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CELLULAR AND MOLECULAR

Cellular G Protein-Coupled Receptor Kinase Levels Regulate Sensitivity of the _2B-Adrenergic Receptor to Undergo Agonist-Induced Down-Regulation

Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas

Received August 11, 2004; accepted September 28, 2004.

	Abstract

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Chronic coactivation of _2B- and ₂-adrenoceptors (AR) was recently reported to down-regulate the _2B-AR at a lower threshold epinephrine (EPI) concentration compared with the activation of _2B-AR alone. This is the result of a modest ₂-AR-dependent up-regulation of G protein-coupled receptor kinase 3 (GRK3). In the present study, we determined that increasing GRK2 or GRK3 levels, independent of ₂-AR activation, decreases the EC₅₀ concentration for agonist-induced down-regulation of the _2B-AR using NG108 cells with or without overexpression (2- to 10-fold) of GRK2 or GRK3. In parental NG108 cells, the EC₅₀ concentration of EPI required for down-regulation of the _2B-AR is 30 µM. A 2- to 3-fold overexpression of GRK3 in NG108 cells, however, reduces the EC₅₀ to 0.2 µM (a 150-fold decrease), whereas a comparable overexpression of GRK2 reduces it to 1 µM (a 30-fold decrease). However, when GRK3 or GRK2 in NG108 cells are overexpressed 8- to 10-fold, the EC₅₀ concentration (0.02 µM EPI) for _2B-AR down-regulation is reduced 1000-fold. These data clearly suggest that a modest (2- to 3-fold) up-regulation of GRK3 is more effective at enhancing the sensitivity of _2B-AR to down-regulation after exposure to EPI than a modest up-regulation of GRK2, but that both GRK2 and GRK3 are equally effective at inducing _2B-AR down-regulation when up-regulated 8- to 10-fold. To our knowledge, this is the first report to systematically demonstrate that GRKs, particularly GRK3, play a pivotal role in modulating the agonist EC₅₀ concentration that down-regulates the _2B-AR and thus adds a new dimension to an already intricate signaling network.

There is significant ambiguity about the role of GRKs in agonist-induced down-regulation of the ₂-AR. In one heterologous expression system, agonist-induced down-regulation of the _2B-AR (Jewell-Motz and Liggett, 1995) and other ₂-AR subtypes was found not to be dependent on GRK-mediated phosphorylation of these receptors (Jewell-Motz et al., 1997), but instead, dependent on palmitoylation state (Eason et al., 1994). In contrast, mutation of potential GRK phosphorylation sites in the _2C-AR blocks the agonist-induced down-regulation of these receptors expressed in OK cells (Deupree et al., 2002). Moreover, in vivo studies also suggest an association between increases in GRK2 levels and down-regulation of the _2A-AR (Ansonoff and Etgen, 2001). Thus, there is no clear consensus on the role of GRK in modulating down-regulation of the ₂-AR.

Studies conducted in our lab have added another level of complexity to the intricate role of GRK in modulating down-regulation of the ₂-AR. We have shown that GRK is not only required for _2A- and _2B-AR down-regulation, but that the sensitivity of the ₂-AR to undergo down-regulation is increased if the level of GRK is increased (Bawa et al., 2003; Desai et al., 2004). This observation is interesting for two reasons. First, previous studies suggest that the ₂-AR requires exposure to supraphysiological agonist concentrations to undergo desensitization or down-regulation compared with other adrenoceptors, especially the -AR (Atkinson and Minneman, 1992; Pleus et al., 1993). Second, there are a number of pathophysiological conditions that are associated with an increase in levels of GRK and abnormalities in GPCR signaling. In a mouse model of cardiac hypertrophy, a marked desensitization of the -AR was associated with a 3-fold increase in the activity of GRK2 (Choi et al., 1997). In cystic fibrosis lung, a decrease in airway -AR density is associated with an increase in GRK2 and GRK5 protein levels (Mak et al., 2002). Age-related desensitization of the -AR is associated with increased expression of GRK2 and GRK3 (Schutzer et al., 2001). Desensitization and down-regulation of the µ-opioid receptor during tolerance is associated with up-regulation of GRK2, GRK3, and -arrestin2 (Hurlé, 2001). In addition, recent studies performed in cultured cells suggest a link between increased GRK levels and the sensitivity of GPCRs for desensitization and down-regulation. Muscarinic receptors M₂ (Tsuga et al., 1998) and M₄ (Holroyd et al., 1999) and adenosine A_2A receptors (Mundell et al., 1998) are rendered more sensitive to desensitization, internalization, or down-regulation when the cellular levels of GRK are increased. Furthermore, prolonged activation of µ- or ORL1-opioid receptors resulted in desensitization of these receptors due to agonist-induced up-regulation of GRK2 and GRK3 (Thakker and Standifer, 2002). Despite mounting evidence for a role of GRKs in down-regulation of GPCRs in general and the ₂-AR in particular, current understanding of the effect of changing levels of GRK on the sensitivity of the ₂-AR to undergo desensitization and down-regulation is limited.

