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CELLULAR AND MOLECULAR
2B- and 
2-Adrenoceptor Activation Sensitizes the 2B-Adrenoceptor for Agonist-Induced Down-Regulation
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas
Received April 15, 2004; accepted June 10, 2004.
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Abstract |
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2A-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 µM) in BE(2)-C cells that express both 2- and 
2-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by 2-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the 2A-versus 2B-ARs have been reported, the present study examines the effects of simultaneous activation of 2B- and 2-ARs on 2B-AR number and signaling. We studied NG108 cells that naturally express 2B-ARs, and BN17 cells, NG108 cells transfected to express the human 2-AR. In NG108 cells, 2B-AR desensitization and down-regulation require treatment with 20 µM EPI or NE; GRK expression was not changed. In BN17 cells expressing 2-ARs, the threshold EPI concentration for 2B-AR desensitization and down-regulation was reduced to 0.3 µM; 10 µM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The -AR antagonist alprenolol (1 µM) and GRK3 antisense (but not sense) DNA blocked 0.3 µM EPI- and 10 µM NE-induced desensitization and down-regulation of the 2B-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of 2B- and 2-ARs results in a 67-fold decrease in the threshold concentration of EPI required for 2B-AR down-regulation. This lower threshold for down-regulation is associated with 2B- and 2-AR dependent up-regulation of GRK3 expression.
2-adrenoceptor signaling. Primarily using transfected receptors in cell lines that do not endogenously express the 2-adrenoceptor, Liggett and coworkers have conducted a series of experiments examining the short-term and long-term desensitization of the three 2-adrenoceptor subtypes (2A/D,2B and 2C) (Liggett, 1998
). Through these studies, structural components of the 2-adrenoceptor have been identified as sites for GRK phosphorylation that are important for the short-term desensitization of these receptors. For example, four serines in the third intracellular loop are reported to be critical for short-term desensitization of the 2A-adrenoceptor (Eason et al., 1995). However, there are structural differences within the 2-adrenoceptor family that may lead to heterogeneity in the regulation of the three 2-subtypes (Eason and Liggett, 1992). For example, significantly higher agonist concentrations are reportedly required for desensitization of 2A compared with 2B-adrenoceptors (Heck and Bylund, 1997). Moreover, studies have suggested that phosphorylation sites critical to short-term 2-adrenoceptor desensitization are not important for long-term desensitization and down-regulation (Jewell-Motz and Liggett, 1995; Jewell-Motz et al., 1997). Data regarding long-term regulation of the 2C-adrenoceptor also are controversial. A recent study by Bylund and coworkers suggests that GRK-mediated phosphorylation is critical for long-term agonist-induced down-regulation and desensitization of the 2C-adrenoceptor (Deupree et al., 2002). They observed that the opossum kidney 2C-adrenoceptor undergoes down-regulation, whereas the human receptor does not, and this difference seemed related to structural differences in the third intracellular loop of the receptor in these species. In contrast to this, down-regulation of the human 2C-adrenoceptor in Hep2G cells was reported after long-term agonist exposure and was demonstrated to be the result of increased receptor degradation (Cayla et al., 1999). In summary, the role of GRK-mediated phosphorylation and receptor down-regulation in the long-term modulation of the 2-adrenoceptor signaling is not well established.
