treatment prevented the increase in GRK3 mRNA. In BN17 cells that endogenously express 2B-AR and are transfected to express 2-AR, EPI produced an increase in GRK3 mRNA similar to that observed in BE(2)-C cells, and this also was prevented by U0126 (10 µM; 2 h) pretreatment (Fig. 1B). Incubation of BE(2)-C or BN17 cells with 0.3 µM EPI for 5 min activates ERK1/2 phosphorylation in both cell lines (Fig. 1C; data shown for BN17 cells only), and pretreatment with U0126 (10 µM, 2 h) is sufficient to block ERK1/2 phosphorylation as detected by western blotting (Fig. 1C; data shown for BN17 cells only). MEK1/2 inhibitor treatment by itself had no effect on GRK3 mRNA in either the BE(2)-C or BN17 cells (data not shown). GRK2 mRNA expression did not change upon EPI treatment in either BE(2)-C (Fig. 2A) or BN17 (Fig. 2B) cells.
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2-AR and 2-AR. Therefore, we investigated the role of these receptors in the increase in GRK3 mRNA and activation of ERK1/2 by EPI. In both BE(2)-C and BN17 cells, blockade of 2-AR with propranolol (1 µM) eliminated the EPI-induced increase in GRK3 mRNA (Fig. 3, A and B). Likewise, blockade of 2-AR with phentolamine (10 µM) prevents the EPI-induced increase in GRK3 mRNA expression in both BE(2)-C and BN17 cells (Fig. 3, A and B). Moreover, in NG108 cells that endogenously express only 2-AR, we did not observe an increase in GRK3 mRNA expression in response to treatment with 0.3 µM EPI (Fig. 4). These results suggest that, as previously observed for GRK3 protein up-regulation, EPI-induced increase in GRK3 mRNA requires simultaneous activation of both 2-AR and 2-AR.
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Since there have been previous reports that GRK2 expression is regulated by PKC (Ramos-Ruiz et al., 2000), we also examined the effect of EPI on GRK3 mRNA in the presence of the PKC inhibitor, chelerythrine chloride. Pretreatment of BE(2)-C cells or BN17 cells with chelerythrine chloride (10 µM) had no effect on the response to EPI (0.3 µM) treatment (Fig. 5, A and B).
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). Therefore, we examined the nuclear translocation of these transcription factors in response to EPI (0.3 µM) treatment. We detected increased Sp-1 and Ap-2 protein accumulation in nuclear extracts of BE(2)-C cells treated with EPI (0.3 µM) for 5 and 15 min compared with vehicle-treated cells (Fig. 6, A and B). Nuclear accumulation of Sp-1 protein in the BE(2)-C cells was increased by 13 and 35% (expressed as percentage of vehicle) after 5 and 15 min of EPI treatment, respectively. Nuclear accumulation of Ap-2 protein was increased 36 and 40% after 5 and 15 min, respectively. Nuclear translocation of Sp-1 and Ap-2 in response to EPI treatment also was investigated by immunofluorescence (Fig. 7, A and B). BE(2)-C cells were treated with EPI (0.3 µM) or vehicle for 5 and 15 min. After the treatment, cells were processed for immunofluorescence as described previously (Desai et al., 2006). In separate samples, Sp-1 or Ap-2 were visualized with Alexa-488, whereas cell nuclei were stained with DAPI for visualization (blue). In vehicle-treated cells, Sp-1 (red) appeared as a punctate signal dispersed throughout the cytosol with limited nuclear localization (blue) (Fig. 7A). In cells treated with EPI, the intensity of the Sp-1 signal in the nuclei increased, indicating increased nuclear localization with increasing time (5–15 min). Moreover, as nuclear localization of Sp-1 increased, the Sp-1 signal disappeared from the cytosol. Similar results depicting increased nuclear translocation upon EPI treatment were obtained for the transcription factor, Ap-2 (Fig. 7B).