To address this issue, we utilized NG108-15 (NG108) cells, a model in which the requirement of supramaximal concentration of agonist for ₂-AR desensitization and down-regulation has been demonstrated (Thomas and Hoffman, 1986; Convents et al., 1989). NG108 cells were transfected to express different levels of GRK2 or GRK3, and the EC₅₀ concentration of EPI required to desensitize and down-regulate the _2B-AR was determined. Information generated from this study helps to discern the significance of changes in GRK levels in _2B-AR regulation.

	Materials and Methods

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Materials.The following drugs were purchased from the indicated sources: (-)EPI, phenylmethylsulphonylfluoride, phentolamine, cAMP, prostaglandin E₁ (PGE₁), Dulbecco's modified Eagle's medium (DMEM), adrenal cortex extract, hydroxyapatite, HAT (0.1 mM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine) supplement, sodium orthovanadate, sodium pyrophosphate, pepstatin, leupeptin, aprotinin, isobutylmethylxanthine, sodium metabisulphite, theophylline, HEPES, bovine serum albumin, and poly-L-lysine hydrobromide from Sigma-Aldrich (St. Louis, MO); (-)NE from Sigma/RBI (Natick, MA); [³H]cAMP and [³H]RX821002 [(1,4-[6,7(n)-³H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride] from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK); G418 sulfate (Geneticin) from Calbiochem (San Diego, CA); fetal bovine serum and penicillin-streptomycin from Atlanta Biologicals (Norcross, GA); TEMED and ammonium persulphate from BioRad (Hercules, CA); anti-GRK3 rabbit IgG, anti-GRK2 rabbit IgG, goat anti-rabbit IgG horseradish peroxidase, goat anti-mouse IgG horseradish peroxidase, and enhanced chemiluminescence reagent from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and mouse antirabbit glyceraldehyde phosphate dehydrogenase (GAPDH) IgG from Research Diagnostics (Flanders, NJ). NG108 cells were obtained from Dr. Graeme Milligan, University of Glasgow, Glasgow, Scotland, UK. GRK2 and GRK3 plasmids (pcDNA3.1 carrying the neomycin resistance cassette) were obtained from Dr. Brian Knoll, University of Houston, Houston, Texas.

Transfection of NG108 Cells with GRK2 or GRK3. Stable transfection of GRK2 or GRK3 in NG108 cells was carried out using FuGENE 6 transfection reagent. Cells in 100-mm tissue culture plates (at 30–40% confluence) were incubated at 37°C with a transfection mixture composed of serum-free DMEM-H (HEPES-buffered DMEM) containing 11 µg of DNA/plate and 17 µl of FuGENE. After 48 h, the cells were split (1:12, 1:24, or 1:50) into 100-mm tissue culture plates, and the medium was supplemented with G418. Surviving colonies were isolated and expanded into cell lines. Whole cell lysates were assayed for the expression of GRK2 or GRK3 protein by Western blotting. NG108 cells transfected to overexpress either GRK2 or GRK3 are described as K2/# or K3/# respectively, where # is the -fold overexpression of GRK2 or GRK3 in the cells compared to the parental cell line.

Cell Culture. NG108 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated fetal calf serum, penicillin, streptomycin, and HAT (0.1 mM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine) supplement. NG108 cells transfected to express either GRK2 or GRK3 were maintained similarly except that the media contained G418 (0.4 mg/ml) to retain selection pressure. All the cell lines were grown either in 75-cm² flasks or 150-cm² plates. Flasks or plates of cells that were more than 80% confluent were used throughout the study.