Recently, we have demonstrated that 0.3 µM epinephrine (EPI) produces desensitization of 2A-adrenoceptors after 24-h treatment of BE(2)-C human neuroblastoma cells in culture, whereas 1 µM norepinephrine (NE) has no effect (Bawa et al., 2003). These cells endogenously express both 2A- and 2-ARs. The desensitization by EPI was accompanied by 2A-adrenoceptor down-regulation, and both desensitization and down-regulation were prevented by -adrenoceptor blockade during the EPI treatment. Furthermore, the 2A-adrenoceptor desensitization and down-regulation was accompanied by a selective up-regulation of GRK3 expression. -Adrenoceptor blockade or GRK3 antisense treatment prevented the GRK3 up-regulation and also prevented the 2A-adrenoceptor down-regulation by EPI treatment. The present study was initiated to investigate the potential cross talk regulation between the 2-adrenoceptor and another 2-adrenoceptor subtype, the 2B. The model system chosen for this study was the NG108 cell, a neuroblastoma/glioma hybrid cell line that naturally expresses 2B-adrenoceptors and has been used previously to study regulation of 2-adrenoceptor signaling (Thomas and Hoffman, 1986; Convents et al., 1989). We also used the BN17 cell line, derived from the transfection of the human 2-adrenoceptor gene into NG108 cells (Adie and Milligan, 1994). Our results suggest that simultaneous activation of the 2B- and 2-adrenoceptors enables the 2B-adrenoceptor to be down-regulated by 67-fold lower EPI concentrations than when only the 2-adrenoceptor is activated. This difference is due to the selective up-regulation of GRK3 produced by this treatment. In addition, we report here that in the absence of 2-adrenoceptor activation, higher agonist concentrations produce 2B-adrenoceptor down-regulation without up-regulation of GRK3 expression.
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Materials and Methods |
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Cells and Plasmids. NG108 and BN17 cells were obtained from Dr. Graeme Milligan (University of Glasgow, Glasgow, Scotland, UK). BN17 cells are NG108 cells transfected using plasmid pJM16, carrying a copy of the neomycin resistance gene and the cDNA encoding human 2-AR, to express the human 2-AR at about 300 fmol/mg protein (Adie and Milligan, 1994).
Cell Culture. The neuroblastoma/glioma hybrid NG108 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and HAT supplement. BN17 cells were grown in the same way as NG108 cells except that the media contained G418 (0.6 mg/ml) to maintain selection pressure. The NG108 and BN17 cells were grown in either 75-cm2 flasks or 150-cm2 plates coated with poly-L-lysine. Flasks or plates of cells that were more than 80% confluent were used throughout the study.
Pretreatment. NG108 cells and BN17 cells were pretreated with vehicle (serum-free medium containing 0.1 mM ascorbate and 1 µM sodium metabisulfite), or vehicle containing 0.3-20 µM NE or EPI for 24 h. In BN17 cells, NE or EPI pretreatment also was performed in the presence or absence of the -AR antagonist alprenolol (1 µM). BN17 cells were also pretreated with vehicle, 1 µM ISO or 1 µM ISO in the presence of 0.3 µM NE for 24 h. For the antisense ODN pretreatment, BN17 cells were pretreated with 1 µM GRK3 antisense (5'-CCCGGTGTCTGCTTTCCT-3') or sense (5'-AGGAAAGCAGACACCGGG-3') ODN in serum-free media for 24 h followed by 24 h in the presence of serum, with or without the addition of 0.3 µM EPI (Dautzenberg et al., 2001; Thakker and Standifer, 2002).
2-AR and 2-AR Agonist Concentration Response Curves. At the end of NE or EPI pretreatment, medium containing the drugs was aspirated, and the cells were harvested by pipetting fresh drug-free medium against the cells. Intact cells were sedimented by centrifugation at 1500g for 10 min, washed once with phosphate-buffered saline (PBS), and resuspended in Hanks' balanced salt solution (137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.4 mM KH2PO4, 4 mM NaHCO3, 6 mM D-glucose 6, 0.5 mM MgCl2, 0.4 mM MgSO4, and 1 mM CaCl2) containing 0.1 mM ascorbate and the phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM). To determine the effect of 2-AR agonist NE (10-9-10-5 M) on PGE1-induced cAMP accumulation, intact cells were first incubated for 10 min at 37°C in Hanks' balanced salt solution buffer. Then, PGE1 (10 nM), NE, and cells were added to assay tubes, and the tubes were incubated for an additional 10 min at 37°C. In all the experiments, the -AR antagonist alprenolol (1 µM) also was included in this step. All assays were performed in duplicate in a total volume of 0.5 ml. The assay was terminated by removing the tubes to a boiling water bath for 5 min. After boiling, samples were centrifuged for 5 min at 14,000g, and cAMP levels from the supernatant fractions were determined in a [3H]cAMP (0.8 pmol) binding assay as described previously (Standifer et al., 1994). Preferential 2-adrenoceptor agonists were not used in this study because NG108 cells express imidazole receptors, the activation of which inhibits cAMP accumulation (Greney et al., 2000). Because all preferential 2-adrenoceptor agonists would activate both 2 and imidazole receptors in NG108 cells, this would significantly complicate any data interpretation.
