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Endocrinology, doi:10.1210/en.2005-0376
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Endocrinology Vol. 147, No. 1 441-450
Copyright © 2006 by The Endocrine Society

G Protein-Coupled Receptor Kinase 2 Involvement in Desensitization of Corticotropin-Releasing Factor (CRF) Receptor Type 1 by CRF in Murine Corticotrophs

Kazunori Kageyama, Komaki Hanada, Takako Moriyama, Takeshi Nigawara, Satoru Sakihara and Toshihiro Suda

Department of Endocrinology, Metabolism, and Infectious Diseases, Hirosaki University School of Medicine, Hirosaki, Aomori 036-8562, Japan

Address all correspondence and requests for reprints to: Dr. Kazunori Kageyama, Department of Endocrinology, Metabolism, and Infectious Diseases, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan. E-mail: kkageyama{at}hkg.odn.ne.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Materials
 Results
 Discussion
 References
 
Hypothalamic CRF stimulates synthesis and secretion of ACTH via CRF receptor type 1 (CRFR1) in the anterior pituitary gland. After agonist-activated stimulation of receptor signaling, CRFR1 is down-regulated and desensitized. Generally, it is thought that G protein-coupled receptors may be desensitized by G protein-coupled receptor kinases (GRKs). However, the role of GRKs in corticotropic cells has not been determined. In this study we focused on involvement of GRKs in desensitization of CRFR1 by CRF in corticotropic cells. We found that GRK2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). To determine the role of GRK2 in CRF-induced desensitization of CRFR1 in mouse corticotrophs, AtT-20 cells were transfected with a dominant-negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRFR1. Desensitization of CRFR1 by CRF was significantly less in AtT-20 cells transfected with the dominant-negative mutant GRK2 construct compared with desensitization in control (an empty vector-transfected) AtT-20 cells. Furthermore, pretreatment with a protein kinase A inhibitor also partially blocked desensitization of CRFR1 by CRF. These results suggest that GRK2 is involved in CRF-induced desensitization of CRFR1 in AtT-20 cells, and the protein kinase A pathway may also have an important role in desensitization of CRFR1 by CRF seen in corticotropic cells.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Materials
 Results
 Discussion
 References
 
CRF, A 41-AMINO acid polypeptide originally isolated from ovine hypothalamus, plays a central role in controlling the hypothalamic-pituitary-adrenal axis under stress (1). Hypothalamic CRF stimulates the synthesis and secretion of ACTH in the anterior pituitary gland via CRF receptor type 1 (CRFR1). CRF generally exerts its biological actions by binding to CRFRs (2). As a group, CRFRs belong to the seven-transmembrane domain G protein-coupled receptor (GPCR) superfamily. Recent studies have identified two major subtypes, namely CRFR1 and CRFR2, and their variants (3, 4). In pituitary corticotropic cells, CRFR1 is the major subtype responsible for regulating the synthesis and secretion of ACTH, which, in turn, stimulates glucocorticoid release from the adrenal cortex (5). Therefore, it is helpful to clarify the regulatory activity of CRFR1 on corticotropic cells as a step toward understanding overall functional regulation of the hypothalamic-pituitary-adrenal axis.

Alterations in hypothalamic-pituitary-adrenal axis function cause dynamic changes in CRFR1 levels in the anterior pituitary (6, 7). CRFR1 mRNA and protein levels are influenced by multiple factors, such as CRF, arginine vasopressin, glucocorticoids, and cytokines, all of which are released under stress (8, 9). It has been documented in corticotropic cells that CRF induces proopiomelanocortin transcription and ACTH secretion through a cAMP-protein kinase A (PKA) pathway. In addition, it has been shown in a rat anterior pituitary model that CRF down-regulates CRFR1 mRNA levels (10, 11). We reported that down-regulation of CRFR1 mRNA by CRF was induced via a cAMP-PKA pathway and possibly also a cAMP response element-binding protein pathway (12). Furthermore, down-regulation of CRFR1 mRNA levels is caused by posttranscriptional activity, including mRNA degradation (Moriyama T., K. Kageyama, Y. Kasagi, Y. Iwasaki, T. Nigawara, S. Sakihara, and T. Suda, unpublished observations).

