Heterogeneity in beta -Adrenergic Receptor Kinase Expression in the Lung Accounts for Cell-specific Desensitization of the beta 2-Adrenergic Receptor*

(Received for publication, September 13, 1996, and in revised form, November 26, 1996)

Dennis W. McGraw and Stephen B. Liggett Dagger

From the Departments of Medicine (Pulmonary) and Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0564

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The principal mechanism of homologous desensitization of the beta -adrenergic receptor (beta 2AR) is phosphorylation of the receptor by the beta AR kinase (beta ARK) or other closely related G protein-coupled receptor kinases (GRKs). However, within a single organ such as the lung where many cell types express the receptor, the presence or extent of beta 2AR desensitization in different cells has been noted to be highly variable. We hypothesized that such variability in desensitization is due to significant cell-type differences in beta ARK expression and/or function. To approach this, in situ hybridization was carried out in the lung and indeed revealed heterogeneity in beta ARK gene expression. Quantitative studies using ribonuclease protection assays with cell lines revealed that the level of beta ARK mRNA in airway smooth muscle cells was ~20% of that in bronchial epithelial cells and ~11% of that in mast cells (6.65 ± 0.96 versus 32.6 ± 4.0 and 60.7 ± 1.5 relative units, respectively, p < 0.001). beta ARK2 gene expression was not detected in any of these cells. At the protein level, beta ARK expression in airway smooth muscle cells was nearly undetectable, being ~10-fold less than that expressed on mast cells. The activities of the GRKs in cell extracts were assessed in vitro by quantitating their ability to phosphorylate rhodopsin in the presence of light. Consistent with the gene and protein expression results, a marked discrepancy in activities was observed between extracts derived from mast cells (90.7 ± 0.5 relative units) as compared to airway smooth muscle cells (9.28 ± 0.6 relative units, p < 0.001). In contrast, the activities of protein kinase A (the other kinase that phosphorylates beta 2AR) in these extracts were not different. We predicted, then, that airway smooth muscle beta 2AR would undergo minimal short-term (5 min) agonist-promoted desensitization as compared to the beta 2AR expressed on mast cells. Mast cell cAMP reached maximal levels after 90 s and did not further increase over time, indicative of receptor desensitization in this cell. In contrast, cAMP levels of airway smooth muscle cells did not plateau, increasing at a rate of 103 ± 9% per min, consistent with little desensitization over the study period. We conclude that there is significant cell-type variation in expression of beta ARK and that such variation is directly related to the extent of short-term agonist-promoted desensitization of the beta 2AR.


INTRODUCTION

Many G protein-coupled receptors display a waning of signal transduction during continuous activation. This phenomenon, termed desensitization, is an important component in maintaining homeostasis under normal physiologic conditions, may contribute to or act to compensate in pathologic states, and may limit the effectiveness of therapeutic agonists (1-3). Of the G protein-coupled receptors, desensitization of the beta 2-adrenergic receptor (beta 2AR)1 has been one of the most extensively studied (2, 3). Agonist-promoted desensitization of beta 2AR has been demonstrated in in vitro reconstituted systems, a variety of naturally and recombinantly expressing cell lines, and in intact animals. The earliest component of agonist-promoted desensitization of the beta 2AR is phosphorylation of the receptor by a cAMP independent kinase, termed the beta AR kinase (beta ARK).2 Such phosphorylation ultimately results in partial uncoupling of the agonist-occupied form of the receptor from the stimulatory guanine nucleotide-binding protein Gs, thereby limiting receptor function. Over the past few years, it has become clear that beta ARK is one of several related kinases that serve to phosphorylate the agonist-occupied forms of a number of G protein-coupled receptors (4, 5). This family of kinases, termed G protein-coupled receptor kinases (GRKs) consist of the following mammalian isoforms: rhodopsin kinase (GRK1), beta ARK (GRK2), beta ARK2 (GRK3), GRK4 (initially termed IT-11), and two kinases denoted as GRK5 and GRK6 (6-11). The potential for these other kinases to phosphorylate beta 2AR has only been explored to a limited extent and their roles in agonist-promoted desensitization of the receptor at the cellular level are not well established. On the other hand, multiple lines of evidence (see "Discussion") have definitively shown that beta ARK mediated phosphorylation represents a key process in homologous desensitization of the receptor.

While desensitization in the aforementioned model systems has been largely internally consistent, physiologic studies evaluating a variety of responses suggest that desensitization of beta 2AR may not occur at all, or to the same extent, in different organs or tissues. For example, repetitive administration of beta 2AR agonists to asthmatics appears to result in desensitization of responses thought to be mediated by the pulmonary mast cell beta 2AR, but not the bronchodilatory response of beta 2AR expressed on bronchial smooth muscle (Ref. 12 and reviewed in Ref. 13). One potential explanation for the apparent cell-type differences in beta 2AR desensitization is a heterogeneity in the expression of GRKs, such as beta ARK, which play critical roles in the desensitization process. The possibility of cell-specific expression of beta ARK within an organ has been largely unexplored, except for a single report in brain (14). That differences in beta ARK expression can indeed alter beta AR signal transduction has been demonstrated in recombinant cells overexpressing beta ARK (15) and in transgenic mice overexpressing beta ARK in the heart (16).