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Discussion |
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2-AR to desensitization and down-regulation in neuronal cells (Bawa et al., 2003; Desai et al., 2005). The results of the present study suggest that this increase in GRK3 expression is the result of an ERK1/2-dependent increase in GRK3 mRNA by EPI. Moreover, this transcriptional activation seems to require the simultaneous activation of 2- and 2-AR. Finally, we provide evidence that activation of GRK3 transcription by EPI is accompanied by increased nuclear localization of the transcription factors Ap-1 and Sp-2, both of which have been suggested to bind to the putative GRK3 promoter.
The results presented herein provide additional evidence that the expression of GRK3 is regulated independently from GRK2 and that GRK3 expression can be up-regulated without changes in GRK2 expression. We had reported previously that EPI can selectively increase GRK3 protein expression in neuronal cells without changes in the cellular content of GRK2 protein. We now have demonstrated that EPI also selectively increases GRK3 mRNA without increasing GRK2 mRNA in both BE(2)-C and BN17 cells. These results are consistent with those of Hauger and coworkers, who also have reported selective increases in GRK3 mRNA in response to agonist treatment. In Y79 retinoblastoma cells, treatment with CRF increased GRK3 mRNA, whereas there was no change in GRK2 mRNA (Dautzenberg et al., 2002). In the same cell line, this group reported that the pituitary adenylyl cyclase-activating polypeptide receptor type 1 agonist, pituitary adenylyl cyclase-activating polypeptide 38, also produced a selective increase in GRK3 mRNA with no change in GRK2 mRNA (Dautzenberg and Hauger, 2001). Similarly, numerous reports of increases in GRK2 mRNA without increases in GRK3 mRNA have been reported. For example, in ventricular tissue from patients with dilated cardiomyopathy, GRK2 levels were significantly elevated, whereas GRK3 levels were unchanged (Dzimiri et al., 2004). Likewise, GRK2 but not GRK3 mRNA was found to be elevated in peripheral lung tissue samples from patients with cystic fibrosis (Mak et al., 2002). Therefore, several different approaches have provided evidence of the differential regulation of GRK2 versus GRK3 expression in various cell types.
Another objective of the present study was to determine the receptors and the signaling pathways responsible for the EPI-induced increase in GRK3 expression. We had previously observed in both BE(2)-C and BN17 cells that up-regulation of GRK3 protein expression required simultaneous activation of both 2-AR and 2-AR. Therefore, we investigated whether this was true for the increase in GRK3 mRNA expression. Pretreatment with either the nonselective -adrenoceptor antagonist, phentolamine, or the nonselective -adrenoceptor antagonist, propranolol, was sufficient to eliminate the EPI-induced increase in GRK3 mRNA expression. Our results also indicate that ERK1/2 activation is required for the EPI-induced increase in GRK3 mRNA expression because this increase is eliminated in the presence of the MEK1/2 inhibitor, U0126. These two observations, when combined, suggest the interesting possibility that activation of either 2-or 2-ARs alone does not produce sufficient activation of ERK1/2 to increase GRK3 transcription. This is somewhat surprising because in other cells, several investigators have reported 2-adrenoceptor-dependent activation of ERK1/2 (Schmitt and Stork, 2000). Even 2-AR-dependent activation of ERK1/2 has been reported (Della Rocca et al., 1997). It is possible that, at the EPI concentrations studied, activation of either receptor individually does not sufficiently activate ERK1/2 to produce an increase in GRK3 mRNA, whereas the summation of the activation by both receptors is sufficient. Although this possibility is not ruled out by our results, one observation suggests that this is unlikely. In NG108 cells, from which BN17 cells are derived by transfection with the 2-AR, concentrations of EPI up to 10 µM do not produce an increase in GRK3 mRNA expression. Another possibility is that activation of 2-AR and 2-AR simultaneously stimulates a unique signaling pathway not activated by either receptor individually. An example of such signaling has been reported recently for the dopamine D1 and D2 receptors (Lee et al., 2004). When either receptor is activated individually, no intracellular calcium signal is generated, but simultaneous activation of the two receptors results in a PKC-dependent increase in intracellular calcium. Coincidentally, the D1 and D2 receptors, when activated independently, activate Gi/Go- and Gs-mediated signaling pathways, respectively, neither of which contributes to the Gq-mediated activation of PKC when the D1 and D2 receptors are activated simultaneously. Furthermore, it has been demonstrated that this unique signaling pathway is the result of heterodimerization of the D1 and D2 receptors (Lee et al., 2004). Heterodimerization of 2C-AR and 2-AR has been reported recently in human embryonic kidney 293 cells (Prinster et al., 2006). However, the relevance of this observation to the present study may be limited due to the significant differences in signaling/trafficking between the 2C-AR and the 2A- and 2B-AR present in our neuronal cells. Future studies will examine whether a similar 2A/B-/2-AR heterodimer in our neuronal cells contributes to EPI-induced activation of ERK1/2 and increased GRK3 mRNA expression.