Prolonged agonist activation of adrenergic receptors leads to a loss of responsiveness, or desensitization, of the receptor, and a ß-adrenergic receptor kinase dominant-negative mutant attenuates desensitization of the receptor (13), suggesting that GPCR kinases (GRKs) contribute to desensitization of GPCR. CRFR1 is also down-regulated and desensitized after agonist-activated stimulation of receptor signaling. In retinoblastoma cells, phosphorylation of CRFR1 by GRK3 is known to contribute to homologous desensitization of CRFR1, because inhibition of GRK3 causes parallel reductions in homologous receptor desensitization (14). GRK3 expression levels are increased during receptor desensitization (15). In HEK 293 cells, GRK3 and GRK6 are the main isoforms that interact with CRFR1{alpha}, and recruitment of GRK3 requires Gß{gamma}-subunits (16). Therefore, GRK3 and GRK6 can participate in desensitization of CRF receptors in specific cell types (16). In addition, in human myometrial and retinoblastoma cells, agonist stimulation of receptor-activated signaling kinases, predominantly PKA or protein kinase C (PKC), can mediate both homologous and heterologous desensitization of CRFR1 (17, 18), whereas forskolin may not be involved in desensitization of CRFR1 in Y-79 cells (14). However, the expression and role of GRKs in corticotropic cells have not been determined. In this study, therefore, we focused on the involvement of GRKs and a protein kinase pathway in desensitization of CRFR1 by CRF in corticotropic cells.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Materials
 Results
 Discussion
 References
 
Animals
Adult male Wistar rats were obtained from CLEA Japan (Tokyo, Japan). Rats were housed in an air-conditioned room with a controlled cycle (lights on at 0800 h, off at 2000 h) and provided rat chow and water ad libitum. Animal maintenance and all animal experiments were carried out in accordance with the Guidelines for Animal Experimentation, Hirosaki University.


   Materials
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 Abstract
 Introduction
 Materials and Methods
 Materials
Results
 Discussion
 References
 
Rat CRF was purchased from the Peptide Institute (Osaka, Japan). The PKA inhibitor 14–22 amide (PKAi) and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Calbiochem (San Diego, CA), and astressin was purchased from Sigma-Aldrich Corp. (St. Louis, MO). The cDNA encoding bovine GRK2 (pRK5-GRK2) and dominant-negative mutant GRK2-K220R (pRK5-GRK2-K220R) constructs were provided by Dr. R. J. Lefkowitz (Duke University, Durham, NC). An empty vector, pRK5, was used as a control (pRK5).

Cell culture and transfection
Anterior pituitary cells obtained from adult male Wistar rats (weighing 200–250 g) were dispersed as previously described (12). In brief, pituitaries were cut into small pieces and incubated in 5 ml sterile HEPES-buffered saline containing 0.4% collagenase (Invitrogen Life Technologies, Inc., Gaithersburg, MD), 0.002% deoxyribonuclease (Sigma-Aldrich Corp.), 0.04% dispase (Godo Shusei Co., Tokyo, Japan), and 2% BSA (Nakarai Tesque, Kyoto, Japan) for 30 min at 37 C. Then dispersed cells were washed and suspended in HEPES-buffered DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin (Invitrogen Life Technologies, Inc.). Aliquots of 1.2 x 106 cells were placed in six-well (35-mm diameter) culture dishes (Iwaki Glass, Funabashi, Japan) and cultured at 37 C in a humidified atmosphere consisting of 5% CO2 and 95% air. On the fourth day, cells were washed and starved overnight by incubation in HEPES-buffered DMEM supplemented with 0.2% BSA, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. On the fifth day, cells were preincubated for 30 min with or without medium containing astressin, then incubated in medium containing CRF.

The murine pituitary corticotroph tumor cell line AtT-20 was obtained from American Type Culture Collection (Manassas, VA). AtT-20 cells were placed in six-well (35-mm diameter) or 24-well (15-mm diameter) culture dishes (Iwaki Glass, Funabashi, Japan) and incubated at 37 C in a humidified atmosphere of 5% CO2 and 95% air in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. Cells were initially plated at a density of 104 cells/cm2 for 7 d before each experiment, with medium changed every 48 h. On the sixth day, cells were washed and starved overnight with DMEM supplemented with 0.2% BSA. On the seventh day, cells were incubated in medium with added vehicle or CRF after preincubation with or without medium containing astressin for 30 min. At the end of incubation, total cellular RNA or protein was collected and stored at –80 C. All treatments were performed in triplicate and repeated three times.