In the current study, we initially examined beta ARK gene expression in the lung and found significant differences in expression among different cell types. This was then further explored by quantitating beta ARK mRNA and protein expression in three human cell lines representing physiologically relevant functions in the lung: bronchial epithelial cells, mast cells, and airway smooth muscle cells. The BEAS-2B cell line was derived from normal human bronchial epithelial cells transformed by infection with Ad12-SV40 virus (17). BEAS-2B cells have the characteristics of epithelial cells by light and electron microscopy and stain positively for keratin (17). Furthermore, BEAS-2B cells have a physiologic number of beta 2AR comparable to that of freshly dissociated human bronchial epithelial cells (18). The human mast cell line HMC-1 was derived from a patient with mast cell leukemia (19) and is the only established cell line that is phenotypically similar to normal human mast cells. On the basis of neutral protease contents (HMC-1 cells contain tryptase but not chymase) and other markers, HMC-1 cells resemble the MCT subset of human mast cells (20) which corresponds to the lung mast cell (21). The airway smooth muscle line is a primary culture of smooth muscle obtained at autopsy from an individual without lung disease. These cells maintain their morphologic characteristics over several passages (22). Marked differences in expression and activity of beta ARK were indeed observed, particularly between mast cells and airway smooth muscle, which was correlated to the extent of agonist-promoted desensitization.


EXPERIMENTAL PROCEDURES

Cell Culture

BEAS-2B cells were maintained in Ham's F-12 medium supplemented with 10% fetal calf serum, 5 µg/ml insulin, 25 ng/ml epidermal cell growth factor, 5 µg/ml human transferrin, 1 µg/ml hydrocortisone, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C, 5% CO2. Primary cultures of human airway smooth muscle (HASM) cells, prepared as described previously (22), were grown as monolayers in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C, 5% CO2. HMC-1 cells were maintained as suspension cultures in RPMI medium supplemented with 10% fetal calf serum and 2 mM L-glutamine at 37 °C, 5% CO2.

RNA Probes

A template for the synthesis of beta ARK riboprobes was prepared by subcloning a 295-base pair EcoRI-SacI restriction fragment from the human beta ARK cDNA (23) into pGEM 4Z (Promega). For beta ARK2, an AvaI-HindIII restriction fragment from the bovine cDNA (8) was subcloned into the same sites of pGEM 4Z. Orientation of subcloned fragments was confirmed by restriction analysis and dideoxy sequencing. Plasmids were linearized with the appropriate restriction enzyme, and in vitro transcription reactions were carried out with either T7 or SP6 RNA polymerase to generate antisense or sense RNA probes. The probes were labeled with 35S-UTP for in situ hybridization experiments and [32P]UTP for ribonuclease protection assays.

In Situ Hybridization

Frozen sections from cryoprotected, paraformaldehyde-fixed monkey lungs were subjected to in situ hybridization (24). Cryosections were rehydrated, treated with proteinase K, fixed with 4% paraformaldehyde, acetylated with acetic anhydride, and dehydrated. Sections were then incubated overnight at 52 °C with hybridization solution that contained 5-7 × 104 cpm/µl of sense or antisense beta ARK riboprobe labeled with 35S-UTP. The sections were washed under high stringency conditions, treated with RNase A, subjected to a second high stringency wash, and dehydrated. Dried slides were dipped in Kodak NTB-2 autoradiographic emulsion and exposed at 4 °C for 8 weeks.

RNA Analysis

Expression of beta ARK and beta ARK2 mRNA was measured by ribonuclease protection assays (25). Total cellular RNA was isolated from BEAS-2B, HASM, and HMC-1 cells by the rapid acid guanidinium thiocyanate-phenol-chloroform method (26). Ribonuclease protection assays were performed as previously reported (27) using 20 µg of total RNA and the riboprobes described above. The reaction products were separated by electrophoresis on 6% polyacrylamide gels containing 8 M urea. Radiographic bands were visualized with a PhosphorImager (Molecular Dynamics) and conventional autoradiography. Quantitation of the phosphorimage was performed with the ImageQuant software package (Molecular Dynamics).

Western Analysis

The detection of beta ARK protein in whole cell lysates was carried out by standard SDS-PAGE and immunoblotting techniques as described previously (28). The cells were washed with phosphate-buffered saline and homogenized in hypotonic lysis buffer (5 mM Tris, pH 7.4, 2 mM EDTA, containing the protease inhibitors aprotinin (10 µg/ml), benzamidine (5 µg/ml), and soybean trypsin inhibitor (5 µg/ml)). The homogenates were diluted with 2 × Laemmli sample buffer, subjected to 10% SDS-PAGE, and electroblotted onto a nitrocellulose membrane (Protran, Schleicher & Schull). The membranes were blocked with 5% nonfat dry milk in Tris saline buffer (50 mM Tris, pH 7.4, 200 mM NaCl) containing 0.1% Tween, and then incubated with a monoclonal antibody raised against purified beta ARK diluted 1:200 in the blocking buffer. After washing with Tris saline/Tween buffer, the filters were incubated with an anti-mouse horseradish peroxidase-conjugated second antibody and developed using enhanced chemiluminescence (DuPont NEN). The radiographic film was scanned and quantitated using Scan Analysis (Biosoft).

