From the
Interaction of the rat A
The multiple physiological effects of adenosine are mediated by
activation of cell surface adenosine receptors
(ARs)(
Agonist-induced desensitization, or refractoriness, is a universal
feature of G-protein-coupled receptors, including the various AR
subtypes(9) . Several studies have indicated that, for many
receptors, desensitization can be divided into two temporally and
mechanistically distinct phases: short term agonist exposure can induce
receptor phosphorylation, which in turn can impair receptor/G-protein
interaction(9, 10) . Longer agonist treatment times can
result in down-regulation of the receptor and/or its associated
G-protein, as well as the up-regulation of components controlling
opposing signaling pathways(9) .
Rapid, homologous functional
desensitization of A
With these observations in
mind, the goals of the present study were: (a) to determine
the identity of the G-proteins with which the A
The following primary
antibodies were used for immunoblotting at the concentrations indicated
in parentheses: 982 (1 in 4000 dilution of serum) for detection of
G
After solubilization in
electrophoresis sample buffer, equivalent amounts of membrane protein
(typically 75 µg/sample) were resolved by SDS-PAGE using 10% (w/v)
polyacrylamide resolving gels. Resolved proteins were transferred to
PVDF membranes and nonspecific protein binding sites blocked by a
60-min incubation at room temperature in blocking buffer (5% (w/v) skim
milk in phosphate-buffered saline containing 0.2% (v/v) Triton X-100
and 0.02% (w/v) thimerosal). Membranes were then incubated with the
appropriate dilution of primary antiserum in fresh blocking buffer for
either 2 h at room temperature or overnight at 4 °C. After removal
of antiserum and extensive washing with three changes of blocking
buffer, the membrane was incubated for 60 min at room temperature with
a 1 in 5000 dilution of horseradish peroxidase-conjugated protein A in
a high detergent skim milk solution. The series of washes described
above was then repeated and followed by two further washes in
phosphate-buffered saline alone. Reactive proteins were visualized by
an enhanced chemiluminescence protocol in accordance with the
manufacturer's instructions (Renaissance, DuPont NEN).
Quantitation of immunoblots was by densitometric scanning of
autoradiographs using a Bio-Rad model 620 densitometer with analysis by
the 1-D Analyst software package. Preliminary experiments demonstrated
that the amounts of membrane protein and primary antibody dilutions
employed produced signals within the linear response range of our
detection methods (data not shown).
Immunoprecipitations
using the indicated anti-G-protein subunit antisera described above
were performed essentially as described
previously(23, 24) .
Recent
experiments performed on recombinant G-protein
Analysis of total membranes after photolabeling indicated that under
conditions where the addition of 10 µM NECA produced a
2-3-fold increase in the incorporation of label into the 41-kDa
band (which presumably consists of G
The adaptive responses of cells to sustained hormone exposure
operate at several different levels. While rapid covalent modifications
of receptors by various kinases are most likely responsible for
initiating short term desensitization, prolonged agonist treatment
invokes distinct processes. A common phenomenon exhibited by many
G-protein-coupled receptors is that of down-regulation, defined as a
loss in the total number of receptors expressed by the cell.
Such
receptor-specific events are presumably responsible for the homologous
desensitization exhibited by many receptors, but cannot explain
heterologous desensitization phenomena, whereby exposure to a given
agonist can desensitize responses elicited by other hormone receptors.
One potential mechanism of heterologous desensitization is that of
regulation of G-protein function and expression, since changes at this
level via one receptor would be expected to affect the signaling
capacity of any other receptor that utilizes the particular G-protein
to activate its appropriate effector. Evidence in support of this
hypothesis has accumulated over recent years, due to the availability
of antisera capable of discriminating among the multiple G-protein
subunits expressed in many cell
types(11, 12, 13, 33) . In particular,
many studies have demonstrated that chronic cellular exposure to
specific agonists results in a rapid down-regulation of the G-protein
which that receptor might be expected to activate upon agonist
occupancy; these effects are generally independent of second messenger
generation despite the requirement for receptor activation (33).
Additionally, receptor-independent, constitutive activation of G
With these observations in mind, the present study was undertaken.
