(Received for publication, July 27, 1995; and in revised form, October 2, 1995)
From the
A adenosine receptor (A
AR) activation
contributes to both the cardioprotective and antihypertensive effects
of adenosine. To date, no studies have examined the mechanisms by which
this receptor undergoes rapid homologous desensitization. Therefore, a
functional hemagglutinin epitope-tagged A
AR has been stably
expressed in Chinese hamster ovary cells, and its regulation by the AR
agonist 5`-N-ethylcarboxamidoadenosine (NECA) has been
studied. Cellular exposure to NECA induces rapid (t
=
1 min) A
AR phosphorylation on
serine and threonine residues. This is associated with a functional
desensitization and a 30-40% reduction in the number of high
affinity agonist binding sites as determined by radioligand binding
assays. Activation of second messenger-regulated kinases could not
mimic the effect of NECA, suggesting a role for G-protein-coupled
receptor kinases (GRKs). In vitro phosphorylation assays
demonstrate that phosphorylation of agonist-occupied A
ARs
is enhanced by GRK2 and that cellular pretreatment with NECA
dramatically inhibits subsequent GRK2-mediated phosphorylation in
vitro. Therefore, the A
AR is phosphorylated in
situ by a kinase similar or identical to GRK2, and this may be
involved in rapid functional desensitization of the A
AR.
The multiple physiological effects of adenosine are mediated by
the activation of cell surface adenosine receptors (ARs). ()Biochemical and molecular cloning studies have
demonstrated the existence of four AR subtypes designated
A
, A
, A
, and
A
(1) . A
AR cDNA clones were initially
isolated from rat testis and brain libraries(2, 3) ,
but subsequently isolated cDNAs from sheep and human sources, which
encode proteins with a 70% identity to the rat protein, have also been
designated as A
ARs(4, 5) .
Characterization of the pharmacological properties of the recombinant
rat A
AR led to the realization that the A
AR is
the ``atypical'' AR expressed in a rat mast cell-derived
tumor cell line RBL-2H3(6, 7) .
Despite its
relatively recent discovery, AAR activation has already
been implicated in contributing to several important physiological
effects of adenosine, including vasodilation, bronchoconstriction, and
cardioprotection(8) . Moreover, evidence has been presented to
suggest that these effects are initiated by A
AR activation
of mast cells, thereby leading to the release of allergic mediators
that are directly responsible for the observed phenomena(8) .
Therefore, an understanding of how A
AR signaling is
regulated would be a significant advance toward controlling these
events.
The initial characterization of the AAR
expressed in RBL-2H3 cells demonstrated that agonist-stimulated calcium
mobilization is subject to a rapid, homologous
desensitization(6, 7) . However, the molecular events
responsible for this effect are currently unknown. Rapid termination of
signaling by G-protein-coupled receptors is typically initiated by
receptor phosphorylation events catalyzed by either second
messenger-activated kinases or G-protein-coupled receptor kinases
(GRKs)(9) . The latter constitute a growing family of proteins
that specifically phosphorylate agonist-occupied receptors. To date,
six such kinases, termed GRKs 1 through 6, have been cloned from
mammalian sources but, with the exception of GRK1 (rhodopsin kinase),
the spectrum of receptor substrates for each GRK in vivo remains unknown(10) .
To examine a role of receptor
phosphorylation in regulating AAR signaling, a functional
hemagglutinin epitope-tagged rat A
AR has been expressed in
Chinese hamster ovary cells. We report the first visualization of a
recombinant A
AR and the first detailed characterization of
adenosine receptor phosphorylation both in situ and in
vitro. Moreover, evidence is presented that supports a role for a
specific GRK isoform in mediating A
AR phosphorylation and
desensitization.
Chinese hamster ovary
(CHO) cell lines stably expressing the epitope-tagged AAR
cDNA were generated by co-transfecting cells with
pCMV5/HA-A
AR and pSV2Neo in a 20:1 ratio using a modified
calcium phosphate precipitation/glycerol shock procedure previously
described(15) . After selection in G418, resistant colonies
were isolated, expanded, and screened for receptor expression by
radioligand binding using 1 nM
I-AB-MECA(11) . Cells were propagated in
T-75 flasks with Ham's F-12 medium supplemented with 10% (v/v)
fetal bovine serum, penicillin (100 units/ml), and streptomycin (100
µg/ml) in a 37 °C humidified atmosphere containing 5%
CO
.