Bovine Rod Outer Segments (ROS) Phosphorylation Assay

Cytosolic beta ARK activity was measured by phosphorylation of rhodopsin derived from rod outer segments (7). Urea-treated ROS were prepared from dark-adapted calf retinas by stepwise sucrose gradient centrifugation (29). The ROS consisted of ~90% rhodopsin as assessed by Coomassie Blue staining and had no significant endogenous kinase activity. To prepare cell lysates, HASM and HMC-1 cells were homogenized in 10 mM Tris, pH 7.4, 5 mM EDTA buffer containing the aforementioned protease inhibitors. The homogenates were centrifuged at 100,000 × g for 30 min, and the supernatants were concentrated with a Centricon 10 cartridge (Amicon). For each phosphorylation reaction, 15 µg of cytosolic proteins were incubated with 85 pmol of rhodopsin in buffer containing 20 mM Tris, pH 7.4, 2 mM EDTA, 6 mM MgCl2, and 100 µM [gamma -32P]ATP (~2,000 cpm/pmol). Incubations were carried out in the presence of light for 30 min at 30 °C. Reactions were stopped by the addition of 1 ml of cold 100 mM sodium phosphate, pH 7.0, 5 mM EDTA buffer and centrifuged at 100,000 × g for 30 min. The pellets were resuspended in Lammeli sample buffer and subjected to 10% SDS-PAGE. Dried gels were exposed on a PhosphorImager for quantitation. Light dependence of the phosphorylation reaction was confirmed using purified beta ARK as a control (data not shown). One micromolar of the beta ARK inhibitor heparin (30), 1 µM of a protein kinase A inhibitor peptide (31), or 1 µM of a protein kinase C inhibitor peptide (32) were added to some reactions to confirm that rhodopsin phosphorylation was beta ARK-dependent.

PKA Activity

An assay that measures the phosphorylation of Kemptide (33), a peptide substrate for PKA, was used to determine the phosphotransferase activity of PKA in extracts from HASM and HMC-1 cells. Cell lysates, prepared as described above, were incubated in a reaction mixture that contained 20 mM MOPS, pH 7.2, 25 mM beta -glycerol phosphate, 5 mM EGTA, 1 mM sodium vanadate, 15 mM MgCl2, 125 µM [gamma -32P]ATP (~4,000 cpm/pmol), and 125 µM Kemptide for 10 min at 30 °C. The reactions were stopped by spotting the assay mixture onto P81 phosphocellulose paper. The filters were washed three times with 0.75% phosphoric acid and once with acetone. Bound radioactivity was measured by liquid scintillation counting. PKA activity was defined as the amount of phosphate incorporated in the presence of 5 µM PKC inhibitor peptide (32).

Radioligand Binding

Membranes from HASM and HMC-1 cells were prepared and radioligand binding carried out as described previously (22, 34). Briefly, cells were washed three times with cold phosphate-buffered saline, resuspended in hypotonic lysis buffer (5 mM Tris, 2 mM EDTA, pH 7.4) containing the aforementioned protease inhibitors, disrupted with a Polytron (Brinkman) homogenizer, and centrifuged at 40,000 × g for 10 min at 4 °C. The resulting pellets were resuspended in 10 volumes of lysis buffer, centrifuged again, and resuspended in assay buffer (75 mM Tris, 12.5 mM MgCl2, 2 mM EDTA, pH 7.4). To determine total receptor density, membranes were incubated in a total volume of 250 µl at room temperature for 120 min with concentrations of 125I-CYP ranging from 3.125 to 400 pM. Nonspecific binding was determined in the presence of 1 µM propranolol. Assays were stopped by dilution with cold wash buffer (10 mM Tris, pH 7.4) and vacuum filtration through Whatmann GF/C glass fiber filters. The bound radioactivity was measured with a gamma -counter. Competition experiments with 40 pM 125I-CYP and varying concentrations of ICI 118,551 or CGP 20712A were also performed using the conditions described above to determine the proportion beta 2AR as compared to other beta AR subtypes expressed on the membranes. Protein concentration was determined by the copper-bicinchoninic acid method (35) with bovine serum albumin used as the standard. Data from the saturation binding and competition experiments were analyzed by nonlinear least squares techniques using the Prism software program (GraphPad). Curves were modeled to a one-site fit unless the two-site fit was significantly better (p < 0.5 by F test).

cAMP Assays

cAMP content of HASM and HMC-1 cells was measured by an acetylated radioimmunoassay method. All cells were treated with 0.1 mM isobutylmethylxanthine for 30 min at 37 °C, washed, and incubated in 500 µl of serum-free media at 37 °C in the presence or absence of 1 µM isoproterenol or 100 µM forskolin for the indicated times. Reactions were stopped every 30 s by the addition of 50 µl of 1.0 M HCl. The cAMP formed was acetylated and measured by radioimmunoassay using a polyclonal anti-cAMP antibody as described (36). Results were expressed as a percentage of the nonstimulated values.

Materials

Tissue culture supplies were purchased from JRH Biosciences. Radioisotopes were from DuPont NEN. ICI 118,551 was purchased from Research Biochemicals Int. CGP 20712A was a gift from Ciba Geigy. Heparin was from Sigma. PKA and PKC inhibitor peptides and Kemptide were from Upstate Biotechnology Inc. HMC-1 cells were obtained from J. Butterfield, Mayo Clinic, Rochester, MN. BEAS-2B cells were provided by C. Harris, National Institutes of Health, Bethesda, MD. GRK cDNAs, purified beta ARK, and the beta ARK antibody were provided by J. Benovic, Thomas Jefferson University, Philadelphia, PA.


RESULTS

To examine the possibility that beta ARK is differentially expressed among different cell types within the lung, we performed in situ hybridization experiments using cryosections of monkey lung (Fig. 1). Panel A, a phase-contrast micrograph depicts the cellular architecture in a region of a moderate sized bronchus of the lung with some adjacent alveoli. Hybridization with the antisense beta ARK probe gave a specific signal that was greatest over bronchial epithelial cells and cells lining the alveolar space (Fig. 1B). A minimal signal was also present over bronchial smooth muscle. No specific signal was detected when sections were hybridized with the sense probe (data not shown).