Pretreatment. NG108 cells and NG108 cells transfected to overexpress either GRK2 or GRK3 were pretreated with vehicle (medium containing 0.1 mM ascorbate and 1 µM sodium metabisulphite) or vehicle containing 0.003 to 200 µM EPI for 24 h.

₂-AR Agonist Concentration Response Curves. After EPI pretreatment, media containing the drugs were aspirated, and the cells were harvested by pipetting fresh drug-free medium against the cells. Intact cells were harvested and assayed for cAMP accumulation as described (Desai et al., 2004). Briefly, 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. All assays were performed in duplicate in a total volume of 0.5 ml, and 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 in the supernatant fractions were determined in a [³H]cAMP (0.8 pmol) binding assay as previously described (Standifer et al., 1994). Preferential ₂-AR agonists were not used in this study because NG108 cells express imidazoline receptors, the activation of which inhibits cAMP accumulation (Greney et al., 2000). Since all preferential ₂-AR agonists would activate both the ₂- and imidazoline-receptors in the NG108 cells, this would significantly complicate data interpretation.

Membrane Preparation for Receptor Binding. To prepare membranes for receptor binding, the cells were first washed three times with phosphate-buffered saline (pH 7.4) and harvested 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 100 mM NaCl, 10 mM Na₂EDTA, and 0.1 mM phenylmethylsulphonylflouride 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 binding assay.

Radioligand Binding Assay to Determine Receptor Number. To determine the number of _2B-ARs, binding was performed using the ₂-AR antagonist [³H]RX821002. The membranes (0.25–0.30 mg of protein/ml) were incubated with [³H]RX821002 (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 by using 100 µM phentolamine. At the end of the incubation period, the 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 three times with 3 to 4 ml of the filtration buffer (50 mM Tris-HCl pH 8.0). The amount of radioactivity in the filter paper was determined by scintillation spectroscopy in a Beckman LS6000 liquid scintillation counter.

Western Blot Analysis. Some of the cells collected for _2B-AR response assay or receptor binding were used to prepare samples for Western blot analysis. Cell pellets were washed once with 1x phosphate-buffered saline 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 phenylmethylsulfonylfluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin), vortexed, and incubated for 30 min in an ice bath. The resultant cell lysate was diluted with 2x Laemmli buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.1 mg/ml bromo-phenol blue) and resolved on SDS-polyacrylamide gel electrophoresis (10% gel). The resolved proteins were electrophoretically transferred to a polyvinylidene difluoride membrane (Amersham Biosciences UK, Ltd.), and the GRK2 or GRK3 expression levels were determined by immunoblotting using anti-GRK2 or -GRK3 (1:1000; Santa Cruz Biotechnology, Inc.) in 2.5% nonfat milk as described (Desai et al., 2004). The blots were stripped and reprobed for GAPDH as a loading control using mouse anti-rabbit GAPDH (1:8000; Research Diagnostics).

Protein Estimation. Protein concentrations were determined by the Lowry's method (Lowry et al., 1951).

Data Analysis. B_max, EC₅₀, and maximal response to the ₂-AR agonist were determined by nonlinear regression analysis using GraphPad Prism version 3.0 (GraphPad Software, Inc., San Diego, CA). Prism also was utilized to estimate the EC₅₀ concentrations for ₂-AR down-regulation. In these estimations of EC₅₀, the iterative curve-fitting process was constrained by setting the minimum value for receptor down-regulation at 0. For comparison between groups, the values were expressed as mean ± S.E.M. Between group comparisons 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.), and the groups were considered significantly different if p < 0.05.