For cAMP assay, AtT-20 cells were placed in 24-well (15-mm diameter) culture dishes. AtT-20 cells were transfected following the manufacturer’s instructions using the FuGene 6 Transfection Reagent Kit (Roche, Indianapolis, IN). We used 3 µl FuGene/1 µg DNA. For each well, the total amount of DNA was 0.5 µg.

Western blot analysis
After treatment with CRF, AtT-20 cells were washed twice with PBS and lysed with Laemmli sample buffer. Cell debris was pelleted by centrifugation, and supernatant was recovered. Samples were boiled and subjected to electrophoresis on a gradient polyacrylamide gel (4–20%). Proteins were transferred to a polyvinylidene difluoride membrane (Daiichi Kagaku, Tokyo, Japan). After the membrane was blocked by Detector Block (Kirkegaard & Perry Laboratories, Gaithersburg, MD), the membrane was incubated for 1 h with a rabbit anti-GRK2 antibody, anti-GRK3 antibody, anti-GRK5 antibody, anti-GRK6, or anti-ß2-microglobulin (anti-B2M) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), washed with PBS containing 0.05% Tween 20, and incubated with horseradish peroxidase-labeled antirabbit IgG (Daiichi Kagaku). Detection was performed using a chemiluminescent substrate Super-Signal WestPico (Pierce Chemical Co., Rockford, IL), and the membrane was exposed to a BioMax film (Eastman Kodak Co., Rochester, NY).

RNA extraction
Cellular total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Then, cDNAs were synthesized from total RNA (0.5 µg) using random hexamer as a primer with the SuperScript First-Strand Synthesis System for RT-PCR Kit (Invitrogen Life Technologies, Inc., Carlsbad, CA) according to the manufacturer’s instructions.

RT-PCR
PCR was carried out in a programmable thermal controller (Bio-Rad Laboratories, Hercules, CA) with the following oligonucleotide primers: GRK2 forward (5'-GATGAGGAGGACACAAAAGGAATC-3'), GRK2 reverse (5'-TCAGAGGCCGTTGGCACTGCCACGCTG-3'), GRK3 forward (5'-AATTGAGGCCAGGAAGAAGGCTA-3'), GRK3 reverse (5'-TCAGAGGCCGCTGCTATTTCTGTGACA-3'), GRK4 forward (5'-CAAGATGTGTTCCTCCATTC-3'), GRK4 reverse (5'-TCAGTGTTCTGTAGGCTCCC-3'), GRK5 forward (5'-GAACCGCCAAAGAAAGGGCTG-3'), GRK5 reverse (5'-CTAGCTGCTTCCAGTGGAG-3'), GRK6 forward (5'-TTTGGGCTGGATGGGTCTGTTC-3'), GRK6 reverse (5'-GCAGTTCCCACAGCAATCTTG-3'), mouse B2M forward just1(5'-CCGAACATACTGAACTGCTA-3'); mouse B2M reverse (5'-TGCTATTTCTTTCTGCGTGC-3'), and rat B2M forward (5'-AACTTCCTCAACTGCTACGT-3'), rat B2M reverse (5'-TGAAGAAGATGGTGTGCTCA-3').

Conditions for the GRK2 and GRK3 isoforms were one cycle of 94 C for 5 min; 30 cycles of 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min; and one cycle of 72 C for 10 min. Conditions for the GRK4, GRK5, and GRK6 isoforms were one cycle of 94 C for 5 min; 35 cycles of 94 C for 1 min, 60 C for 30 sec, and 72 C for 1 min; and one cycle of 72 C for 10 min. Products were sorted by electrophoresis on a 1.2% agarose gel containing ethidium bromide. The expected sizes of PCR products for GRK2 and GRK3 were 606 and 463 bp, respectively. The expected sizes of PCR products for GRK5 and GRK6 were 144 and 117 bp, respectively. The expected sizes of PCR products for mouse and rat B2M were 342 and 357 bp, respectively.

Real-time RT-PCR
The resulting cDNAs were subjected to real-time PCR as follows. The expression level of rat GRK2 mRNA was evaluated, using quantitative real-time PCR based on specific sets of primers and probes (Assays-on-Demand Gene Expression Products, Applied Biosystems, Foster City, CA). B2M was used as a housekeeping gene to normalize values. Each reaction consisted of 1x TaqMan Universal PCR Master Mix (Applied Biosystems), 1x Assays-on-Demand Gene Expression Products (Rn00563688 m1 for rat GRK2, Rn00560865 m1 for rat B2M, Mm00497878 m1 for mouse GRK2, and Mm00437762 m1 for mouse B2M), and 2 µl cDNA in a total volume of 50 µl using the following parameters with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems): 95 C for 10 min and 40 cycles at 95 C for 15 sec and 60 C for 1 min. The above assays involve specific sets of primers and TaqMan probe spanning exon/exon junctions and should not therefore be influenced by DNA contamination. Data were collected and recorded using ABI PRISM 7000 SDS software (Applied Biosystems) and were expressed as a function of the threshold cycle (CT). Using diluted samples, the amplification efficacies for each gene of interest and the housekeeping gene amplimers were found to be identical.