Fig. 1. Distribution of beta ARK mRNA in the lung by in situ hybridization. Serial lung sections from an adult macaque monkey are shown. Slides were exposed for 8 weeks. Panel A is a phase-contrast photomicrograph of a section hybridized with the beta ARK antisense probe. Bronchial epithelium (EP), airway smooth muscle (SM), cartilage (CT), and alveoli (AL) are identified. (The section is positioned with the lumen of the airway (AW) on the right.) Panel B is a dark-field photomicrograph of the section in panel A. Grain density is heaviest over airway epithelium and alveolar cells with fewer grains noted over smooth muscle. No specific binding was observed when sections were hybridized with the sense probe (not shown).
[View Larger Version of this Image (90K GIF file)]


To assess beta ARK expression in a more quantitative fashion, we performed ribonuclease protection assays using RNA from three human cell types that are physiologically relevant to lung function and are targets for therapeutic beta -agonists: airway epithelial cells (BEAS-2B), airway smooth muscle cells (HASM), and mast cells (HMC-1). Although a band corresponding to the 295-base pair fragment expected for beta ARK mRNA could be detected in all three cell types, there was a substantial difference in the level of expression among the different cell types (Fig. 2A). Analysis of the gels showed that beta ARK mRNA content in HASM cells (6.65 ± 0.96 relative units) was only ~11% of that in HMC-1 cells (60.7 ± 1.5 relative units, n = 4, p < 0.001) and ~17% of that in epithelial cells (32.6 ± 4.1 relative units, n = 4, p < 0.001). We also used ribonuclease protection assays to assess beta ARK2 gene expression in these cell lines. Although a strong band was observed in the positive control, no significant signal for beta ARK2 was detected in BEAS-2B, HASM, or HMC-1 cells (Fig. 2B). We subsequently focused our experiments on the role of beta ARK in regulating beta 2AR desensitization in these cell types.


Fig. 2. Quantitative analysis of beta ARK and beta ARK2 mRNA expression in BEAS-2B, HASM, and HMC-1 cells. Ribonuclease protection assays were performed using 20 µg of total RNA and the described beta ARK or beta ARK2 antisense probe. A, a representative autoradiogram using the beta ARK antisense probe. Full-length probe is on the left. beta ARK mRNA was detected in all three cell lines but the signal intensity from HASM cells was significantly less than that of HMC-1 and BEAS-2B cells. The data from three independent experiments are summarized in the bar graph (mean ± S.E.). Relative units represent the pixel density of each sample from the phosphorimage expressed as a percentage of the total pixel density for all samples on the gel. *, p < 0.001 compared to HASM cells. B, a representative autoradiogram using the beta ARK2 antisense probe. Full-length, undigested probe is on the left. A strong signal was noted from a sample of mRNA from COS-7 cells transfected with an expression plasmid encoding bovine beta ARK2, which was used as a positive control. However, no significant signal was observed in BEAS-2B, HASM, or HMC-1 cells.
[View Larger Version of this Image (23K GIF file)]


We next measured beta ARK protein content by Western blotting. A distinct band with a molecular mass of ~80 kDa corresponding to beta ARK was detected in BEAS-2B, HASM, and HMC-1 cell extracts (Fig. 3). beta ARK levels were lowest in HASM cells (6.2 ± 1.3 relative units), as compared to that of HMC-1 cells (56.5 ± 3.5 relative units, n = 3, p < 0.001) and that of BEAS-2B cells (37.3 ± 5.0 relative units, n = 3, p < 0.001). Thus beta ARK protein expression closely paralled mRNA levels in the three subtypes, with HASM cells expressing about one-tenth the level of beta ARK as compared to HMC-1 cells and one-fifth of that of BEAS-2B cells.


Fig. 3. Western analysis of beta ARK expression in BEAS-2B, HASM, and HMC-1 cells. 20 µg of protein isolated from whole cells were subjected to Western analysis using a monoclonal antibody directed against beta ARK. A shows a representative blot. The signal in HASM cells is faint compared to that for BEAS-2B and HMC-1 cells. The data from three independent experiments (mean ± S.E.) are summarized in B. Relative pixel density was derived from the analysis of scanned autoradiograms as described for ribonuclease protection assays. *, p < 0.001 compared to HASM cells.
[View Larger Version of this Image (20K GIF file)]


To confirm the above observations and to determine if increased beta ARK content resulted in increased kinase activity in vitro, we measured the ability of extracts from HASM and HMC-1 cells to phosphorylate ROS (Fig. 4). Initial studies were carried out to assure the specificity of the reactions for assessing GRK-mediated phosphorylation by the use of purified bovine beta ARK and various inhibitors. Using purified beta ARK, light dependence of ROS phosphorylation was demonstrated (data not shown). beta ARK-dependent phosphorylation was then assessed by carrying out reactions in the presence of heparin (1 µM), a PKA inhibitor peptide (1 µM), or a PKC inhibitor peptide (1 µM). ROS phosphorylation was blocked by the beta ARK inhibitor heparin in both cell types, whereas inhibitors of PKC and PKA had no effect (Fig. 4A). Using this in vitro assay, we determined the activities of extracts from both cell types (Fig. 4B). As can be seen, there were marked differences in activities, with the kinase activity of HMC-1 cells being nearly 10-fold greater (90.7 ± 0.6 relative units) than that of HASM cells (9.2 ± 0.6 relative units, n = 4, p < 0.001). We also assessed protein kinase A activities in these extracts since this kinase also phosphorylates beta 2AR in response to elevated cAMP, and any cell-type differences might confound interpretation of functional desensitization studies. In contrast to the marked differences in beta ARK activities between the two cell types, PKA activities from extracts of HASM and HMC-1 cells were not different (Fig. 5).