The A
Chronic agonist exposure of A
The profound loss of
The selective loss of G
In conclusion, we have assessed the ability of the
A
A
We thank Dr. Mark Olah for donating the
A
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
adenosine receptor
(A
AR) with G-proteins has been assessed using a stably
transfected Chinese hamster ovary cell system. The non-selective AR
agonist 5`-N-ethylcarboxamidoadenosine (NECA) increased the
labeling of a 41-kDa membrane protein by
4-azidoanilido-[
-
P]guanosine
5`-triphosphate (AA-[
P]GTP), a photolabile GTP
analogue. Subsequent immunoprecipitation of G
-subunits indicated that NECA stimulated incorporation of
label into both G
-2 and G
-3.
Additional experiments revealed an A
AR stimulation of label
into G
and/or G
-subunits, albeit to a
lesser degree than that elicited by endogenous P
purinergic receptors. No interaction with G
could be
detected. Sustained cellular exposure to NECA induced A
AR
desensitization and specific down-regulation of G
-3
and G-protein
-subunits without changing levels of
G
-2, G
, or G
-subunits. Therefore the A
AR can interact with
G
-2, G
-3, and, to some extent,
G
-like proteins, but sustained agonist exposure
down-regulates only one of the G-proteins with which it interacts. This
is the first description of the differing specificities of
A
AR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the
A
AR could elicit the heterologous desensitization of
signaling events mediated by G
3.
)
(1) . Biochemical and molecular
cloning studies have facilitated classification of these
G-protein-coupled receptors into four subtypes, denoted as
A
, A
, A
, and A
(2,
3). The A
AR cDNA clone was initially isolated from a rat
brain cDNA library and was classed as a distinct AR subtype due to its
distinct agonist potency series compared with A
and
A
AR subtypes and, most intriguingly, its insensitivity to
inhibition by alkylxanthine compounds(4) . The isolation of an
A
AR cDNA has implicated this receptor in mediating some
physiological effects of adenosine. In particular, it has been
demonstrated that the A
AR is the AR responsible for
enhancing antigen-stimulated secretion in a rat mast cell line, RBL-2H3
(5, 6). Subsequently isolated AR cDNAs from sheep and human sources,
which exhibit a 70% amino acid identity with the rat A
AR,
have also been designated as A
ARs, although differences in
their pharmacological properties versus the rat protein make
it unclear as to whether these proteins are species homologues or
constitute a distinct AR subtype(7, 8) .
AR-stimulated Ca
mobilization has been reported in RBL-2H3
cells(5, 6) . However, the effects of chronic agonist
exposure on A
AR signaling have not been investigated. In
this regard, it has been suggested that chronic agonist exposure can
result in the specific down-regulation of the G-protein with which a
receptor preferentially couples. This phenomenon has been studied
extensively in rat adipocytes both in the intact animal (11) and
in primary adipocyte cultures (12, 13) and suggests that
chronic stimulation of the rat A
AR results in the
heterologous desensitization of other anti-lipolytic hormone responses
due to the down-regulation of G
proteins(13) . These
changes are not the result of reduced gene transcription, as mRNA
levels for each of the three G
-subunits expressed in
adipocytes are unaffected by agonist treatment(11) . Similar
phenomena have since been described for several G-protein-coupled
receptors, including those coupled to stimulation of adenylyl cyclase
via G
(14) and phospholipase C via G
and
G
(15, 16, 17) . In the few cases
examined, it has been proposed that agonist occupation of the
appropriate receptor reduces the half-life of the G-protein with which
it interacts, thereby resulting in
down-regulation(18, 19) .
AR
interacts, and (b) to determine whether chronic
A
AR activation could modulate expression of these proteins.
Materials
Cell culture supplies were from Life
Technologies, Inc. NECA was the generous gift of Dr. Ray Olsson
(University of South Florida, Tampa, FL). I-AB-MECA was
synthesized and purified to homogeneity by reverse phase high
performance liquid chromatography as described previously(20) .
4-Azidoanilido-[
-
P]GTP
(AA-[
P]GTP) was the generous gift of Dr. John
Raymond (Duke University and VA Medical Centers, Durham, NC) and was
prepared by the method of Offermans et al.(21) .
Protein A-Sepharose was from Pharmacia Biotech Inc. PVDF membranes and
horseradish peroxidase-conjugated recombinant protein A were from
Pierce. UTP was from Sigma. Sources of other materials have been
described elsewhere (20, 22).