Relative quantitative gene expression
Relative quantitative gene expression was calculated with the 2–{Delta}{Delta}CT method (19). In brief, for each sample assayed, the CT for reactions amplifying a gene of interest (mouse/rat GRK2) and a housekeeping gene (mouse/rat B2M) was determined. The gene of interest CT for each sample was corrected by subtracting the CT for the housekeeping gene ({Delta}CT). Untreated controls were chosen as reference samples, and the {Delta}CT for all experimental samples was subtracted by the average {Delta}CT for the control samples ({Delta}{Delta}CT). Finally, experimental mRNA abundance relative to control mRNA abundance was calculated with use of the formula 2–{Delta}{Delta}CT.

cAMP assay
Serum-starved AtT-20 cells were preincubated for 20 min with 0.1 mM IBMX in assay medium and then treated at 37 C for 20 min with the indicated concentration of each peptide. The medium was aspirated, and cells were extracted with 1 ml 95% ethanol containing 0.1 N HCl. The cAMP content was measured in supernatants using commercial cAMP enzyme immunoassay (EIA) kits (Amersham Biosciences, Little Chalfont, UK).

Desensitization studies
Cells were stimulated first with CRF for varying lengths of time, then washed with serum-free medium and restimulated with CRF (100 nM) or forskolin (50 µM) in the presence of IBMX (1 mM) for 30 min in this collection phase. Desensitization was defined as the percent decrease in cAMP production in response to this subsequent CRF stimulation compared with the level of cAMP production in response to initial CRF stimulation of control cells.

Statistical analysis
All values are expressed as the mean ± SEM in three independent experiments. Statistical analyses of data were performed using one-way ANOVA or two-way ANOVA on repeated measures, with time/dose and treatment as factors (followed by Scheffé’s F post hoc test). P < 0.05 was accepted as statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Materials
 Results
Discussion
 References
 
Expression of GRK2 mRNA and protein in murine pituitary cells
GRK2, GRK5, and GRK6 mRNAs were expressed in both anterior pituitary and AtT-20 cells (Fig. 1AGo), whereas GRK3 and GRK4 mRNAs were undetectable in both types of cells (not shown). GRK2 protein, but not GRK3 or GRK5 protein, was expressed in both anterior pituitary and AtT-20 cells, whereas GRK6 protein was expressed only in AtT-20 cells (Fig. 1Go B).



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  		FIG. 1. Expression of GRKs mRNA and protein in pituitary cells. A, A representative image of RT-PCR for GRK2, GRK5, and GRK6 mRNA in anterior pituitary and AtT-20 cells. C1, Negative control for anterior pituitary cells; C2, negative control for AtT-20 cells. B, A representative Western blot of GRK2 and GRK6 proteins in anterior pituitary and AtT-20 cells.

 
Effects of CRF on GRK2 protein levels in murine pituitary cells
Figure 2AGoGo shows the effects of CRF on GRK2 protein levels in AtT-20 cells. As shown in Fig. 2A-aGoGo, incubation with CRF increased GRK2 protein levels 8 h after the addition of 100 nM CRF. Incubation with 10 or 100 nM CRF for 8 h significantly increased GRK2 protein levels (Fig. 2A-bGoGo). A CRFR antagonist, astressin, suppressed the CRF effect of increasing GRK2 protein levels (Fig. 2A-cGoGo). Figure 2BGoGo shows the effects of CRF on GRK2 protein levels in anterior pituitary cells. As shown in Fig. 2B-aGoGo, incubation with CRF increased GRK2 protein levels 2 h after the addition of 100 nM CRF. Incubation with CRF for 2 h significantly increased GRK2 protein levels in a dose-dependent manner (Fig. 2B-bGoGo). A CRFR antagonist, astressin, suppressed the CRF effect of increasing GRK2 protein levels (Fig. 2B-cGoGo).