Fig. 4. In vitro assessment of beta ARK activity in HASM and HMC-1 cells. ROS phosphorylation assays were performed using cytosolic proteins (15 µg) from HASM and HMC-1 cells. A shows that phosphorylation of ROS by both cell types was inhibited by heparin, an inhibitor of beta ARK, whereas inhibitors of PKC and PKA had no effect. B, a representative autoradiogram illustrating the marked difference in activity between HASM and HMC-1 cells. Purified beta ARK (10 ng) was used as a positive control. C, summary of data from four experiments comparing activities between HASM and HMC-1 cells (mean ± S.E.). Quantitative analysis of gels was performed with a PhosphorImager as described under "Experimental Procedures." *, p < 0.001 compared to HASM cells.
[View Larger Version of this Image (15K GIF file)]



Fig. 5. PKA activity in HASM and HMC-1 cells. PKA activity was defined as the phosphorylation of Kemptide in the presence of a PKC inhibitor peptide. Results are reported as the mean ± S.E. of three experiments. PKA activity of HASM and HMC-1 cells was not significantly different.
[View Larger Version of this Image (17K GIF file)]


The results of the above experiments clearly showed that beta ARK was differentially expressed among lung cells, and that increased beta ARK content was associated with increased activity in vitro. We speculated that beta 2AR from cells with the highest levels of beta ARK activity might be most subject to desensitization. We thus compared short-term desensitization in HASM cells, which had low levels of beta ARK, to HMC-1 cells which we found to have substantially higher levels of beta ARK. We have previously shown that the beta AR of the HASM cells consists entirely of the beta 2AR subtype (22). The beta AR of HMC-1 cells has not been previously characterized. Briefly, we found that the beta AR radioligand 125I-CYP bound to a population with high affinity (22.7 ± 1.8 pM, n = 3). In competition studies with subtype-specific antagonists, these cells were found to express a single population of beta AR with a high affinity for ICI 118,551, consistent with this receptor being of the beta 2AR subtype. Expression of the beta 2AR in these HMC-1 cells as measured in saturation binding experiments was 8.5 ± 1.3 fmol/mg of protein (n = 3). We then assessed agonist-promoted desensitization in HASM and HMC-1 cells using a previously described intact cell paradigm, where the kinetics of cAMP accumulation were determined following exposure to 1 µM isoproterenol (36). cAMP levels were determined every 30 s for 5 min. In HMC-1 cells, cAMP levels initially increased but reached a maximum within 90 s, and the levels remained relatively constant (rate = -7.9 ± 4.3% per min) for the remaining 210 s, indicative of agonist promoted desensitization of the beta 2AR on this cell (Fig. 6). In marked contrast, HASM cell cAMP continued to increase at a rate of 103 ± 9.7%/min throughout the course of the study, reflective of substantially less desensitization of these receptors as compared to those of HMC-1 cells (Fig. 6). Over a similar time period, the cAMP responses to forskolin were found to be linear for both HMC-1 cells (r2 = 0.93 ± 0.02, n = 3) and bronchial smooth muscle cells (r2 = 0.97 ± 0.01, n = 3), pointing toward the desensitization of the isoproterenol response observed in the former cells to be receptor specific.


Fig. 6. Time course of isoproterenol-stimulated cAMP production in HASM and HMC-1 cells. Following treatment with 1 mM isobutylmethylxanthine for 30 min, cells were incubated in 0.5 ml of serum-free media containing 0.1 mM ascorbic acid, 1 mM isobutylmethylxanthine, and 1 µM isoproterenol. Reactions were stopped at the indicated times by addition of HCl, and cAMP was measured by radioimmunoassay. As is shown, after 90 s of agonist exposure cAMP levels of HMC-1 cells plateaued (rate = -7.9 ± 4.3% per min from 90 s to 300 s), indicative of receptor desensitization. In contrast, cAMP levels continued to increase in HASM cells (rate = 103 ± 9.7% per min) consistent with little desensitization over the time period studied. Data shown are the mean ± S.E. from four independent experiments each performed in duplicate.
[View Larger Version of this Image (17K GIF file)]



DISCUSSION

During continuous exposure of beta 2AR to agonist, a number of regulatory events occur which act to limit the cellular responsiveness. The most rapid process (seconds to minutes) is phosphorylation of the receptor by beta ARK (and potentially other GRKs) leading to the binding of an arrestin-like moiety (termed beta -arrestin) which results in depressed coupling to Gs (2, 3). Phosphorylation of the receptor also occurs via protein kinase A, whenever intracellular cAMP is increased due to receptor activation by agonist, or by other means. After more prolonged agonist exposure, an internalization of receptors occurs which results in a loss of some proportion of cell surface receptors. This process, termed sequestration, has also been considered to be another mechanism of desensitization, but recent studies have suggested that its major role in short-term regulation of the receptor may be in resensitization (37, 38), since it appears that the sequestered pool is the site of dephosphorylation of the receptor. After hours of agonist exposure, a net loss of cellular receptors occurs (denoted down-regulation) via several mechanisms that are independent of receptor phosphorylation.