Cell Culture and Transfections
Cell lines stably
expressing the pCMV5-rat AAR and pBC12BI-canine
A
AR constructs have been previously described and
characterized(20, 22) . Cells were maintained in
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
. Cells were
grown as monolayers in T-75 flasks and used just prior to reaching
confluence.
Membrane Preparation
Cells from one T-75 flask
were washed, scraped into 5 ml of lysis buffer (5 mM Hepes, 2
mM EDTA, pH 7.5, containing 10 µg/ml soybean trypsin
inhibitor, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, and 0.1
mM phenylmethylsulfonyl fluoride) and disrupted by Dounce
homogenization on ice (20 strokes). After centrifugation at 48,000
g for 10 min, the crude membrane pellet was
resuspended in lysis buffer to a concentration of approximately 1 mg of
protein/ml and aliquoted for storage at -80 °C.
Antibodies and Immunoblotting
The generation and
specificities of all but one of the anti-peptide antisera used in this
study have been demonstrated
previously(23, 24, 25) . Antiserum 457 was
generated against a decapeptide whose sequence is identical to that of
the COOH termini of G and
G
(26) . Reactivity with G
and
G
and a lack of cross-reactivity with inhibitory
G-protein
-subunits have been assessed using recombinant proteins
in immunoblotting studies (data not shown). Additionally, although
antiserum 982 was raised against the carboxyl-terminal decapeptide
sequence common to rod and cone transducins, as well as
G
-1 and G
-2, the restricted
expression of the transducins and the lack of expression of
G
-1 in CHO cells (23, 25) means that
this antiserum can be used as a specific tool for identification of
G
-2 in this system.
-2, 977 (1 in 4000 dilution of serum) for detection
of G
-3, 951 (1 in 8000 dilution of serum) for
detection of G
, 457 (1 in 1000 dilution of protein A
affinity-purified IgG) for detection of G
and
G
, and 987 (1 in 4000 dilution of serum) for
detection of G-protein
-subunits.
G-protein Photolabeling
These were performed as
described previously (24) except that freshly isolated crude
cell membranes (prepared as described above) were used and adenosine
deaminase was added to a concentration of 1 unit/ml. Quantitation was
by densitometric scanning of autoradiographs.
Radioligand Binding and Adenylyl Cyclase
Assays
Binding studies using the high affinity AAR
radioligand
I-AB-MECA and adenylyl cyclase assays were
performed and analyzed as described previously(20) .
Expression of the A
To examine AAR in CHO
Cells
AR interaction with and
regulation of G-proteins, a transfected CHO cell system was chosen,
thereby allowing the use of non-transfected CHO cells as an appropriate
negative control. The level of expression of the recombinant
A
AR in this system was determined using the high affinity
A
AR agonist radioligand
I-AB-MECA in
saturation binding experiments (Fig. 1A). This ligand
bound to a single saturable high affinity site in membranes from
transfected cells, with K
and B
values of 1.4 ± 0.4 nM and 3.7
± 0.4 pmol/mg membrane protein, respectively (three
experiments). Moreover, the expressed A
AR was functional as
the non-selective AR agonist NECA could elicit a dose-dependent
inhibition of forskolin-stimulated adenylyl cyclase activity in
membranes from transfected cells (Fig. 1B). Neither
specific binding of
I-AB-MECA nor inhibition of
forskolin-stimulated adenylyl cyclase activity was observed in
non-transfected CHO cells (data not shown).
Figure 1:
Stable expression of the rat
AAR in CHO cells. A, saturation isotherm for
I-AB-MECA binding to membranes from
A
AR-transfected CHO cells. Nonspecific binding was defined
by the inclusion of 10 µM NECA in the assay. This is one
of three experiments, composite data from which are given under
``Results.'' B, NECA-mediated inhibition of
forskolin-stimulated adenylyl cyclase activity in membranes from
A
AR-transfected CHO cells. At a concentration of 5
µM, forskolin elevated adenylyl cyclase activity by some
10-12-fold above basal. Each point is the mean of three separate
experiments. Curve-fitting analysis gave an IC
for NECA of
4.7 ± 1.4 µM and a maximal inhibition of 42
± 2% of the forskolin-stimulated
activity.