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  		FIG. 2. Effects of CRF on GRK2 protein levels in pituitary cells. Cells were treated in triplicate, with the average of three independent experiments (an average in triplicate was considered n = 1, n = 3) shown in the figures. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.05; **, P < 0.005 (compared with control). A, Effects of CRF on GRK2 protein levels in AtT-20 cells. a, Time-dependent changes in CRF-induced GRK2 protein levels. Cells were incubated with medium containing 100 nM CRF for the durations shown. b, Dose-dependent changes in CRF-induced GRK2 protein levels. Cells were incubated for 8 h with medium containing increasing concentrations of CRF (1–100 nM). c, Effects of astressin on CRF-induced GRK2 protein changes. Cells were preincubated with medium containing 100 nM astressin or vehicle for 30 min, then incubated for 8 h with medium containing CRF (100 nM) or vehicle. Ast, Cells treated with astressin alone. B, Effects of CRF on GRK2 protein levels in anterior pituitary cells. a, Time-dependent changes in CRF-induced GRK2 protein levels. Cells were incubated with medium containing 100 nM CRF for the durations shown. b, Dose-dependent changes in CRF-induced GRK2 protein levels. Cells were incubated for 2 h with medium containing increasing concentrations of CRF (1 nM to 1 µM). c, Effects of astressin on CRF-induced GRK2 protein changes. Cells were preincubated with medium containing 100 nM astressin or vehicle for 30 min, then incubated for 2 h with medium containing CRF (100 nM) or vehicle.

 


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  		FIG. 2A. Continued

 
Effects of CRF on GRK2 mRNA levels in murine pituitary cells
Figure 3AGo shows the effects of CRF on GRK2 mRNA levels in AtT-20 cells. To examine whether CRF-mediated GRK2 mRNA levels changed in a time- and dose-dependent manner, AtT-20 cells were incubated with CRF. As shown in Fig. 3A-aGo, incubation with CRF significantly decreased GRK2 mRNA levels (by ANOVA, P < 0.0001). GRK2 mRNA levels transiently fell to 59.7 ± 3.4% of the control value within 8 h after treatment with 100 nM CRF, and levels recovered to control values after 24 h. Incubation with 10 or 100 nM CRF for 8 h decreased GRK2 mRNA levels to 62.3 ± 10.8% and 61.0 ± 3.0% of the control value, respectively (Fig. 3A-bGo). A CRFR antagonist, astressin, suppressed CRF-mediated decreases in GRK2 mRNA levels (Fig. 3A-cGo). Figure 3BGo shows effects of CRF on GRK2 mRNA levels in anterior pituitary cells. To examine whether CRF-mediated GRK2 mRNA levels changed in a time- and dose-dependent manner, anterior pituitary cells were incubated with CRF. As shown in Fig. 3B-aGo, incubation with CRF significantly increased GRK2 mRNA levels (by ANOVA, P = 0.012). GRK2 mRNA levels transiently increased to 131.5 ± 4.9% of the control value within 2 h after treatment with 100 nM CRF, and they recovered to control levels after 8 h (Fig. 3B-aGo). Incubation with 100 nM and 1 µM CRF for 2 h increased GRK2 mRNA levels to 130.2 ± 5.8% and 133.9 ± 6.8% of the control value, respectively (Fig. 3B-bGo). A CRFR antagonist, astressin, suppressed CRF-mediated increases in GRK2 mRNA levels in anterior pituitary cells (Fig. 3B-cGo).



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  		FIG. 3. CRF effects on GRK2 mRNA levels in pituitary cells. Cells were treated in triplicate, with the average of three independent experiments (an average in triplicate was considered n = 1, n = 3) shown in the figures. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.05; **, P < 0.005 (compared with control). A, CRF effects on GRK2 mRNA levels in AtT-20 cells. a, Time-dependent changes in CRF-induced GRK2 mRNA levels. Cells were incubated with medium containing 100 nM CRF for the durations shown. b, Dose-dependent changes in CRF-induced GRK2 mRNA levels. Cells were incubated for 8 h with medium containing increasing concentrations of CRF (0.1 nM to 100 nM). c, Effects of astressin on CRF-induced GRK2 mRNA changes. Cells were preincubated with medium containing 100 nM astressin or vehicle for 30 min, then incubated for 8 h with medium containing CRF (100 nM) or vehicle. Ast, Cells treated with astressin alone. B, CRF effects on GRK2 mRNA levels in anterior pituitary cells. a, Time-dependent changes in CRF-induced GRK2 mRNA levels. Cells were incubated with medium containing 100 nM CRF for the durations shown. b, Dose-dependent changes in CRF-induced GRK2 mRNA levels. Cells were incubated for 2 h with medium containing increasing concentrations of CRF (1 nM to 1 µM). c, Effects of astressin on CRF-induced GRK2 mRNA changes. Cells were preincubated with medium containing 100 nM astressin or vehicle for 30 min, then incubated for 2 h with medium containing CRF (100 nM) or vehicle.