	Results

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In NG108 cells, the EC₅₀ for down-regulation of the _2B-AR is 30 µM EPI (Fig. 1A). We previously showed that in NG108 cells transfected to express human ₂-AR, the _2B-AR down-regulates after 24 h of pretreatment with 0.3 µM EPI, a concentration at which the _2B-AR does not down-regulate in the parental NG108 cells. This down-regulation is associated with a modest increase in the cellular levels of GRK3 (Desai et al., 2004). We therefore wanted to determine whether increasing the levels of GRK3, in the absence of ₂-AR activation, would reduce the concentration of EPI required to down-regulate the _2B-AR. Accordingly, NG108 cells were transfected with GRK3 plasmids, and clones overexpressing GRK3 at various levels (2- to 10-fold over basal) were isolated. The degree of overexpression of GRK3 in these clones compared with the parental NG108 cells is shown in Table 1. In NG108 cells transfected to overexpress GRK3 (2- to 3-fold, K3/3), the EC₅₀ for down-regulation of the _2B-AR is 0.2 µM EPI. To insure that the decrease in EC₅₀ for the down-regulation of the _2B-AR in this clone was not the characteristic of a single clone but the result of GRK3 overexpression, the _2B-AR binding sites in another clone (K3/2.4) with comparable levels of GRK3 overexpression were also measured after 24 h of pretreatment with either 0.1 µM (a concentration at which no down-regulation was observed in K3/3) or 0.3 µM EPI (the minimum concentration at which significant down-regulation of the _2B-AR was observed in K3/3), and the results were similar (data not shown). On the other hand, in NG108 cells transfected to overexpress GRK3 approximately 10-fold (K3/9.8), the EC₅₀ for down-regulation of the _2B-AR is 0.02 µM EPI (Fig. 1A). The cellular levels of GRK3 in K3/3, K3/2.4, and K3/9.8 were not altered by pretreatment with EPI (Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 1. The EC50 for down-regulation of the 2B-AR in parental NG108 cells and NG108 cells transfected to overexpress GRK3 (A) or GRK2 (B). A, maximal binding of [3H]RX821002 (30 nM) to 2B-AR was determined using membranes prepared from cells pretreated with vehicle and 0.003 to 200 µM EPI for 24 h. The EC50 for down-regulation of the 2B-AR in NG108 cells is 30 µM EPI, in K3/3 cells is 0.2 µM EPI, and in the K3/9.8 cells is 0.02 µM EPI. B, the EC50 for down-regulation of the 2B-AR in K2/2.9 cells is 1 µM EPI and in the K2/9.5 cells is 0.02 µM EPI. NG108 cells transfected to overexpress either GRK2 or GRK3 are described as K2/# or K3/#, respectively, where # is the -fold overexpression of GRK2 or GRK3 in the cells.

Clearly, increasing the level of GRK3 renders the _2B-AR more sensitive to agonist-induced down-regulation. Previous results indicate that the ₂-AR may be more susceptible to the action of GRK3 than GRK2 (Diverse-Pierluissi et al., 1996; Bawa et al., 2003; Desai et al., 2004). Therefore, the sensitivity of the _2B-AR to increasing levels of GRK2 also was determined. To that end, NG108 cells were transfected with GRK2 plasmids, and a number of clones overexpressing GRK2 at various levels (2- to 10-fold over basal) were isolated. The degree of overexpression of GRK2 in these clones compared with the parental NG108 cells is shown in Table 1. In NG108 cells transfected to overexpress GRK2 (2- to 3-fold, K2/2.9), the EC₅₀ for down-regulation of the _2B-AR is 1 µM EPI (Fig. 1B). Similar results were obtained in a second clone (K2/2) with comparable GRK2 overexpression levels following EPI pretreatment (data not shown). In NG108 cells transfected to overexpress GRK2 about 10-fold (K2/9.5), the EC₅₀ for down-regulation is 0.02 µM EPI (Fig. 1B). Again, we measured the _2B-AR binding sites in another clone (K2/8.5) with comparable levels of GRK2 overexpression after pretreatment with 0.01 µM (a concentration at which no down-regulation was observed in K2/9.5) and 0.03 µM EPI (the minimum concentration at which down-regulation of the _2B-AR was observed in K2/9.5), and the results were similar (data not shown). The cellular levels of GRK2 in K2/2.9, K2/2, K2/9.5, and K2/8.5 were not altered by pretreatment with EPI (Table 2).