A
It has been documented
that the abilities of recombinant AAR-stimulated
G
-protein Labeling
ARs in transfected CHO
cells to inhibit cAMP accumulation and endogenous A
ARs in
RBL-2H3 cells to stimulate phospholipase C are abolished by
pretreatment with pertussis toxin(4, 5) . To determine
the nature of the A
AR/G
-protein interaction, a
photolabile GTP analogue (AA-[
P]GTP) was used to
assess A
AR-stimulated G-protein
activation(22, 23) . Using this approach, it was
determined that NECA could stimulate an increase in the labeling of a
41-kDa protein in membranes from A
AR cDNA-transfected but
not in non-transfected CHO cells; this effect was dose-dependent, with
half-maximal effects occurring at a NECA concentration of 0.2
µM (Fig. 2A). The -fold increase in
labeling over basal at a saturating dose of NECA was between 3- and
6-fold (four experiments). The identity of the G
protein(s)
activated by the A
AR was deduced by immunoprecipitating
each of G
-2 and G
-3 from crude
membranes after AA-[
P]GTP labeling using
subtype-specific antibodies (Fig. 2B). This demonstrated
that NECA increased incorporation of label into each of the
precipitated proteins, thereby demonstrating that the A
AR
is capable of interacting with both G
-2 and
G
-3 in this system.
Figure 2:
AAR-stimulated
photoincorporation of AA-[
P]GTP into
G
-proteins. A, dose-dependent incorporation of
AA-[
P]GTP into a 41-kDa membrane protein by NECA
in membranes from A
AR-transfected CHO cells. Membranes were
preincubated for 10 min at 30 °C with differing concentrations of
NECA prior to the addition of AA-[
P]GTP and
incubation for another 10 min. After UV irradiation, 10% of each sample
was resolved by SDS-PAGE and labeled proteins visualized by
autoradiography. Quantitation was by densitometric scanning. B, after incubation with or without 10 µM NECA,
membranes were photolabeled as described above. The final membrane
pellets were then solubilized in detergent buffer, divided into two
aliquots, and each aliquot immunoprecipitated with antibodies specific
for either G
-2 or G
-3 and protein
A-Sepharose. Immunoprecipitates were then analyzed by SDS-PAGE and
autoradiography.
Assessment of A
It has been recently demonstrated that receptors
thought previously to couple exclusively to GAR Interaction with
G
proteins can
also interact with other signaling systems. One example of this is the
adrenergic receptor, which, as well as coupling to
multiple G
proteins, can also activate G
and
G
(27, 28, 29) .
-subunits have
demonstrated that G
-subunits have a much lower
affinity for AA-[
P]GTP than G
proteins (30). Therefore, despite the fact that no
agonist-stimulated photoincorporation into a 48-kDa band was observed
(the size of G
-subunits in CHO cells as determined by
immunoblotting; Ref. 22), we could not immediately eliminate the
possibility that the A
AR was capable of coupling to
G
. Therefore we assayed cell membranes for effects of
A
AR activation on GTP-stimulated, i.e. ``basal,'' adenylyl cyclase activity. In non-transfected
CHO cells, 50 µM NECA produced a 6 ± 4% activation
of adenylyl cyclase activity above that of GTP alone. In contrast, the
addition of 50 µM NECA to membranes from
A
AR-expressing CHO cells inhibited basal activity by 57
± 7% (three experiments). Under the same conditions, the
addition of 50 µM NECA to membranes from CHO cells
expressing 0.26-0.30 pmol/mg A
AR, a prototypical
G
-coupled receptor(22) , elicited a 19.7 ±
0.5-fold increase over GTP-stimulated activity (10 experiments). To
determine whether A
AR activation of G
was
masking an interaction between the A
AR and G
,
assays were also performed after treatment of CHO cells with 20 ng/ml
PTx for 24 h, an incubation sufficient to ADP-ribosylate the total
cellular pool of G
as determined by subsequent
[
P]ADP-ribosylation experiments on isolated
membranes (22). Under these conditions, we observed a 79 ± 12%
loss of the ability of 50 µM NECA to inhibit
forskolin-stimulated adenylyl cyclase activity (three experiments)
consistent with the inactivation of almost all functional
G
. However, this treatment did not unmask an activation of
adenylyl cyclase activity: in membranes from PTx-treated cells, the
addition of 50 µM NECA to membranes merely attenuated
inhibition of GTP-stimulated activity from 57 ± 7% to 17
± 12% (three experiments).