Membrane Preparation for Receptor Binding. To prepare membranes for receptor binding, the cells were first washed three times with PBS (pH 7.4) and then harvested from the plates by gentle scraping. The cells were sedimented by centrifugation at 3000g for 10 min. The cell pellet was then suspended in 10 volumes of Tris-HCl buffer (50 mM, pH 7.7) containing NaCl (100 mM), Na2EDTA (10 mM), and phenylmethylsulfonyl fluoride (0.1 mM) and homogenized with a Polytron homogenizer (setting 5, 10 s). The membranes were incubated for 15 min at 25°C and sedimented by centrifugation (20,000 rpm) for 30 min at 4°C. The membranes were immediately used for the binding assay.
Radioligand Binding Assay to Determine Receptor Number. The 2B-AR binding was performed with the 2-AR antagonist, [3H]RX821002. For saturation studies, membranes (0.25-0.3 mg protein/ml) were incubated with [3H]RX821002 (0.1-30 nM) in potassium phosphate buffer (25 mM, pH 7.4) at 23°C for 30 min. Assays were performed in triplicate, and nonspecific binding was defined using 100 µM phentolamine. At the end of the incubation period, reaction was terminated by adding Tris-HCl buffer (50 mM, pH 8.0 at 4°C) and filtration over Whatman GF/B paper (Brandel Inc., Gaithersburg, MD). The filter paper was washed thrice with 3 to 4 ml of the filtration buffer (50 mM Tris-HCl, pH 8.0, 4°C). The amount of radioactivity in the sample was determined by scintillation spectroscopy in an LS6000 liquid scintillation counter (Beckman Coulter, Fullerton, CA).
Western Blot Analysis. Some of the cells collected either for the 2B-AR response assay or receptor binding were used to prepare samples for Western blot analysis. Cell pellets were washed once with 1x PBS buffer (pH 7.4), lysed immediately in 100 to 150 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.02% sodium azide, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin), vortexed, and digested for 30 min in an ice bath. The resultant cell lysate was diluted with 2x Laemmli gel loading buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 0.1 mg/ml bromphenol blue). The proteins in the cell lysate were separated by SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel. The separated proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Amersham Biosciences UK, Ltd.) using a Mini Trans-Blot transfer gel unit (Bio-Rad). The polyvinylidene difluoride membrane was then incubated for 1 h in Tris-buffered saline, pH 7.6 (TBS-T; 20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20) containing 5% nonfat milk protein. After removal of blocking buffer, the membrane was washed with TBS-T and incubated overnight at 4°C with rabbit polyclonal antisera (primary antibody) directed against either Gi2 (1:1000; Upstate Biotechnology, Lake Placid, NY), GRK2, or GRK3 (1:1000; Santa Cruz Biotechnology, Inc.) in 2.5% nonfat milk. The blots were then washed with TBS-T to remove the primary antibody and incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2500; Santa Cruz Biotechnology, Inc.) at room temperature for 45 min (Gi2) or 1 h 30 min (GRK2/3) in 2.5% nonfat milk. Levels of GAPDH protein served as loading control. Therefore, the same blot was stripped with 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol in TBS-T, blocked, and reprobed by incubating either for 1 h at room temperature or overnight at 4°C with mouse anti-rabbit GAPDH antibody (1:8000; Research Diagnostics) in 2.5% nonfat milk. After removing the primary antibody, the blot was incubated with an anti-mouse horseradish peroxidase (1:10,000; Santa Cruz Biotechnology, Inc.) at room temperature for 45 min in 2.5% nonfat milk. Immunoreactive bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Inc.). The intensity of each immunoreactive band was determined using either a FluorChem imaging system (Alpha Innotech Corp., San Leandro, CA) or Nucleovision Imaging Workstation (Nucleotech Corp., San Carlos, CA) and normalized to the GAPDH loading control.