Figure 1:
Functional
expression of HA-AAR in CHO cells. A,
representative saturation isotherm for
I-AB-MECA binding
to membranes from HA-A
AR cDNA-transfected CHO cells. Inset, Scatchard transformation of the specific binding data
from the same experiment. B, membranes from transfected CHO
cells were assayed for adenylyl cyclase activity in the presence of 5
µM forskolin and increasing concentrations of IB-MECA as
described under ``Experimental Procedures.'' Basal and
forskolin-stimulated activities in this experiment were 2.24 and 21.30
pmol/min/mg protein, respectively. C, nontransfected and
HA-A
AR-expressing CHO cells were sequentially treated with
periodate and biotin LC-hydrazide prior to membrane preparation,
solubilization, and immunoprecipitation with 12CA5. Following SDS-PAGE,
resolved proteins were transferred to a polyvinylidene difluoride
membrane for probing with horseradish peroxidase-conjugated
streptavidin and visualization of reactive proteins by enhanced
chemiluminescence as described under ``Experimental
Procedures.''
To identify the HA-AAR protein, we utilized the
presence of three predicted sites for N-linked glycosylation
within the A
AR sequence(3) . Cell surface
carbohydrate residues were covalently labeled with biotin by sequential
treatment of cell monolayers with periodate and biotin LC-hydrazide.
After immunoprecipitation with 12CA5 and SDS-PAGE, immunoprecipitated
glycoproteins were visualized by probing blots with horseradish
peroxidase-conjugated streptavidin. 12CA5 specifically
immunoprecipitated a 50-70-kDa glycoprotein from
HA-A
AR-expressing cells but not from nontransfected CHO
cells, demonstrating that this labeled protein represents the expressed
HA-A
AR (Fig. 1C). This also demonstrates
that HA-A
AR is appropriately processed with respect to
utilization of glycosylation sites and is expressed on the cell
surface, thereby rendering it accessible to agonist.
Figure 2:
Agonist-dependent phosphorylation of
HA-AAR. A, nontransfected and
HA-A
AR-expressing CHO cells were metabolically labeled with
[
P]orthophosphate, exposed to 10 µM NECA or vehicle for 10 min at 37 °C, and then lysed for
solubilization and immunoprecipitation with 12CA5. Immunoprecipitates
were analyzed by SDS-PAGE and autoradiography as described under
``Experimental Procedures.'' B, transfected cells
were treated with agonist and immunoprecipitated with 12CA5 as
described for A. Following SDS-PAGE, proteins were transferred
to a polyvinylidene difluoride membrane, and the region corresponding
to the phosphorylated HA-A
AR was excised for phosphoamino
acid analysis as described under ``Experimental Procedures.''
The migration of ninhydrin-stained phosphoamino acid standards in this
TLC buffer system is indicated. C, HA-A
AR
phosphorylation in response to various stimuli. After labeling with
[
P]orthophosphoric acid, transfected cells were
incubated for 10 min at 37 °C in the absence of any ligand (None), 10 µM NECA, 100 nM phorbol
12-myristate 13-acetate (PMA), 10 µM calcium
ionophore A23187 in the presence of 1.8 mM calcium chloride (A23187), 100 µM forskolin (Forskolin),
and 100 µM 8-bromo-cyclic GMP (8BrcGMP).
Membranes were then prepared for solubilization and immunoprecipitation
with 12CA5 as described under ``Experimental
Procedures.''
Figure 3:
Concentration and time dependences of
agonist-stimulated HA-AAR phosphorylation. A,
P-labeled transfected CHO cells were incubated for 10 min
at 37 °C with the indicated concentrations of NECA. Membranes were
then prepared for solubilization and immunoprecipitation with 12CA5 as
described under ``Experimental Procedures.'' Quantitative
analysis is from data pooled from three such experiments. B,
P-labeled transfected CHO cells were incubated for the
indicated times at 37 °C with 10 µM NECA. Membranes
were then prepared for solubilization and immunoprecipitation with
12CA5 as described under ``Experimental Procedures.''
Quantitative analysis is from data pooled from three such
experiments.
Figure 4:
Effects of agonist treatment on
HA-AAR function. A, transfected cells were
incubated with 1 unit/ml adenosine deaminase in the absence (
) or
presence (
) of 10 µM NECA for 10 min at 37 °C.
Membranes were then prepared for radioligand binding with increasing
concentrations of
I-AB-MECA as described under
``Experimental Procedures.'' Scatchard transformations are
shown of the binding data from one of four such experiments. B, transfected cells were treated as described for A,
and membranes were prepared for assay of adenylyl cyclase assay in the
presence of 5 µM forskolin and increasing concentrations
of IB-MECA as described under ``Experimental Procedures.''
Composite data from multiple experiments are given in Table 1.