A
To assess potential
AAR Interaction with
G
-like Proteins
AR interaction with G
and G
,
membranes were photolabeled with AA-[
P]GTP
followed by immunoprecipitation with antibody 457
(anti-G
). Preliminary immunoblotting experiments
with 457 demonstrated that under these conditions 457 specifically
immunoprecipitates a 42-kDa band that co-migrates with G
-subunits on SDS-PAGE, and that these immunoprecipitates are
devoid of detectable G
-2 and G
-3
proteins, as determined by immunoblotting of the immunoprecipitates
with antisera 982 and 977, respectively (data not shown). As a positive
control for the photolabeling experiments, we made use of the
endogenous P
purinergic receptor expressed by CHO-K1
cells. Agonist occupation of this receptor raises intracellular
Ca
concentrations in a manner that is resistant to
modulation by PTx treatment, indicating that the response is mediated
by G-proteins belonging to the G
family(31) .
-2 and
G
-3; Fig. 2), no such increase was noted after
the addition of 100 µM UTP, a P
purinergic
receptor agonist (Fig. 3A). However, after membrane
solubilization and immunoprecipitation with antiserum 457, a single
42-kDa labeled band is observed (Fig. 3B), which
presumably consists of a mixture of G
and
G
, both of which are expressed in CHO
cells(32) . The agonist-modulated labeling of the
immunoprecipitated 42-kDa band is quite distinct from that observed for
the 41-kDa band in total membranes (Fig. 3, A and B). The addition of UTP increases the labeling of the 42-kDa
band by some 2-2.5-fold, whereas 10 µM NECA
increases labeling by 1.3-1.5-fold (ranges of values from two
experiments). Therefore agonist-occupied A
ARs are capable
of increasing the labeling of G
-like proteins in membranes
from transfected CHO cells, albeit to a lesser degree than that
elicited by endogenous activated P
purinergic receptors.
Figure 3:
AAR and P
purinergic receptor-stimulated incorporation of
AA-[
P]GTP into G
-subunits. A, membranes were preincubated for 10 min at
30 °C with the indicated agonists prior to the addition of
AA-[
P]GTP and incubation for another 10 min.
After UV irradiation, 10% of each sample was resolved by SDS-PAGE and
labeled proteins visualized by autoradiography. B, membranes
were photolabeled as described above prior to solubilization and
immunoprecipitation with antiserum 457 (anti-G
-subunits) and protein A-Sepharose. Immunoprecipitates were
then analyzed by SDS-PAGE and
autoradiography.
A
To determine whether the activated
AAR Regulation of G-protein
Expression
AR could regulate the expression of the G-proteins with
which it interacts, cells were treated with 10 µM NECA for
up to 24 h and membranes prepared for comparative immunoblotting
experiments. These demonstrated that while the expression of
G
-2 and G
-subunits are
unaffected by prolonged agonist occupancy of A
ARs,
G
-3 undergoes a profound down-regulation (Fig. 4, A and B; ). This effect
was absolutely dependent on the expression of the A
AR since
parallel treatment of non-transfected CHO cells failed to alter levels
of G
-3 (Fig. 4C). Additionally this
figure also demonstrates that constitutive expression of the
A
AR did not significantly alter the levels of expression of
G
-3 in transfected versus non-transfected CHO
cells (Fig. 4C). Expression levels of G
were not greatly affected by agonist treatment (). However,
levels of the
-subunits common to all G-proteins decreased by
approximately 60% ( Fig. 5and ); as for
G
-3, this effect could not be observed in
non-transfected CHO cells (data not shown). Under these conditions, the
A
AR underwent a functional desensitization, as manifested
by an increase in the IC
value for NECA-mediated
inhibition of forskolin-stimulated adenylyl cyclase activity (Fig. 6).
Figure 4:
Selective agonist-mediated down-regulation
of G-3 in A
AR-transfected CHO cells. 75
µg of crude membrane protein from cells treated in the absence (Control) or presence (Treated) of 10 µM NECA for 24 h were resolved by SDS-PAGE and transferred to PVDF
membranes for immunoblotting with antisera 982, specific for
G
-2 (panelA), or 977, specific for
G
-3 (panelB). C,
non-transfected and A
AR-transfected CHO cells were treated
with or without 10 µM NECA for 24 h prior to membrane
preparation and immunoblotting with antiserum 977
(anti-G
-3).