Protein Estimation. Protein concentrations were determined by the Lowry method (Lowry et al., 1951).
Data Analysis. Kd, Bmax, EC50, and maximal response to the 2-AR agonist was determined by nonlinear regression analysis using GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA). 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 ANOVA 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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2B-ARs, 24-h pretreatment with 20 µM NE was required to desensitize the 2-AR-mediated inhibition of cAMP accumulation (Fig. 1A); 10 µM NE was insufficient to produce desensitization of the 2B-ARs. Similarly, 20 µM but not 10 µM, EPI produced 2-adrenoceptor desensitization (Fig. 1B). Neither NE nor EPI, at concentrations that resulted in 2-adrenoceptor desensitization, produced any changes in the cellular expression of GRK2 or GRK3 (Fig. 2). However the desensitization was accompanied by down-regulation of specific 2B-AR binding (Fig. 3).
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In BN17 cells that express both 2B- and 2-ARs, 24-h pretreatment with only 0.3 µM EPI resulted in desensitization of the 2B-ARs (Fig. 4). This concentration was 67-fold lower than the minimal concentration of EPI that resulted in 2B-AR desensitization in NG108 cells. Pretreatment with 0.1 µM EPI had no significant effect on 2B-AR responsiveness. Also, in BN17 cells, the concentration of NE (10 µM NE) required to desensitize the 2B-AR (Fig. 5) was reduced by only 2-fold compared with the NG108 cells. More importantly, the effect of EPI or NE on 2B-AR response was antagonized by the presence of the -AR antagonist alprenolol (1 µM) during the EPI or NE pretreatment (Fig. 6).
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In addition to desensitization of the 2B-AR, pretreatment of BN17 cells with 0.3 µM EPI also resulted in down-regulation of the 2B-ARs that was dependent on coactivation of the 2B- and the 2-AR (Fig. 7A). The pretreatment did not change the affinity of [3H]RX821002 for the 2B-AR (Veh, 3.2 ± 0.8 nM; 0.3 µM NE, 3.6 ± 0.5 nM; 0.3 µM EPI, 3.2 ± 0.7 nM; vehicle + 1 µM alprenolol (Alp), 3.1 ± 0.4 nM; and 0.3 µM EPI + 1 µM Alp, 2.8 ± 0.3 nM). Similarly, pretreatment of BN17 cells with 10 µM NE resulted in a 2-AR-dependent down-regulation of the 2B-AR (Fig. 7B).
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The effects of EPI and NE pretreatments on cellular levels of GRK3 also were different in BN17 cells, compared with NG108 cells. In BN17 cells, pretreatment with EPI or NE that resulted in 2B-AR desensitization and down-regulation also produced a selective approximately 2-fold up-regulation of GRK3 (Fig. 8A). This increase in GRK3 expression also was prevented when the pretreatment was done in the presence of 1 µM alprenolol (Fig. 8A). No effect on GRK2 expression was observed with either treatment (Fig. 8B).