In NG108 cells, 24 h of pretreatment with 20 µM EPI, the minimum concentration at which significant down-regulation of the _2B-AR is observed, decreases the responsiveness of the _2B-AR (Fig. 2). Pretreatment with 10 µM EPI for 24 h, a concentration at which significant down-regulation of the _2B-AR is not observed, does not decrease the responsiveness of the _2B-AR. The basal cAMP accumulation in NG108 cells is: Veh, 30.5 ± 2.3 pmol/mg protein; 10 µM EPI, 29.6 ± 1.7 pmol/mg protein; and 20 µM EPI, 31.3 ± 2.9 pmol/mg protein. In NG108 and BN17 cells, we always have observed a decrease in responsiveness of the _2B-AR at the minimum concentration of EPI that causes down-regulation of the _2B-AR. Therefore, we determined whether the loss of _2B-AR binding sites corresponds with the loss of _2B-AR responsiveness following 24 h of pretreatment with EPI in NG108 cells transfected to overexpress GRK2 or GRK3. Prior to that, the _2B-AR responsiveness in naive GRK2 or GRK3 overexpressing cells also was measured. The acute response to _2B-AR activation was not altered in cells overexpressing GRK3 (Fig. 3A) or GRK2 (Fig. 3B) either 2- to 3-fold or 8- to 10-fold over basal levels. The basal cAMP accumulation (32.5 ± 4.2 pmol/mg protein) in all the GRK3 or GRK2 overexpressing clones was similar to that seen in the NG108 cells.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Chronic (24-h) pretreatment with 20 µM but not with 10 µM EPI desensitizes the 2B-AR in NG108 cells. NE-induced inhibition of 10 nM PGE1-stimulated cAMP accumulation was studied in cells pretreated with vehicle, 10 µM EPI, or 20 µM EPI for 24 h. The pretreatment did not alter the potency of NE or the basal cAMP levels in the cells. However, the maximal percentage of inhibition in cells pretreated with 20 µM EPI (32.8 ± 4.2) was significantly different (*) from that in the cells pretreated with either vehicle (50.6 ± 3.0) or 10 µM EPI (49.5 ± 3.7), p < 0.05. n = 3–6.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Acute 2B-AR signaling is not altered in NG108 cells overexpressing either GRK3 (A) or GRK2 (B). NE-induced inhibition of 10 nM PGE1-stimulated cAMP accumulation was studied in NG108 cells transfected to express either GRK2 or GRK3. The basal cAMP accumulation and potency of NE were similar among all the cell lines. The maximal percentage of inhibition was NG108 cells (54.7 ± 3.6), K3/3 (53 ± 4.6), K3/2.4 (52.6 ± 4.4), K3/9.8 (60.2 ± 3.6), K2/2 (54 ± 3.7), K2/2.9 (51.9 ± 3.1), K2/8.5 (51.9 ± 4.3), and K2/9.5 (46.4 ± 4.6); p > 0.05 (n = 3).

Pretreatment of K3/3 and K3/2.4 for 24 h with 0.3 µM EPI desensitizes _2B-AR signaling (Fig. 4A). Similarly, a 24-h pretreatment with 0.03 µM EPI desensitizes the _2B-AR signaling in K3/9.8 cells (Fig. 4B). The _2B-AR signaling also is desensitized in both K2/2.9 and K2/2 following 24 h of pretreatment with 3 µM EPI (Fig. 5A). A 24-h pretreatment with 0.03 µM EPI, the minimum concentration at which significant down-regulation of the _2B-AR is observed, desensitizes the _2B-AR signaling in both K2/8.5 and K2/9.5 (Fig. 5B).