 
Effects of dominant-negative GRK2 on CRF-induced cAMP production in AtT-20 cells
Figure 4Go shows the time course of cAMP production due to CRF stimulation of AtT-20 cells transfected with pRK5, pRK5-GRK2, or pRK5-GRK2-K220R. Basal levels of cAMP were similar in AtT-20 cells transfected with pRK5, pRK5-GRK2, or pRK5-GRK2-K220R. Stimulation of either pRK5 or pRK5-GRK2 cells resulted in similar attenuation of CRF responsiveness, with peak levels of cAMP achieved in 45 min. In contrast, increases in cAMP were significantly higher in the pRK5-GRK2-K220R cells compared with the others (pRK5 vs. pRK5-GRK2-K220R, P = 0.0005; pRK5-GRK2 vs. pRK5-GRK2-K220R, P = 0.0072).



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  		FIG. 4. Effects of the dominant-negative GRK2 construct on CRF-induced cAMP production in AtT-20 cells. Cells were treated in triplicate, with the average of three independent experiments (an average in triplicate was considered n = 1, n = 3) shown in the figures. The level of cAMP was measured by cAMP EIA. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post hoc test. **, P < 0.005 (compared with the pRK5 control).

 
Effects of dominant-negative GRK2 on CRF-induced desensitization in AtT-20 cells
Desensitization of CRFR1 was examined after preincubation with CRF for varying durations. As illustrated in Fig. 5AGo, a time-course study showed that AtT-20 cells transfected with a dominant-negative mutant GRK2 construct (pRK5-GRK2-K220R) reduced CRF-induced desensitization of CRFR1 compared with the control (pRK5 vs. pRK5-GRK2-K220R, P < 0.0001; pRK5-GRK2 vs. pRK5-GRK2-K220R, P < 0.0001). As illustrated in Fig. 5BGo, a dose-dependent study also showed that AtT-20 cells transfected with pRK5-GRK2-K220R had decreased CRF-induced desensitization of CRFR1 compared with the controls (pRK5 vs. pRK5-GRK2-K220R, P = 0.006; pRK5-GRK2 vs. pRK5-GRK2-K220R, P = 0.006).



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  		FIG. 5. Effects of the dominant-negative GRK2 construct on CRF-induced desensitization in AtT-20 cells. Cells were treated in triplicate, with the average of three independent experiments (an average in triplicate was considered n = 1, n = 3) shown in the figures. The level of cAMP was measured by cAMP EIA. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post hoc test. *, P < 0.05; **, P < 0.005 (compared with the pRK5 control). A, Time-dependent effects of the dominant-negative GRK2 construct on CRF-induced desensitization in AtT-20 cells. AtT-20 cells (transfected with pRK5, pRK5-GRK2, or pRK5-GRK2-K220R) were incubated with medium-added vehicle or CRF (100 nM) for the durations shown, then restimulated with 100 nM CRF (plus IBMX) for 30 min. B, Dose-dependent effects of the dominant-negative GRK2 construct on CRF-induced desensitization in AtT-20 cells. AtT-20 cells (transfected with pRK5, pRK5-GRK2, or pRK5-GRK2-K220R) were incubated for 30 min with medium-added vehicle or CRF at the concentration shown, then restimulated with 100 nM CRF (plus IBMX) for 30 min.

 
Effects of a PKAi on CRF-induced desensitization in AtT-20 cells
To determine whether a PKA pathway might be involved in the desensitization of CRFR1, AtT-20 cells were preincubated with PKAi (1 µM), a specific PKA inhibitor, for 30 min before incubation with CRF. As shown in Fig. 6Go, PKAi blocked desensitization of CRFR1 by CRF [control vs. PKAi, P < 0.0001 (Fig. 6AGo) and P = 0.049 (Fig. 6BGo)]. The effects of CRF or forskolin on CRF- or forskolin-induced desensitization were next examined. Figure 6CGo shows that 52% and 67% desensitization (P < 0.0001) of CRF- and forskolin-stimulated cAMP accumulations were observed in AtT-20 cells exposed to CRF and forskolin, respectively. However, forskolin treatment did not cause desensitization in AtT-20 cells preincubated with CRF.



