(Received for publication, September 13, 1996, and in revised form, November 26, 1996)
From the Departments of Medicine (Pulmonary) and Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0564
The principal mechanism of homologous
desensitization of the -adrenergic receptor (
2AR) is
phosphorylation of the receptor by the
AR kinase (
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
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
ARK expression and/or
function. To approach this, in situ hybridization was
carried out in the lung and indeed revealed heterogeneity in
ARK
gene expression. Quantitative studies using ribonuclease protection
assays with cell lines revealed that the level of
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).
ARK2 gene
expression was not detected in any of these cells. At the protein
level,
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
2AR) in these extracts were not different. We predicted,
then, that airway smooth muscle
2AR would undergo
minimal short-term (5 min) agonist-promoted desensitization as compared
to the
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
ARK and that such
variation is directly related to the extent of short-term
agonist-promoted desensitization of the
2AR.
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 2-adrenergic receptor
(
2AR)1 has been one of the
most extensively studied (2, 3). Agonist-promoted desensitization of
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
2AR is
phosphorylation of the receptor by a cAMP independent kinase, termed
the
AR kinase (
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
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),
ARK (GRK2),
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
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
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 2AR may not
occur at all, or to the same extent, in different organs or tissues.
For example, repetitive administration of
2AR agonists to asthmatics appears to result in desensitization of responses thought
to be mediated by the pulmonary mast cell
2AR, but not the bronchodilatory response of
2AR expressed on
bronchial smooth muscle (Ref. 12 and reviewed in Ref. 13). One
potential explanation for the apparent cell-type differences in
2AR desensitization is a heterogeneity in the expression
of GRKs, such as
ARK, which play critical roles in the
desensitization process. The possibility of cell-specific expression of
ARK within an organ has been largely unexplored, except for a single
report in brain (14). That differences in
ARK expression can indeed
alter
AR signal transduction has been demonstrated in recombinant
cells overexpressing
ARK (15) and in transgenic mice overexpressing
ARK in the heart (16).
In the current study, we initially examined ARK gene expression in
the lung and found significant differences in expression among
different cell types. This was then further explored by quantitating
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
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
ARK
were indeed observed, particularly between mast cells and airway smooth
muscle, which was correlated to the extent of agonist-promoted
desensitization.
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 ProbesA template for the synthesis of ARK
riboprobes was prepared by subcloning a 295-base pair
EcoRI-SacI restriction fragment from the human
ARK cDNA (23) into pGEM 4Z (Promega). For
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.
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 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.
Expression of ARK and
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).
The detection of 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
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).
Cytosolic 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
[
-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
ARK as a
control (data not shown). One micromolar of the
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
ARK-dependent.
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 -glycerol phosphate, 5 mM EGTA, 1 mM sodium vanadate, 15 mM MgCl2,
125 µM [
-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).
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 -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
2AR as compared to other
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 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.
MaterialsTissue 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 ARK, and the
ARK antibody were provided by J. Benovic, Thomas Jefferson University, Philadelphia, PA.
To examine the possibility that 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
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).
To assess 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
-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
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
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
ARK2 gene expression in these cell lines. Although
a strong band was observed in the positive control, no significant
signal for
ARK2 was detected in BEAS-2B, HASM, or HMC-1 cells (Fig.
2B). We subsequently focused our experiments on the role of
ARK in regulating
2AR desensitization in these cell
types.
We next measured ARK protein content by Western blotting. A distinct
band with a molecular mass of ~80 kDa corresponding to
ARK was
detected in BEAS-2B, HASM, and HMC-1 cell extracts (Fig.
3).
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
ARK protein
expression closely paralled mRNA levels in the three subtypes, with
HASM cells expressing about one-tenth the level of
ARK as compared to HMC-1 cells and one-fifth of that of BEAS-2B cells.
To confirm the above observations and to determine if increased 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
ARK and
various inhibitors. Using purified
ARK, light dependence of ROS
phosphorylation was demonstrated (data not shown).
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
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
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
ARK activities
between the two cell types, PKA activities from extracts of HASM and
HMC-1 cells were not different (Fig. 5).
The results of the above experiments clearly showed that ARK was
differentially expressed among lung cells, and that increased
ARK
content was associated with increased activity in vitro. We
speculated that
2AR from cells with the highest levels
of
ARK activity might be most subject to desensitization. We thus compared short-term desensitization in HASM cells, which had low levels
of
ARK, to HMC-1 cells which we found to have substantially higher
levels of
ARK. We have previously shown that the
AR of the HASM
cells consists entirely of the
2AR subtype (22). The
AR of HMC-1 cells has not been previously characterized. Briefly, we
found that the
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
AR with a high affinity for ICI 118,551, consistent with this
receptor being of the
2AR subtype. Expression of the
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
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.
During continuous exposure of 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
ARK (and potentially other GRKs)
leading to the binding of an arrestin-like moiety (termed
-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 ARK-mediated phosphorylation of
2AR in
short-term agonist-promoted desensitization has been elucidated using multiple approaches (15, 16, 36, 39-42). In recombinant cell lines,
mutated
2AR lacking
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
ARK inhibitor heparin results in a loss
of receptor desensitization and phosphorylation (40, 41), and
expression of a dominant-negative
ARK in cells that natively express
2AR inhibits agonist-promoted desensitization (42).
Overexpression of
ARK has been found to enhance agonist-promoted desensitization and phosphorylation in Chinese hamster ovary cells overexpressing
2AR (15). While in vivo
agonist-promoted desensitization was not assessed per se,
AR of cardiac membranes from transgenic mice expressing a
dominant-negative
ARK display increased coupling, while receptors in
transgenic mice overexpressing
ARK display decreased coupling (16).
Although some in vitro studies have been carried out with
the
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 2AR undergoes
rapid agonist-promoted desensitization, but the
3AR appears to be relatively resistant to such regulation (43). Similarly,
the human
2AAR displays desensitization after brief exposure to agonist, while the human
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
2AR has not been explored.
We approached this issue by examining ARK gene and protein
expression, kinase activity, and
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
2AR as compared to other
AR subtypes. Second,
in vivo the
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
2AR desensitization (or lack thereof) in these cell
types in vivo. Finally,
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 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
2AR expression, since the receptor
densities are similar between the two cell types (22, 48). We then
explored quantitatively the expression of
ARK, and the closely
related isoform
ARK2, in three cell lines. For
ARK, airway smooth
muscle mRNA was clearly less than that of mast cells and bronchial
epithelial cells. (
ARK2 transcripts could not be detected in any of
the three lines.) Consistent with these results, expression of
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
ARK expression, the mast cell and
the smooth muscle cell. To assess the activity of
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
ARK expression, where smooth
muscle cells were found to express
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
2AR lacking phosphorylation sites (36) and with a dominant-negative
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
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 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
2AR regulation in which their levels may be in excess,
physiologic variation in the level of
ARK may have a distinct effect
on signal transduction. These findings also support functional studies
in man examining in vivo desensitization of pulmonary
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
ARK levels may indeed result in
alterations of receptor function. For example, in chronic heart
failure,
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
ARK in the locus ceruleus (50). The
extents of the changes in
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
2AR in natively expressing, physiologically relevant
cells, can in part be ascribed to the level of expression of
ARK.
Furthermore, these studies reveal that
ARK expression is indeed
highly variable among different cell-types, and supports the concept
that dynamic regulation of the kinase can significantly alter
AR
signal transduction. Finally, given that
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.
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
(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.