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Because desensitization and down-regulation of the 2B-ARs in BN17 cells were accompanied by a selective up-regulation of GRK3 expression, and because all of these effects were prevented by -AR blockade with alprenolol, we determined whether 2B-AR desensitization and down-regulation in BN17 cells was dependent upon GRK3 up-regulation. For this purpose, we performed the EPI pretreatment in cells treated with GRK3 antisense ODN to prevent the up-regulation of GRK3. Treatment with EPI in the presence of GRK3 antisense ODN prevented desensitization (Fig. 9A) and down-regulation (Fig. 9B) of the 2B-AR, as well as prevented GRK3 up-regulation (Fig. 10A). Pretreatment with GRK3 antisense ODN or GRK3 sense ODN alone did not affect the acute 2B-AR response in the BN17 cells (data not shown; see Fig. 9 legend). The GRK3 antisense ODN treatment also did not alter basal GRK3 (Fig. 10A) or GRK2 expression (Fig. 10B).
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Finally, because desensitization and down-regulation of the 2B-AR at low agonist concentrations was dependent upon 2-AR activation in BN17 cells, we determined whether 2-AR activation alone was sufficient to produce 2B-AR desensitization by pretreating for 24 h with isoproterenol (1 µM). Isoproterenol pretreatment had no effect either on 2B-AR responsiveness (Fig. 11A) or level (Fig. 11B) in BN17 cells, suggesting that simultaneous activation of the 2B- and 2-AR is necessary for 2-AR desensitization at lower agonist concentrations in BN17 cells. Treating BN17 cells with 1 µM isoproterenol in the presence of 0.3 µM NE further tested this requirement of simultaneous activation of the 2B- and 2-AR. The NE and isoproterenol cotreatment desensitized (Fig. 11A) and down-regulated (Fig. 11B) the 2B-AR. Also, pretreatment of the BN17 cells with 1 µM isoproterenol did not alter the cellular levels of either GRK2 or GRK3, but the cotreatment with 1 µM isoproterenol and 0.3 µM NE selectively increased the level of GRK3 (data not shown).
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Discussion |
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2B- and 2-adrenoceptors results in down-regulation of the 2B-adrenoceptor by EPI at concentrations 
70-fold lower than those required when the 2-adrenoceptor is not activated. The mechanism responsible for this effect of EPI at lower agonist concentrations is a selective up-regulation of GRK3 expression that requires simultaneous 2B- and 2-adrenoceptor activation. Prevention of GRK3 up-regulation by -adrenoceptor blockade or GRK3 antisense ODN treatment during the EPI pretreatment eliminates the 2B-adrenoceptor down-regulation at low EPI concentrations (0.3 µM). Without simultaneous activation of both 2B- and 2-adrenoceptors, EPI and NE still produced 2B-adrenoceptor down-regulation, but significantly higher agonist concentrations (20 µM) are required.
The results of this study reveal an intriguing difference between the threshold concentrations of the physiological agonists NE and EPI required to induce 2B-adrenoceptor down-regulation. This result is surprising in the context of the potency and efficacy of NE and EPI at the 2-adrenoceptor. It has repeatedly been reported that these agonists are equipotent and equiefficacious at this receptor (Jasper et al., 1998). Therefore, one would not expect the observed difference in the minimal concentration of EPI versus NE required to induce down-regulation. However, the contribution of the 2-adrenoceptor to this process immediately reveals the reason for this difference. In BN17 cells expressing both 2B- and 2-adrenoceptors, 0.3 µM EPI or 10 µM NE was the minimal concentration required to produce 2B-adrenoceptor down-regulation, whereas in NG108 cells that express only the 2B-adrenoceptor, the minimal concentration required to produce down-regulation was the same for NE or EPI, 20 µM. The difference between the minimal concentration of NE versus EPI required in BN17 cells is 33-fold, similar to the 50- to 70-fold greater potency for EPI versus NE at the 2-adrenoceptor (Lands et al., 1967). Most likely, the greater degree of 2B-adrenoceptor activation produced by NE at 10 µM compared with 0.3 µM, combined with the 2B-adrenoceptor activation by NE at 10 µM, enabled NE to down-regulate the 2B-adrenoceptor in BN17 cells at a slightly lower concentration than that required in NG108 cells. Therefore, the lower threshold concentration of EPI versus NE required to produce 2B-adrenoceptor down-regulation, although surprising, is in good agreement with the pharmacology of EPI versus NE at the 2-adrenoceptor.