A final factor considered in the present investigation is the identity of the transcription factor(s) that might be activated to increase GRK3 gene expression in response to EPI. Unlike the GRK2 gene, where the promoter region has been cloned and expressed as a promoter/reporter construct in vascular smooth muscle cells, the GRK3 gene promoter has not been characterized. The promoter is proposed to be located in the 2-kb region of DNA just 5' to the GRK3 gene transcription start site, and within this region are the consensus-binding sites for several transcription factors (Barrett et al., 2003). Given the fact that ERK1/2 activation was required to increase GRK3 mRNA expression, we considered two transcription factors known to be activated by ERK1/2 and whose binding sites are present in the putative GRK3 promoter region, namely Sp-1 and Ap-2. EPI treatment increased Sp-1 and Ap-2 protein accumulation in nuclear extracts. This suggests the possible involvement of Sp-1/Ap-2 in the EPI-induced increase in GRK3 gene expression. An increase in the DNA binding activity of Sp-1 and Ap-2 following ERK1/2 activation was previously reported (Milanini et al., 1998).
The regulation of GRK3 expression may have important implications in bipolar and other affective disorders based upon recent observations. A genome-wide linkage study suggested that the chromosome 22q11 region contained a susceptibility locus for bipolar disorder (BPD) in families of northern European Caucasian ancestry (Lachman et al., 1997; Kelsoe et al., 2001). The gene for GRK3 is found in this region, and sequence analysis identified a single nucleotide polymorphism in the putative promoter region of the GRK3 gene (Barrett et al., 2003). A subset of patients with BPD express this polymorph, and lymphocytic cell lines derived from these BPD patients exhibit a selective decrease in GRK3 expression; the reduced levels of GRK3 were inversely related to the severity of psychosis and mania (Niculescu et al., 2000). A significant increase in GRK3 mRNA in the prefrontal cortex following amphetamine administration in rats also has been reported (Barrett et al., 2003), suggesting that GRK3 plays an important role in the brain's homeostatic response to dopamine and possibly other neurotransmitters. These observations suggest that control of GRK3 expression may have significant implications in mood disorders.
In conclusion, the present study demonstrates that simultaneous activation of 2 and 2-adrenoceptors results in an ERK1/2-dependent increase in GRK3 mRNA expression accompanied by the translocation of Sp-1 and Ap-2 to the nucleus. Future studies will examine the possible contribution of receptor heterodimers in the activation of ERK1/2 as well as characterization of the promoter for the GRK3 gene.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GRK, G-protein-coupled receptor kinase; GPCR, G-protein-coupled receptor; AR, adrenoceptor; CRF, corticotropin-releasing factor; NE, norepinephrine; ERK, extracellular signal-regulated kinase; EPI, epinephrine; DMEM, Dulbecco's modified Eagle's medium; MEK, mitogen-activated protein kinase/ERK kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene); RT, reverse transcriptase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; NG108-15 (NG108), neuroblastoma/glioma hybrid cell; BE(2)-C, human neuroblastoma cell(s); DAPI, 4',6-diamidino-2-phenylindole; CC, chelerythrine chloride; PBS, phosphate-buffered saline; PBSS, PBS containing 1.2% sucrose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PKC, protein kinase C; BPD, bipolar disorder.
1 Current affiliation: Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma. 
Address correspondence to: Dr. Douglas C. Eikenburg, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5037. E-Mail: deikenburg{at}uh.edu
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