View larger version (20K): [in this window] [in a new window]   Fig. 4. Chronic (24-h) pretreatment with 0.3 µM EPI desensitizes the 2B-AR in K3/3 and K3/2.4 cells (A), whereas chronic pretreatment with 0.03 µM EPI desensitizes the 2B-AR in K3/9.8 (B). A, NE-induced inhibition of 10 nM PGE1-stimulated cAMP accumulation was studied in cells pretreated with vehicle or 0.3 µM EPI for 24 h. The pretreatment did not alter the potency of NE or the basal cAMP levels in the cells. However, the maximal percentage of inhibition in K3/3 cells pretreated with 0.3 µM EPI (33.0 ± 2.8) was significantly different (*) from that in the cells pretreated with vehicle (50.0 ± 2.7), p < 0.05. The maximal percentage of inhibition in K3/2.4 cells pretreated with 0.3 µM EPI (35.7 ± 3.7) was significantly different (*) from that in the cells pretreated with vehicle (51.2 ± 3.0), p < 0.05. n = 3. B, NE-induced inhibition of 10 nM PGE1-stimulated cAMP accumulation was studied in K3/9.8 cells pretreated with vehicle or 0.03 µM EPI for 24 h. The pretreatment did not alter the potency of NE or the basal cAMP levels in the cells. However, the maximal percentage of inhibition in cells pretreated with 0.03 µM EPI (28.7 ± 2.5) was significantly different (*) from that in the cells pretreated with vehicle (55.2 ± 2.3), p < 0.05. n = 3.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Chronic (24-h) pretreatment with 3 µM EPI desensitizes the 2B-AR in K2/2.9 or K2/2 cells (A), whereas chronic pretreatment with 0.03 µM EPI desensitizes the 2B-AR in K2/9.5 cell. A, NE-induced inhibition of 10 nM PGE1-stimulated cAMP accumulation was studied in K2/2.9 or K2/2 cells pretreated with vehicle or 3 µM EPI for 24 h. The pretreatment did not alter the potency of NE or the basal cAMP levels in the cells. However, the maximal percentage of inhibition in K2/2.9 cells pretreated with 3 µM EPI (27.4 ± 1.6) was significantly different (*) from that in the cells pretreated with vehicle (44.3 ± 2.1), p < 0.05. n = 3. The maximal percentage of inhibition in K2/2 cells pretreated with 3 µM EPI (31.0 ± 2.2) was significantly different (*) from that in the cells pretreated with vehicle (48.7 ± 1.7), p < 0.05. n = 3. B, NE-induced inhibition of 10 nM PGE1-stimulated cAMP accumulation was studied in cells pretreated with vehicle or 0.03 µM EPI for 24 h. The pretreatment did not alter the potency of NE or the basal cAMP levels in the cells. However, the maximal percentage of inhibition in K2/9.5 cells pretreated with 0.03 µM EPI (33.9 ± 2.8) was significantly different (*) from that in the cells pretreated with vehicle (49.0 ± 2.1), p < 0.05. n = 3. The maximal percentage of inhibition in K2/8.5 cells pretreated with 0.0 3 µM EPI (34.2 ± 2.0) was significantly different (*) from that in the cells pretreated with vehicle (48.3 ± 2.2), p < 0.05. n = 3.

	Discussion

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This study demonstrates that increasing the cellular levels of GRK3 or GRK2 increases the sensitivity of the _2B-AR to agonist-induced down-regulation. To our knowledge, this is the first evidence that levels of GRK3 or GRK2 in a given cell can regulate the EC₅₀ concentration of agonist that down-regulates the _2B-AR. Our report also is the first to demonstrate that when overexpressed at modest levels (2- to 3-fold) GRK3 is more effective than GRK2 in modulating the _2B-AR; however, when overexpressed at high levels (8- to 10-fold), this difference between GRK3 and GRK2 is eliminated, and both are equally effective.

The results of the present study provide evidence indicating that GRK3 is more effective than GRK2 in the long-term regulation of ₂-AR signaling. Both GRK3 and GRK2 previously have been shown to phosphorylate and desensitize the _2A-AR equieffectively (Jewell-Motz and Liggett, 1996). However, in that study, GRK3 and GRK2 were overexpressed at very high levels (more than 15-fold); our data suggests that the subtle differences observed at lower, more physiological levels of GRK3 and GRK2 overexpression were not apparent when GRKs were expressed at high levels. There is substantial evidence in the literature to suggest that GRK3 may be a more important player than GRK2 in regulating the ₂-AR upon prolonged agonist exposure. Introduction of recombinant, purified GRKs or synthetic blocking peptides into individual embryonic sensory neurons has demonstrated the involvement of a GRK3-like protein in the desensitization of the ₂-AR (Diverse-Pierluissi et al., 1996). We also have previously demonstrated that endogenous GRK3 rather than GRK2 plays an important role in the modulation of the _2A- and _2B-AR after prolonged exposure to agonist (Bawa et al., 2003; Desai et al., 2004).