The present study also clearly defines the role of the 2-adrenoceptor in the down-regulation of the 2B-adrenoceptor, i.e., to contribute to up-regulation of GRK3. However, it is important to recognize that neither 2- nor 2-adrenoceptor activation alone produces GKR3 up-regulation. First, 2-adrenoceptor activation by NE at low concentrations in BN17 cells or by higher concentrations of NE or EPI in NG108 cells does not produce GRK3 up-regulation. Second, isoproterenol at concentrations that cause similar degrees of 2-adrenoceptor activation as 0.3 µM EPI does not cause up-regulation of GKR3. Finally, up-regulation of GRK3 by EPI in BN17 cells is prevented by -adrenoceptor blockade. Hence, simultaneous activation of both the 2- and 2-adrenoceptors was required for GRK3 up-regulation. The mechanisms responsible for GRK3 up-regulation were not studied further because this was not the focus of the present project. However, in a previous study of 2A-adrenoceptor regulation, it was observed that activation of p42/44 MAP kinase by simultaneous 2- and 2-adrenoceptor activation was required for GRK3 up-regulation and that inhibition of protein synthesis or GRK3 antisense ODN prevented GRK3 up-regulation by EPI (K. M. Standifer, personal communication). Although these observations are suggestive of a regulation of GRK3 expression by a mitogen-activated protein kinase substrate, this is pure speculation because very limited information is available about the regulation of the expression of GRK3 or GRK2 (Penela et al., 2003).
Another somewhat surprising component of the results in the present study is the critical role of GRK3 in enabling EPI to produce 2-adrenoceptor down-regulation at significantly lower agonist concentration. A role of GRKs in the short-term desensitization of the 2B-adrenoceptor is clearly suggested by the work of Liggett and coworkers. These investigators have reported GRK-mediated phosphorylation of the 2B-adrenoceptor and correlated this phosphorylation with desensitization of agonist-mediated inhibition of cAMP accumulation (Jewell-Motz and Liggett, 1995). Moreover, they have reported that mutations within the third intracellular loop of this receptor not only reduce GRK-mediated phosphorylation but also eliminate short-term agonist-induced desensitization of the 2B-adrenoceptor. The physiological importance of this phosphorylation is suggested by recent reports of a polymorphism of the human 2B-adrenoceptor that exhibits reduced agonist-induced phosphorylation and desensitization (Small et al., 2001) and is associated with an increased risk of myocardial infarction in human (Snapir et al., 2003). However, the role of GRK mediated phosphorylation in long-term regulation of the 2B-adrenoceptor is more tenuous. For example, site-directed mutagenesis of the third intracellular loop of the 2B-adrenoceptor, which reduced phosphorylation and eliminated short-term agonist-induced desensitization, was reported to have no effect on long-term agonist-induced receptor down-regulation (Jewell-Motz and Liggett, 1995). Nevertheless, the results of the present study support a critical role of GRK3 in 2B-adrenoceptor down-regulation.
The important role of the GRKs in 2B-adrenoceptor down-regulation suggested by the present study is in agreement with several recent reports regarding the role of GRKs in the down-regulation of other G protein-coupled receptors (GPCRs). For example, the 
-opioid receptor has been shown to undergo down-regulation in N18TG2 cells after exposure to etorpine in a GRK-dependent manner (Shapira et al., 2001). Similarly, the human 
-opioid receptor has been shown to undergo down-regulation in a GRK2-dependent manner in Chinese hamster ovary cells (Li et al., 2000). The expression of the metabotropic glutamate receptor 5 also was reported to be regulated by GRK2 (Sorensen and Conn, 2003). This effect of GRK2 requires the presence of a threonine residue (Thr 840) in the carboxyl terminal tail of metabotropic glutamate receptor 5, suggesting a role for GRK-mediated phosphorylation in down-regulating the receptor. Finally, a recent report suggests that GRK2 overexpression reduces the threshold agonist concentration required for down-regulation of the human muscarinic M2 receptor (Tsuga et al., 1998). Therefore, there is considerable evidence that down-regulation of several GPCRs is regulated by the GRKs.