Figure 5:
GRK-stimulated agonist-dependent
HA-AAR phosphorylation in vitro. In vitro phosphorylations were performed as described under
``Experimental Procedures'' using membranes from
nontransfected and transfected CHO cells incubated with or without 10
µM NECA in the presence or absence of 50 nM GRK2
at 30 °C for 5 min as indicated. Following the addition of stop
solution, membranes were pelleted for solubilization and
immunoprecipitation with 12CA5. Analysis was by SDS-PAGE and
autoradiography. This is one of multiple
experiments.
Figure 6: Effect of NECA pretreatment in situ on GRK2-stimulated phosphorylation in vitro. A, transfected cells were treated with 1 unit/ml adenosine deaminase in the absence (Vehicle) or presence of 10 µM NECA for 10 min at 37 °C. Membranes were then prepared for in vitro phosphorylation assays with the indicated additions, as described under ``Experimental Procedures'' followed by immunoprecipitation with 12CA5 and analysis by SDS-PAGE and autoradiography. B is a quantitative analysis of three such experiments. Phosphorylation is expressed relative to that observed in membranes from control cells in the presence of NECA but in the absence of any added GRK2 (set at 100%).
Despite the growing appreciation of the contribution of
AAR activation toward mediating some of the physiological
effects of adenosine, only limited information is available on how
A
AR function is regulated. We have recently demonstrated
that chronic exposure of A
AR-expressing CHO cells to the
agonist NECA induces a functional desensitization that is associated
with the specific down-regulation of G
-3 and G-protein
-subunits(13) . However, the time-course of G-protein
down-regulation (t
=
6 h) would suggest
that although this event may be an important adaptive mechanism to
prolonged agonist exposure, it cannot account for the rapid functional
desensitization observed for the native rat A
AR in response
to acute agonist treatment(6, 7) . By examining the
effects of agonist exposure in CHO cells expressing a recombinant
epitope-tagged A
AR, the current study now provides a
testable working model with which to explain the phenomenon of rapid
A
AR desensitization. The validity of the model system we
have chosen is proven by two observations. Firstly, both native and
recombinant epitope-tagged A
ARs undergo a rapid functional
desensitization (Fig. 4B and (6) and (7) ). Secondly, in membranes from transfected CHO cells (Fig. 4A) and RBL-2H3 cells, (
)this
desensitization is associated with a 30-40% reduction in the
number of agonist binding sites recognized by
I-AB-MECA
in radioligand binding assays. Therefore, it is likely that similar
adaptive processes operate to diminish A
AR signaling in CHO
and RBL-2H3 cells.
Several lines of evidence support a role for
G-protein-coupled receptor kinase involvement in AAR
desensitization. Firstly, the A
AR is rapidly phosphorylated
in an agonist-dependent manner, and this cannot be mimicked by simple
activation of second messenger-regulated kinases alone. Moreover, the
EC
value for NECA-stimulated A
AR
phosphorylation (
0.15 µM) is essentially the same as
its K
value for displacing
I-AB-MECA
binding from the A
AR(11) . Such a correlation
between extents of receptor occupancy and phosphorylation coupled with
the lack of any effect of second messenger-regulated kinases strongly
suggest that one or more GRK isoforms is responsible for
A
AR phosphorylation, because these kinases specifically
phosphorylate agonist-occupied receptors(9, 10) . A
specific role for GRK2 or a related kinase is suggested from the in
vitro phosphorylation experiments, which demonstrated that GRK2
was capable of enhancing the agonist-dependent A
AR
phosphorylation observed in isolated membranes. Although it is
theoretically possible that GRK2 mediates its stimulatory effect on
A
AR phosphorylation in vitro indirectly via
interaction with nonreceptor proteins present in the membrane
preparation, this is unlikely considering the high degree of substrate
specificity that GRKs exhibit toward G-protein-coupled
receptors(17, 18, 19, 22, 23) .