The results of the present study provide additional insights into the mechanisms that enable EPI to desensitize and down-regulate the _2B-AR at lower concentrations than NE in cells that express ₂- and ₂-AR. We previously reported that the _2B-ARs endogenously expressed in BN17 cells (NG108 cells transfected to express human ₂-AR) desensitize and down-regulate after exposure to low concentration of EPI (0.3 µM) due to an agonist-induced selective 2-fold upregulation of GRK3 (Desai et al., 2004). This concentration of EPI was the minimum concentration that down-regulated the _2B-AR and up-regulated GRK3. One of the confounding factors of our results in the BN17 cells was that ₂-AR activation was required for both the up-regulation of GRK3 and the down-regulation of the _2B-AR at low EPI concentration. Therefore, we could not separate the roles of the ₂-AR and GRK3 in the enhanced sensitivity of the _2B-AR to down-regulation by EPI. Increasing the level of GRK in the absence of ₂-AR, as achieved in the present study, allowed us to distinguish between these possibilities. The data from NG108 cells transfected to overexpress GRK3 or GRK2 suggests that a modest increase in cellular levels of GRK by itself is sufficient to produce a dramatic decrease in the EC₅₀ for agonist-induced down-regulation of the _2B-AR.

A critical role for GRKs in down-regulation of the ₂-AR following prolonged agonist exposure suggested by the present study is in agreement with some studies but not others. Mutation of potential GRK phosphorylation sites in the third intracellular loop prevents agonist-induced down-regulation of the _2C-AR expressed in OK cells (Deupree et al., 2002). In the human neuroblastoma cell line BE(2)-C, endogenous _2A-AR undergo down-regulation in a GRK3-dependent manner (Bawa et al., 2003). In contrast, in Chinese hamster ovary cells, a mutant _2A-AR lacking potential GRK phosphorylation sites in the third intracellular loop does not undergo short-term desensitization but is down-regulated by 24-h agonist treatment (Jewell-Motz et al., 1997). One explanation for these conflicting results could be the difference in level of receptor expression between the different studies. In the OK cells, the wild-type or mutant _2C-AR were expressed at about 40 or 300 fmol/mg protein (Deupree et al., 2002), and in BE(2)C cells, the _2A-AR were expressed at approximately 40 fmol/mg protein (Bawa et al., 2003). In Chinese hamster ovary cells where GRK was reported to play no role in down-regulation of the _2A-AR, the receptors were expressed at 700 or 2000 fmol/mg protein. Another explanation could be that different cell types have different mechanisms for agonist-induced down-regulation of GPCRs. There are a number of reports in the literature that suggest cell-specific differences in the mechanism of the down-regulation of GPCRs. For example, the OR undergoes down-regulation either via a GRK-dependent mechanism or a mitogen-activated protein kinase- and tyrosine kinase-dependent mechanism depending on the cell type studied (Shapira et al., 2001). Coincidentally in this report, the GRK-dependent mechanism for down-regulating the OR was dominant in NT18G cells where the receptors are expressed endogenously at low levels (about 130 fmol/mg protein), whereas the mitogen-activated protein kinase and tyrosine kinase mechanism for down-regulation was dominant in human embryonic kidney (HEK293) cells transfected to express the OR at high levels (17 pmol/mg protein). Overall, these data suggest that regulation of GPCRs by GRKs after prolonged exposure to agonist is a physiologically relevant phenomenon and one that warrants further investigation.