The role of beta ARK-mediated phosphorylation of beta 2AR in short-term agonist-promoted desensitization has been elucidated using multiple approaches (15, 16, 36, 39-42). In recombinant cell lines, mutated beta 2AR lacking beta ARK phosphorylation sites display attenuated agonist-promoted desensitization as assessed in intact cell (36) and membrane (39) based assays. In addition, treatment of permeabilzed cells with the beta ARK inhibitor heparin results in a loss of receptor desensitization and phosphorylation (40, 41), and expression of a dominant-negative beta ARK in cells that natively express beta 2AR inhibits agonist-promoted desensitization (42). Overexpression of beta ARK has been found to enhance agonist-promoted desensitization and phosphorylation in Chinese hamster ovary cells overexpressing beta 2AR (15). While in vivo agonist-promoted desensitization was not assessed per se, beta AR of cardiac membranes from transgenic mice expressing a dominant-negative beta ARK display increased coupling, while receptors in transgenic mice overexpressing beta ARK display decreased coupling (16). Although some in vitro studies have been carried out with the beta 2AR and other known GRKs, little is known regarding their potential for mediating desensitization of the receptor as assessed in studies such as discussed above.

While a loss of signaling via G protein-coupled receptors during continuous exposure to agonist has been observed with many members of the superfamily, some receptors do not demonstrate the phenomenon, including those that share the same endogenous agonist and signal transduction pathways. For example, the beta 2AR undergoes rapid agonist-promoted desensitization, but the beta 3AR appears to be relatively resistant to such regulation (43). Similarly, the human alpha 2AAR displays desensitization after brief exposure to agonist, while the human alpha 2C subtype does not (44, 45). In both of the above instances, the lack of desensitization is paralleled by a lack of phosphorylation by GRKs. Mutagenesis studies have delineated the structural determinants within the intracellular regions that define GRK phosphorylation sites within these, and other, receptors (39, 43, 46, 47). Thus one way in which cell-specific desensitization of agonist responsiveness occurs is by selective expression of certain receptor subtypes. Another potential component which may dictate the presence or absence of short-term desensitization by agonist is the level of GRKs expressed in a given cell. We considered that if such heterogeneity of GRK expression was indeed present, the responsiveness of cell-specific signaling to agonist might differ markedly in an organ populated by multiple cell types even though the same receptor subtype is present on the cells. Whether differences in GRK gene expression occur in cell types of a given organ, and whether such correlate with differences in protein expression, kinase activity, and agonist-promoted desensitization of the beta 2AR has not been explored.

We approached this issue by examining beta ARK gene and protein expression, kinase activity, and beta 2AR signal transduction in lung cells. We utilized lung for several reasons. First, this organ has a large number of different cell types, many expressing exclusively the beta 2AR as compared to other beta AR subtypes. Second, in vivo the beta 2AR of these different cell types have clearly defined physiologic functions: bronchial epithelial cell receptors regulate ciliary beat frequency, airway smooth muscle cell receptors regulate relaxation of bronchial smooth muscle, and mast cell receptors regulate inflammatory mediator release (13). These functions have provided for distinct signals in the assessment of the relevance of beta 2AR desensitization (or lack thereof) in these cell types in vivo. Finally, beta 2AR agonists delivered by inhalation are used for the treatment of bronchospasm, which has afforded others the opportunity for studying desensitization in a physiologically relevant setting.

Our initial approach was to screen for gene expression of beta ARK in the lung using in situ hybridization. These results indeed showed a paucity of expression in smooth muscle as compared to epithelial cells. This difference is not simply a reflection of differences in beta 2AR expression, since the receptor densities are similar between the two cell types (22, 48). We then explored quantitatively the expression of beta ARK, and the closely related isoform beta ARK2, in three cell lines. For beta ARK, airway smooth muscle mRNA was clearly less than that of mast cells and bronchial epithelial cells. (beta ARK2 transcripts could not be detected in any of the three lines.) Consistent with these results, expression of beta ARK as assessed using Western blots indicated a significant difference in expression in the cell lines, with mast cell > epithelial cell >> > smooth muscle cell. We then further studied the two cell types with the greatest difference in beta ARK expression, the mast cell and the smooth muscle cell. To assess the activity of beta ARK (and potentially other GRKs) in the two cell lines, extracts were used to phosphorylate rhodopsin in ROS. These studies revealed ~10% activity in smooth muscle cells as compared to mast cells. These results were remarkably similar to those assessing beta ARK expression, where smooth muscle cells were found to express beta ARK at only ~11% of that in mast cells. Based on our hypothesis, then, we expected that the two cell types would exhibit differences in the extent of agonist-promoted desensitization. To approach this, cells were exposed to a saturating concentration (1 µM) of isoproterenol, and cAMP accumulation determined every 30 s for 5 min. Such an approach has been verified with mutant beta 2AR lacking phosphorylation sites (36) and with a dominant-negative beta ARK (42). As shown in Fig. 6, the smooth muscle cells continued to accumulate cAMP over time during exposure to agonist, consistent with little desensitization. In contrast, cAMP accumulation in mast cells had a rapid onset, but the accumulation rate markedly decreased after ~90 s. As described previously (36), this plateau in cAMP levels represents receptor desensitization, which is clearly evident in these cells but not smooth muscle cells. The difference in susceptibility to beta 2AR desensitization did not appear to be due to differences in PKA, as the activity of this kinase was similar in both cell types.