Therefore, it is most likely that the agonist-occupied A
AR
is directly phosphorylated by GRK2 in vitro. More importantly,
pretreatment of transfected cells with agonist reduced the subsequent
level of GRK2-stimulated, agonist-dependent A
AR
phosphorylation observed in vitro. The simplest explanation
for this phenomenon would be that agonist pretreatment induces
A
AR phosphorylation in situ on some of these
residues by GRK2 or a very similar kinase such that they are not
available for subsequent phosphorylation in vitro. However,
interpretation of these results is complicated by the observation that
agonist pretreatment in situ results in an agonist-independent
component of endogenous kinase- and GRK2-mediated receptor
phosphorylation in vitro. Because this effect is reversible in
a time-dependent manner with agonist washout, it cannot be simply due
to the carry-over of residual NECA from the cellular treatment. Because
agonist pretreatment would result in A
AR activation and a
resulting dissociation of G
-proteins in the proximity of
the receptor, it is possible that localized release of
-subunits near the receptor may be sufficient to translocate
sufficient GRK2 such that in the absence of agonist some receptor
phosphorylation can occur. The ability of
-subunits alone to
stimulate low level GRK2-mediated phosphorylation of
antagonist-occupied
-adrenergic receptors in vitro has been previously described(24) . Alternatively, it is
possible that agonist-dependent phosphorylation of the receptor on
specific residues in situ primes the receptor for subsequent
agonist-independent phosphorylation in vitro. Such a
sequential model of protein phosphorylation, whereby phosphorylation at
specific residues is required in order to observe phosphorylation at
other sites, has been well described for the kinases that control the
enzymes involved in glycogen metabolism(25) . Moreover, recent
experiments using synthetic peptide substrates(22) , as well as
a glutathione S-transferase fusion protein containing the
COOH-terminal sequence of the fMet-Leu-Phe receptor (26) , have
indicated that GRK2 may utilize such a sequential phosphorylation
mechanism.
Cellular pretreatment with agonist does not completely
abolish subsequent AAR phosphorylation in vitro.
Therefore, it seems likely that the A
AR may contain
multiple sites for GRK phosphorylation with only a subset of these
being utilized in CHO cells in situ such that incubation with
GRK2 in vitro results in the phosphorylation of some of the
remaining sites. This is not an unexpected finding because studies of
GRK-mediated phosphorylation of rhodopsin and the
-adrenergic receptor have demonstrated that although
high phosphorylation stoichiometries are reported in vitro,
the stoichiometries exhibited by receptors in situ are much
lower(27, 28) .
Although AAR
phosphorylation is associated with the onset of functional
desensitization, we cannot directly determine whether the
phosphorylated receptor is impaired in its ability to signal
downstream. This will require the purification of the receptor and its
reconstitution with inhibitory G-proteins in order to directly measure
the receptor's signaling capacity in the absence of any other
components. It is possible that additional factors, such as arrestins,
are required to elicit a profound desensitization in response to GRK
phosphorylation, as has been shown for rhodopsin and the human
-adrenergic receptor(29) .
Unfortunately,
we were unable to determine the stoichiometry of AAR
phosphorylation. This will be possible if either an A
AR
antagonist radioligand is developed or purified preparations of
HA-A
AR become available to allow us to accurately measure
receptor content in 12CA5 immunoprecipitates. However, a knowledge of
the A
AR sequence (3) and the substrate preference
for GRK2 (22, 23) can allow us to make some
predictions as to where the phosphorylation sites for GRK2 may reside.
Studies utilizing synthetic peptide substrates have demonstrated that
GRK2 prefers to phosphorylate serine and threonine residues flanked on
their NH
-terminal side by acidic amino acids and/or
phosphoamino acids(22, 23) . Inspection of the
A
AR sequence shows that the region of the COOH-terminal
domain immediately following the predicted palmitoylation site is
particularly enriched in such residues (sequence reads as
CQTSDSLDSNLEQTTE). Hence this may be a useful domain
within which to begin mutagenesis experiments to determine which
residues are phosphorylated by receptor kinases in situ.
Perhaps more interestingly, the identification of GRK2 as an
AAR kinase in vitro, and possibly in the intact
cell, as well as the association of receptor phosphorylation with
desensitization, suggests that A
AR responsiveness in a
given cell type may be controlled by overexpressing recombinant wild
type and mutant forms of this kinase. The potential utility of such an
approach has already been demonstrated for recombinant thrombin
receptors co-expressed with GRK3 in Xenopus oocytes (30) and native
-adrenergic receptors in a
human bronchial epithelial cell line after expression of a
catalytically inactive GRK2 mutant that could function as a dominant
negative
-adrenergic receptor kinase(31) .
Therefore it would be of interest to determine the effects of
expressing wild type and mutant GRK2 on the functioning of the native
A
AR in RBL-2H3 cells and, given the synergism exhibited
between the responses elicited via the A
AR and IgE receptor
in these cells(6) , whether expression would affect cellular
responses to antigen.
In conclusion, we have demonstrated for the
first time that the AAR is rapidly phosphorylated in
response to agonist exposure and that this event is associated with the
onset of functional desensitization. Furthermore, in vitro phosphorylation studies strongly support a role for GRK2, or a
kinase of similar substrate specificity, in mediating this
phosphorylation, and these results suggest a potential strategy with
which to manipulate A
AR signaling in the intact cell.
Experiments to determine the usefulness of this approach and to
identify the sites of A
AR phosphorylation are currently
underway.