The present results also suggest that down-regulation of the _2B-AR differs significantly from down-regulation of the ₂-AR with regard to the dose-response relationships for receptor signaling versus down-regulation. For example, the EC₅₀ for adenylyl cyclase activation by fenoterol in BEAS-2B cells (endogenous ₂-AR) is 36 nM. In those cells, fenoterol-induced down-regulation of the ₂-AR is proposed to proceed via low- and high-affinity pathways with EC₅₀ of 163 and 0.53 nM, respectively. The EC₅₀ for the low-affinity pathway is about 10-fold greater than the EC₅₀ for stimulation of adenylyl cylase and similar to the affinity of fenoterol for the ₂-AR (Williams et al., 2000). In contrast, the affinity for EPI at the _2B-AR in NG108 cells is reported to be 3 to 10 nM, and the EC₅₀ for the _2B-AR-induced inhibition of cAMP accumulation in NG108 cells is about 30 nM, whereas the EC₅₀ for down-regulation of the _2B-AR is about 30 µM EPI. At the EC₅₀ concentration for _2B-AR down-regulation, the receptor is maximally activated and near saturation, very different from what is observed for the ₂-AR. Moreover, as the cellular level of GRK3 or GRK2 is increased, the EC₅₀ for down-regulation of the _2B-AR is reduced such that at a 10-fold overexpression of either GRK3 or GRK2, the EC₅₀ for down-regulation of the _2B-AR approaches the EC₅₀ for _2B-AR-induced inhibition of cAMP accumulation. These results are supportive of the concept that in NG108 cells the endogenous levels of GRK3 and GRK2 are a rate-limiting factor for down-regulation of the _2B-AR. We hypothesize that the GRKs contribute to down-regulation of ₂-AR by phosphorylating the receptors, thereby facilitating the trafficking of the receptor to lysosomes for degradation. Furthermore, we propose that a modest increase in the cellular level of GRK3 increases the rate of phosphorylation of the ₂-AR relative to the rate of its dephosphorylation, whereas a modest increase in the level of GRK2 is less able to do so. Hence, a small increase in GRK3 levels renders the ₂-AR more sensitive to agonist-induced down-regulation than a similar increase in GRK2. At high levels of GRK3 and GRK2 (10 fold) overexpression, this difference between the two is eliminated because phosphorylation of the ₂-AR by either GRK dominates over its dephosphorylation. Future studies will attempt to test this hypothesis.

A final important implication of this study relates to the pathophysiology of disease. There are a number of conditions that are associated with a modest change in levels of GRK and abnormalities in GPCR signaling. Hypothyroidism, a condition where -AR signaling is compromised, has been reported to exhibit about a 50% increase in GRK2 in heart and lung tissue (Penela et al., 2001). In a mouse model of cardiac hypertrophy, a marked desensitization of the -AR was associated with a 3-fold increase in the activity of GRK2 (Choi et al., 1997). In cystic fibrosis lung, a decrease in airway -AR density is associated with increases in GRK2 and GRK5 protein levels (Mak et al., 2002). Age-related desensitization of the -AR is associated with a 2-fold increase in expression of GRK2 and GRK3 (Schutzer et al., 2001). Desensitization and down-regulation of the µ-opioid receptor during tolerance is associated with a 2-fold up-regulation of GRK2, GRK3, and -arrestin2 (Hurlé, 2001). An increase in the levels of _2A-AR and a decrease in the levels of GRK2 were observed in platelets of patients suffering from major depression, and their treatment with the ₂-AR antagonist mirtazapine resulted in a 30% up-regulation of GRK2 and a 34% down-regulation of the _2A-AR (Garcia-Sevilla et al., 2004). Our data suggest that the sensitivity of the ₂-AR to undergo agonist-induced down-regulation is dramatically increased by modest changes in the level of GRK2 or GRK3.

In summary, the present study suggests an important role of cellular GRK levels in regulating the sensitivity of the _2B-AR to agonist-induced down-regulation. In addition, the results suggest that small increases in GRK3 expression can have profound effects on _2B-AR regulation; however, the mechanisms whereby GRKs modulate the sensitivity of the ₂-AR for down-regulation remain to be determined.

	Acknowledgements

 
We thank Estrella Foster for advice and excellent technical assistance in the generation of the GRK clones. We also thank Dr. Samina Salim for critical reading in the preparation of this manuscript.

	Footnotes

 
This work was supported by funding to D.C.E. from the Grants to Enhance and Advance Research (GEAR) program of the University of Houston and funding to K.M.S. from the Texas Advanced Research Program (003652-0182-2001) and the National Institute on Drug Abuse at the National Institutes of Health (DA17380).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.076042.

ABBREVIATIONS: AR, adrenoceptor; GPCR, G protein-coupled receptor; NE, norepinephrine; EPI, epinephrine bitartrate; GRK, G protein-coupled receptor kinase; OK, opossum kidney; PGE₁, prostaglandin E₁; DMEM, Dulbecco's modified Eagle's medium; [³H]RX821002, (1,4-[6,7(n)-³H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride; TEMED, N,N,N',N'-tetramethylethylenediamine; GAPDH, glyceraldehyde phosphate dehydrogenase; OR, -opioid receptor.

Address correspondence to: Dr. Douglas C. Eikenburg, Associate Professor and Chair, 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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