Thus, we have demonstrated that there is significant variation in the expression of beta ARK between cell types of the lung, and that this variation correlates with the extent of agonist-promoted desensitization. This implies, that unlike some components involved in beta 2AR regulation in which their levels may be in excess, physiologic variation in the level of beta ARK may have a distinct effect on signal transduction. These findings also support functional studies in man examining in vivo desensitization of pulmonary beta 2AR during treatment with agonists in asthma. Most studies have observed desensitization of the so-called bronchoprotective effects of agonists (implying desensitization of mast cell receptors) while desensitization of the bronchodilatory effects (mediated by smooth muscle receptors) appears to be substantially less, if at all, present (12). Our findings also imply that pathologic conditions that result in changes in beta ARK levels may indeed result in alterations of receptor function. For example, in chronic heart failure, beta ARK mRNA levels are elevated (49), which is consistent with the impaired agonist-mediated receptor function in the heart that is observed in this disease. In addition, opiod-dependence is associated with increases in beta ARK in the locus ceruleus (50). The extents of the changes in beta ARK that have been reported in these types of studies appear to be of sufficient magnitude, based on our current results, to be potentially relevant.

Taken together, then, these studies indicate that the basis of cell-type specificity of agonist-promoted desensitization of the beta 2AR in natively expressing, physiologically relevant cells, can in part be ascribed to the level of expression of beta ARK. Furthermore, these studies reveal that beta ARK expression is indeed highly variable among different cell-types, and supports the concept that dynamic regulation of the kinase can significantly alter beta AR signal transduction. Finally, given that beta ARK phosphorylates other G protein-coupled receptors, it is likely that cellular variation in its expression is important to desensitization of other receptors as well.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants HL45967 and HL41496.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: University of Cincinnati Medical Center, P. O. Box 670564, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835.
1   The abbreviations are: beta AR, beta -adrenergic receptor; beta ARK, beta AR kinase; GRK, G protein-coupled receptor kinase; ROS, rod outer segments; 125I-CYP, 125I-cyanopindolol; PAGE, polyacrylamide gel electrophoresis; PKA, protein kinase A; MOPS, 4-morpholinepropanesulfonic acid; HASM, human airway smooth muscle.
2   In this report, beta ARK refers to EC 2.7.1.126, which has also been termed beta ARK1 and GRK2 (7, 51). beta ARK2 refers to a related isoform which is also known as GRK3 (8).

Acknowledgments

We gratefully appreciate the assistance of S. Wert, and the use of the facilities of J. Whitsett, for the in situ hybridization experiments. We also thank the following individuals who kindly provided the indicated reagents: J. Benovic (beta ARK cDNAs and antibody), J. Butterfield (HMC-1 cells), and C. Harris (BEAS-2B cells). We also acknowledge K. Gouge for assistance in preparation of the manuscript.