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  		FIG. 6. Effects of a PKA inhibitor on CRF-induced desensitization in AtT-20 cells. Cells were treated in triplicate, with the average of three independent experiments (an average in triplicate was considered n = 1, n = 3) shown in the figures. The level of cAMP was measured by cAMP EIA. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post hoc test. A, Time-dependent effects of PKAi on CRF-induced desensitization in AtT-20 cells. AtT-20 cells were incubated with medium-added vehicle or CRF (100 nM) for the durations shown after preincubation in the absence or presence of PKAi (1 µM) for 30 min, then restimulated with 100 nM CRF (plus IBMX) for 30 min. *, P < 0.05; **, P < 0.005 (compared with control). B, Dose-dependent effects of PKAi on CRF-induced desensitization in AtT-20 cells. AtT-20 cells were incubated for 30 min with medium-added vehicle or CRF at the concentration shown after preincubation in the absence or presence of PKAi (1 µM) for 30 min, then restimulated with 100 nM CRF (plus IBMX) for 30 min. *, P < 0.05; **, P < 0.005 (compared with control). C, Effects of forskolin on CRF-induced desensitization in AtT-20 cells. AtT-20 cells were incubated for 30 min with medium-added vehicle, 100 nM CRF, or 50 µM forskolin (Fosk) for 30 min, then restimulated with 100 nM CRF or 50 µM Fosk (plus IBMX) for 30 min. **, P < 0.005 (compared with CRF or Fosk alone).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Materials
 Results
 Discussion
References
 
We report here that GRK2, GRK5, and GRK6 mRNAs were expressed in both anterior pituitary and AtT-20 cells, and GRK2 protein, but not GRK5 protein, was expressed in both anterior pituitary and AtT-20 cells, whereas GRK6 protein was expressed only in AtT-20 cells. Together, GRK2 was expressed in rat anterior pituitary cells and the AtT-20 cell line (mouse corticotroph tumor cells), suggesting a pivotal role for GRK2 in corticotropic cells. We found that CRF increased GRK2 protein levels in both rat anterior pituitary cells and AtT-20 cells. In general, the level of protein depends on the net balance of receptor protein synthesis and degradation (7). CRF may contribute to an increase in GRK2 mRNA translation and/or a decrease in receptor protein degradation. Recent studies have shown regulation of GRK2 during receptor desensitization (20, 21). After activation of a GPCR by an agonist, ß{gamma}-subunits dissociate from the heterotrimeric G protein and bind to GRK2. This interaction with Gß{gamma}-subunits facilitates translocation of the kinase to the plasma membrane as well as synergistically enhances agonist-dependent phosphorylation of the receptor (22, 23). The biological significance of changes in GRK2 protein levels remains to be elucidated. In the anterior pituitary, a ligand-induced reduction in CRF-binding sites (24, 25) is achieved through both internalization of receptors (26) and suppression of new receptor synthesis (7). It would be possible that up-regulation of GRK as well as decreased CRFR1 protein levels may cooperate in desensitization of GPCR. However, other mechanisms in the regulation of GRK2 may contribute to desensitization of CRFR1 in corticotropic cells.

GRK2 mRNA levels increased after CRF stimulation in rat anterior pituitary cells, whereas levels decreased in AtT-20 cells. The expression levels of mRNA are determined mainly by both transcriptional activity, such as synthesis of mRNA, and posttranscriptional activity, such as the rate of mRNA degradation (mRNA stability) (27, 28). In anterior pituitary cells, mRNA synthesis may increase and/or mRNA degradation may decrease in response to CRF. In contrast, changes in mRNA stability after agonist treatment have been reported in other GPCRs, such as the ß-adrenergic receptor (29, 30). Therefore, in contrast to rat anterior pituitary cells, it is possible that in AtT-20 cells, stimulation of translation by CRF increases GRK2 mRNA utilization and the degradation of its mRNA, resulting in decreases in its mRNA levels. Furthermore, despite decreases in its mRNA levels, the protein levels were increased by CRF in AtT-20 cells. Together, these results may suggest that there are differences in transcriptional activity and/or posttranscriptional activity between these two cell types.