Figure 5:
Agonist-mediated down-regulation of
G-protein -subunits in A
AR-transfected CHO cells. 75
µg of crude membrane protein from cells treated in the absence (Control) or presence (Treated) of 10 µM NECA for 24 h were resolved by SDS-PAGE and transferred to PVDF
for immunoblotting with antisera 987 (anti-
-subunits) as described
under ``Experimental Procedures.''
Figure 6:
Agonist-mediated desensitization of
AAR function. After treatment with or without 10 µM NECA for 24 h, membranes were prepared for assay of adenylyl
cyclase activity as described in Fig. 1. In this experiment, the
IC
value for NECA-mediated inhibition of
forskolin-stimulated adenylyl cyclase activity increased from 2.0
± 0.4 µM (Control) to 73 ± 20
µM (Treated). This is one of multiple
experiments, which produced quantitatively similar
data.
The effects on G-protein subunit expression of
treating transfected cells with increasing concentrations of NECA are
shown in Fig. 7. The EC values for loss of
G
-3 and
-subunits are similar (
60 nM and 80 nM, respectively), suggesting an equivalent
dependence of each process on agonist occupation of A
ARs.
However, treatment of transfected CHO cells with 10 µM NECA for various times demonstrates that their respective time
courses of down-regulation are distinct (Fig. 8). While
G
-3 undergoes a steady down-regulation observable at 4
h and maximal by 16 h exposure (Fig. 8, A and C),
-subunit down-regulation is biphasic; a rapid initial
reduction in expression, observable at 2 h, stabilizes until after 8 h,
when the subsequent rate of
-subunit down-regulation more closely
parallels that of G
-3 (Fig. 8, B and C).
Figure 7:
Dose
dependence values of G-protein subunit down-regulation to increasing
concentrations of NECA. 75 µg of membrane protein from
AAR-expressing CHO cells treated with the indicated
concentrations of NECA for 24 h were resolved by SDS-PAGE and
transferred to PVDF membranes for immunoblotting with
anti-G
-3 antiserum 977 (panelA) or
anti-
-subunit antiserum 987 (panelB). PanelC is a quantitative analysis of three separate dose
dependence experiments performed for each of these
proteins.
Figure 8:
Time
courses of G-protein subunit down-regulation. 75 µg of membrane
protein from AAR-expressing CHO cells treated with 10
µM NECA for the indicated times were resolved by SDS-PAGE
and transferred to PVDF membranes for immunoblotting with
anti-G
-3 antiserum 977 (panelA) or
anti-
-subunit antiserum 987 (panelB). PanelC is a quantitative analysis of three separate time
course experiments performed for each of these
proteins.
by cholera toxin-catalyzed ADP-ribosylation induces a rapid loss
of total cellular levels of G
that is independent of the
initial increase in cAMP levels(34, 35) . In S49 cyc
lymphoma cells transfected with an
epitope-tagged G
construct, the cholera
toxin-stimulated down-regulation is due to an enhanced degradation of
the protein, as manifested by a reduced biological half-life, and is
associated with a shift in the subcellular distribution of
G
from the membrane to the cytosol(36) .
AR has been shown to increase
phosphatidylinositol-specific phospholipase C activity in RBL-2H3 cells
(5, 6) and inhibit adenylyl cyclase activity in transfected CHO cells
(4). Both of these effects are abolished by pretreatment with pertussis
toxin, suggesting a role for G
proteins. In the transfected
CHO cell system we have shown, using sequential G-protein photolabeling
and immunoprecipitation with specific antibodies, that the
A
AR is capable of interacting with, and presumably
activating, both G
-2 and G
-3 in
isolated membranes. Interestingly, these are the same two pertussis
toxin substrates that are expressed in RBL-2H3 cells (37) and
suggest that the endogenously expressed A
AR in these cells
is at least capable of interacting with these proteins to produce its
downstream effects. Presumably, the pertussis toxin-sensitive
A
AR-stimulated increase in inositol 1,4,5-trisphosphate
production in RBL-2H3 cells is due to increased levels of dissociated
G
-derived
-subunits activating
phosphatidylinositol-specific phospholipase C-
isoforms, an
interaction that has been demonstrated both in intact cells (38) and with purified components(39) . However, we were
also able to reveal an interaction of the A
AR with
G
-like proteins expressed in CHO cells, and this would
suggest that, at least in some instances, A
AR stimulation
of phospholipase C may have a PTx-insensitive component. The generality
and significance of A
AR/G
interaction in
systems expressing A
ARs endogenously remain to be
determined. No stimulatory effect of A
AR activation on
adenylyl cyclase activity could be determined, suggesting that the
A
AR does not interact significantly with G
.