REFERENCES

  1. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  2. Liggett, S. B., and Lefkowitz, R. J. (1993) in Regulation of Cellular Signal Transduction Pathways by Desensitization and Amplification (Sibley, D., and Houslay, M., eds), pp. 71-97, John Wiley & Sons, London
  3. Liggett, S. B. (1996) in The Lung: Scientific Foundations (Crystal, R., West, J. B., Weibel, E. R., and Barnes, P. J., eds), Raven Press, New York
  4. Lefkowitz, R. J. (1993) Cell 74, 409-412 [Medline] [Order article via Infotrieve]
  5. Premont, R. T., Inglese, J., and Lefkowitz, R. J. (1995) FASEB J. 9, 175-182 [Abstract/Free Full Text]
  6. Lorenz, W., Inglese, J., Palczewski, K., Onorato, J. J., Caron, M. G., and Lefkowitz, R. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8715-8719 [Abstract]
  7. Benovic, J. L., Deblasi, A., Stone, W. C., Caron, M. G., and Lefkowitz, R. J. (1989) Science 246, 235-240 [Medline] [Order article via Infotrieve]
  8. Benovic, J. L., Onorato, J. J., Arriza, J. L., Stone, W. C., Lohse, M., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Caron, M. G., and Lefkowitz, R. J. (1991) J. Biol. Chem. 266, 14939-14946 [Abstract/Free Full Text]
  9. Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1992) Hum. Mol. Genet. 1, 697-703 [Abstract]
  10. Kunapuli, P., and Benovic, J. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5588-5592 [Abstract]
  11. Benovic, J. L., and Gomez, J. (1993) J. Biol. Chem. 268, 19521-19527 [Abstract/Free Full Text]
  12. O'Connor, B. J., Aikman, S., and Barnes, P. J. (1992) N. Engl. J. Med. 327, 1204-1208 [Abstract]
  13. Barnes, P. J. (1995) Am. J. Respir. Crit. Care Med. 152, 838-860 [Medline] [Order article via Infotrieve]
  14. Arriza, J. L., Dawson, T. M., Simerly, R. B., Martin, L. J., Caron, M. G., Snyder, S. H., and Lefkowitz, R. J. (1992) J. Neurosci. 12, 4045-4055 [Abstract]
  15. Pippig, S., Andexinger, S., Daniel, K., Puzicha, M., Caron, M. G., Lefkowitz, R. J., and Lohse, M. J. (1993) J. Biol. Chem. 268, 3201-3208 [Abstract/Free Full Text]
  16. Koch, W. J., Rockman, H. A., Samama, P., Hamilton, R. A., Bond, R. A., Milano, C. A., and Lefkowitz, R. J. (1995) Science 268, 1350-1353 [Medline] [Order article via Infotrieve]
  17. Reddel, K. Y., Gerwin, B. I., Miyashita, M., McMenamin, M., Lechner, J. F., and Harris, C. C. (1988) Differentiation 38, 60-66 [Medline] [Order article via Infotrieve]
  18. Penn, R. B., Kelsen, S. G., and Benovic, J. L. (1994) Am. J. Resp. Cell Mol. Biol. 11, 496-505 [Abstract]
  19. Butterfield, J. H., Weiler, D., Dewald, G., and Gleich, G. J. (1988) Leuk. Res. 12, 345-355 [Medline] [Order article via Infotrieve]
  20. Nilsson, G., Blom, T., Kusche-Gullberg, M., Kjellen, I., Butterfield, J. H., Sundstrom, C., Nilsson, K., and Hellman, L. (1994) Scand. J. Immun. 39, 489-498 [Medline] [Order article via Infotrieve]
  21. Irani, A. A., Schechter, N. M., Craig, S. S., DeBlois, G., and Schwartz, L. B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4464-4468 [Abstract]
  22. Green, S. A., Turki, J., Bejarano, P., Hall, I. P., and Liggett, S. B. (1995) Am. J. Resp. Cell Mol. Biol. 13, 25-33 [Abstract]
  23. Penn, R. B., and Benovic, J. L. (1994) J. Biol. Chem. 269, 14924-14930 [Abstract/Free Full Text]
  24. Wert, S. E., Glasser, S. W., Korfhagen, T. R., and Whitsett, J. A. (1993) Dev. Biol. 156, 426-443 [CrossRef][Medline] [Order article via Infotrieve]
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual (Ford, N., Nolan, C., Ferguson, M., and Ockler, M., eds), pp. 7.71-7.78, Cold Spring Harbor Laboratory Press, New York
  26. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  27. McGraw, D. W., Chai, S. E., Hiller, C., and Cornett, L. E. (1995) Exp. Lung Res. 21, 535-546 [Medline] [Order article via Infotrieve]
  28. Jewell-Motz, E. A., and Liggett, S. B. (1996) J. Biol. Chem. 271, 18082-18087 [Abstract/Free Full Text]
  29. Wilden, U., and Kuhn, H. (1982) Biochemistry 21, 3014-3022 [Medline] [Order article via Infotrieve]
  30. Benovic, J. L., Stone, W. C., Caron, M. G., and Lefkowitz, R. J. (1989) J. Biol. Chem. 264, 6707-6710 [Abstract/Free Full Text]
  31. Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810 [Abstract/Free Full Text]
  32. Smith, M. K., Colbran, R. J., and Soderling, T. R. (1990) J. Biol. Chem. 265, 1837-1840 [Abstract/Free Full Text]
  33. Whitehouse, S., Feramisco, J. R., Casnellie, J. E., Krebs, E. G., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3693-3701 [Abstract/Free Full Text]
  34. Green, S. A., and Liggett, S. B. (1994) J. Biol. Chem. 269, 26215-26219 [Abstract/Free Full Text]
  35. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85 [Medline] [Order article via Infotrieve]
  36. Liggett, S. B., Bouvier, M., Hausdorff, W. P., O'Dowd, B., Caron, M. G., and Lefkowitz, R. J. (1989) Mol. Pharmacol. 36, 641-646 [Abstract]
  37. Yu, S. S., Lefkowitz, R. J., and Hausdorff, W. P. (1993) J. Biol. Chem. 268, 337-341 [Abstract/Free Full Text]
  38. Pippig, S., Andexinger, S., and Lohse, M. J. (1995) Mol. Pharmacol. 47, 666-676 [Abstract]
  39. Hausdorff, W. P., Bouvier, M., O'Dowd, B. F., Irons, G. P., Caron, M. G., and Lefkowitz, R. J. (1989) J. Biol. Chem. 264, 12657-12665 [Abstract/Free Full Text]
  40. Lohse, M. J., Lefkowitz, R. J., Caron, M. G., and Benovic, J. L. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3011-3015 [Abstract]
  41. Lohse, M. J., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1990) J. Biol. Chem. 265, 3202-3209 [Abstract/Free Full Text]
  42. Kong, G., Penn, R., and Benovic, J. L. (1994) J. Biol. Chem. 269, 13084-13087 [Abstract/Free Full Text]
  43. Liggett, S. B., Freedman, N. J., Schwinn, D. A., and Lefkowitz, R. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3665-3669 [Abstract]
  44. Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479 [Abstract/Free Full Text]
  45. Kurose, H., and Lefkowitz, R. J. (1994) J. Biol. Chem. 269, 10093-10099 [Abstract/Free Full Text]
  46. Eason, M. G., Moreira, S. P., and Liggett, S. B. (1995) J. Biol. Chem. 270, 4681-4688 [Abstract/Free Full Text]
  47. Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796-13803 [Abstract/Free Full Text]
  48. Kelsen, S. G., Zhou, Z., Anakwe, O., Mardini, I., Higgins, N., and Benovic, J. L. (1994) Am. J. Physiol. 267, L456-L463 [Abstract/Free Full Text]
  49. Ungerer, M., Parruti, G., Bohm, M., Puzicha, M., Deblasi, A., Erdmann, E., and Lohse, M. J. (1994) Circ. Res. 74, 206-213 [Abstract]
  50. Terwilliger, R. Z., Ortiz, J., Guitart, X., and Nestler, E. J. (1994) J. Neurochem. 63, 1983-1986 [Medline] [Order article via Infotrieve]
  51. Benovic, J. L., Mayor, F., Jr., Staniszewski, E., Lefkowitz, R. J., and Caron, M. G. (1987) J. Biol. Chem. 262, 9026-9032 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.