The role of 2-adrenoceptor mediated GRK3 up-regulation in enabling 2B-adrenoceptor down-regulation by lower EPI concentrations highlights an emerging theme in receptor signal modulation, i.e., heterologous modulation of homologous receptor regulation (Chuang et al., 1996). Phosphorylation of GPCRs by GRKs requires an agonist-occupied receptor. For this reason, mechanisms involving GRKs were initially considered as strictly homologous. However, our results clearly demonstrate that, by contributing to the regulation of GRK3 expression, 2-adrenoceptors heterologously modulate 2B-adrenoceptor down-regulation. These observations are similar to our previous report where 2A-adrenoceptor regulation by simultaneous 2A- and 2-adrenoceptor activation caused selective GRK3 up-regulation and 2A-adrenoceptor down-regulation at low EPI concentrations (Bawa et al., 2003). As discussed in a recent review, accumulating evidence suggests that heterologous regulation of GRK expression may play an important role in the heterologous regulation of GPCR signaling (Penela et al., 2003). In the context of the present study, it is particularly important to note that this heterologous regulation allows long-term agonist-induced receptor down-regulation and desensitization to occur at significantly lower agonist concentrations than would be required for homologous regulation. Considering that the 2-adrenoceptor generally requires micromolar agonist concentrations to produce desensitization compared with the 2-adrenoceptor (Atkinson and Minneman, 1992), and considering the many physiologically important sites where 2- and 2-adrenoceptors are colocalized on cells such as vascular and uterine smooth muscle, fat cells, pancreatic cells, and sympathetic nerve terminals to name a few, such heterologous regulation has the potential to significantly modify long-term regulation of many important functions.
Several investigators previously have reported heterologous regulation of 2-adrenoceptor numbers. In HT29 cells, stimulation of cAMP accumulation led to an increase in the number of 2-adrenoceptors (Sakaue and Hoffman, 1991). This increase was shown to be the result of an increase in gene transcription. By contrast, in cultured astroglia cells, increases in cellular cAMP levels or PKC activation was reported to cause a decrease in the steady-state levels of 2A-adrenoceptor mRNA levels by decreasing gene transcription (Reutter et al., 1998). EPI also produced down-regulation of the 2-adrenoceptor in these cells by a mechanism that involved a decrease in receptor transcription. However, the changes in 2-adrenoceptor number observed in the present study seem to have no contribution from changes in cAMP. First, GRK3 antisense, which had no effect on adrenoceptor signaling, abolished EPI-induced 2-adrenoceptor down-regulation. Second, isoproterenol at concentrations that produced 2-adrenoceptor activation similar to that of EPI, and increased cAMP concentrations in BN17 cells, had no effect of 2-adrenoceptor number. Therefore, the data in the present study are unique in that they emphasize a role for GRK3 expression in this heterologous regulation.
In summary, heterologous up-regulation of GRK3 by simultaneous activation of 2B- and 2-adrenoceptors enables the 2B-adrenoceptor to be down-regulated by significantly lower concentrations of EPI compared with NE. Future studies will focus on the role of GRKs in 2-adrenoceptor down-regulation and the influence of cellular levels of GRK expression on the threshold agonist concentrations required for 2-adrenoceptor down-regulation.
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Footnotes |
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ABBREVIATIONS: GPCR, G protein-coupled receptor; EPI, epinephrine; NE, norepinephrine; PGE1, prostaglandin E1; ODN, oligodeoxynucleotide; AR, adrenoceptor; ISO, isoproterenol; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline/Tween 20; Alp, alprenolol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; [3H]RX821002, (1,4-[6,7)(n)-3H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride.
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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