The translational process may also have an important role in regulation of CRFR1 (7, 31). It has been reported that an upstream open reading frame in the 5'-untranslated region of CRFR1 inhibits receptor expression by inhibiting mRNA translation (32). Our previous results show that CRF significantly decreases the CRFR1 mRNA half-life after addition of actinomycin D, a transcription inhibitor (unpublished observations). Therefore, CRF facilitates CRFR1 mRNA instability or degradation in the anterior pituitary cells. In contrast to rat anterior pituitary cells, it has been reported for AtT-20 and CATH.a cells that CRFR1 mRNA stability is not changed by CRF (27). In these two cell lines, regulation of CRFR1 mRNA levels may be achieved mainly through regulation of gene transcription rather than mRNA stability.

The activity of a CRFR1 response is modified by desensitization and internalization of receptors (7). CRF rapidly desensitizes the cAMP response of CRFR1. Therefore, CRFR1 has an effect on attenuating rapid desensitization of CRFR1-mediated signal transduction. To determine the role of GRK2 in desensitization of CRFR1 by CRF in corticotropic cells, AtT-20 cells were transfected with the dominant-negative mutant GRK2 construct. Reduced desensitization in AtT-20 cells transfected with a dominant-negative mutant GRK2 construct suggests an important role for GRK2 in desensitization of CRFR1 in corticotropic cells.

Intracellular second messengers, such as cAMP, stimulate various protein kinases, resulting in their biological responses (33, 34). Involvement of PKA is suggested to mediate both homologous and heterologous desensitization of some GPCRs (35). In a neuronal cell line that expressed CRFR1 endogenously, CRF down-regulated CRFR1 mRNA levels via a cAMP-mediated pathway (27). In a previous study we found that CRFR1 gene expression levels in rat anterior pituitary cells were down-regulated by CRF via a cAMP-PKA-cAMP response element-binding protein pathway (10, 12). In desensitization of CRFR1, a PKA or PKC pathway is involved in human myometrial and retinoblastoma cells (17, 18), whereas forskolin may not be involved in Y-79 cells (14). The involvement of PKA pathways was examined in the current study with use of a protein kinase inhibitor. Pretreatment with the PKA inhibitor PKAi partially blocked desensitization of CRFR1 by CRF, suggesting that agonist-mediated CRFR1 desensitization in AtT-20 cells is dependent on PKA activation. The effect of forskolin, which activates adenylate cyclase, causing increased cAMP and activation of PKA, on desensitization also supports this idea. These results may suggest that the PKA pathway is involved in the activation of GRK2 through phosphorylation, as shown for the ß-adrenergic receptor (36). Furthermore, it is possible that the PKA pathway may be involved in other signal pathways in addition to GRK2, because inhibition of the PKA pathway was more effective than treatment with dominant-negative GRK2 in preventing desensitization of cAMP responses to CRF. However, forskolin treatment did not cause desensitization in AtT-20 cells preincubated with CRF. Taken together, these findings indicate that the PKA pathway probably has an important role in CRFR1 desensitization by CRF in corticotropic cells, but the homologous desensitization by CRF may require other pathways, directly via CRFR1.

In summary, GRK2 is expressed in rat anterior pituitary cells and AtT-20 cells. GRK2 is involved in desensitization of CRFR1 by CRF in AtT-20 cells, and the PKA pathway may also have an important role in the desensitization of CRFR1 by CRF seen in corticotropic cells.


   Acknowledgments
 
We are grateful to Dr. R. J. Lefkowitz, Duke University, for his gifts of cDNA-encoding bovine GRK2 and dominant-negative mutant GRK2-K220R. We also thank Dr. Y. Iwasaki for helpful discussion.


   Footnotes
 
This work was supported in part by Health and Labor Science Research Grants (Research on Measures for Intractable Diseases) from the Ministry of Health, Labor, and Welfare of Japan; Grant 15590966 from the Ministry of Education, Science, and Culture of Japan (to T.S.); and a grant from the Funds for the Promotion of Aomori Medical Research and the Promotion of International Scientific Research (to K.K.).

First Published Online September 29, 2005

Abbreviations: B2M, ß2-Microglobulin; CRFR1, CRF receptor type 1; CT, threshold cycle; EIA, enzyme immunoassay; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; IBMX, 3-isobutyl-1-methylxanthine; PKA, protein kinase A; PKAi, protein kinase A inhibitor; PKC, protein kinase C.

Received March 31, 2005.

Accepted for publication September 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Materials
 Results
 Discussion
 References
 

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