AR-expressing CHO cells
induces the specific down-regulation of G
-3 and not
G
-2 or G
-subunits, despite
the ability of the receptor to activate each of these proteins. This
effect required agonist occupation of the A
AR, as no
down-regulation was observed in native CHO cells, which express
undetectable levels of functional A
ARs. The loss of
G
-3 was associated with a similarly profound reduction
in the levels of
-subunits common to all G-proteins. The
reductions in expression of these proteins exhibited similar dose
dependence values to increasing concentrations of NECA. Additionally,
the EC
value for NECA stimulation of
AA-[
P]GTP incorporation into G
proteins was also similar to the EC
values for
G-protein subunit down-regulation, suggesting a possible link between
G-protein activation and down-regulation. Under these conditions,
A
AR-mediated inhibition of adenylyl cyclase underwent a
functional desensitization. We cannot determine at the present time the
contribution of G-protein down-regulation to the overall process of
A
AR desensitization; as we have previously demonstrated,
receptor desensitization is a complex phenomenon involving several
distinct processes whose importance to the overall effect varies upon
length of agonist exposure. In particular, receptor down-regulation may
play a role, but we cannot test this due to the unavailability of a
radiolabeled antagonist ligand for the A
AR. Nevertheless,
such a profound down-regulation of G
-3 would be
expected to impair downstream signaling events elicited by any receptor
with which it could interact.
-subunits
was a surprising finding from these studies and is perplexing
considering the quantity of G
-3 expressed in these
cells. Quantitative immunoblotting studies in CHO-K1 cells using E.
coli-derived recombinant proteins as standards have shown that
G
-2 is present at an 8-fold molar excess over
G
-3 (4.8 versus 0.6 pmol/mg membrane
protein)(23, 25) . Transfection of CHO cells with the
A
AR cDNA does not alter the steady-state levels of these
proteins compared with non-transfected cells (Fig. 4C and data not shown). Therefore the 70% reduction in levels of
G
-3 represents a loss of approximately 0.4 pmol of
G
-3/mg of membrane protein. Assuming that 1 mol of
-subunits is down-regulated with 1 mol of G-protein
-subunit,
the loss of 0.4 pmol/mg
-subunits would be undetectable since it
represents a small proportion of the total pool of
-subunits.
Therefore, additional
-subunits, not associated with the loss of
G
-3, must be down-regulated in order to account for
the 60% loss measured by immunoblotting (). This indicates
that additional processes distinct from those involved in
G
-3 down-regulation are involved in regulating
-subunit expression. Such a scenario is also suggested by the
distinct time courses displayed for down-regulation of each of these
proteins (Fig. 8).
-3
over G
-2 and G
-subunits
despite their ability to each couple with activated A
ARs
may be due to any of several reasons. While we are not able to
quantitate the amount of G
-subunits expressed
in CHO cell membranes, it is possible that large molar excesses of
G
-2 or G
-subunits over
G
-3 mask an equivalent down-regulation such that it is
not detectable: for example, a molar reduction in levels of
G
-2 equivalent to that of G
-3 (0.4
pmol/mg membrane protein) would represent less than 10% of the total
pool of G
-2 and would probably not be detected by
immunoblotting. Also, the importance of receptor/G-protein
stoichiometry in determining extent of down-regulation has not been
thoroughly examined, although a recent study on cell lines expressing
different levels of
-adrenergic receptor has suggested
that increasing the receptor:G-protein ratio enhances observation of
agonist-mediated down-regulation of G
-subunits(40) . Therefore, if a receptor couples to
more than one G-protein with equal efficiency, it is most likely that
the least abundant G-protein would be more susceptible to
down-regulation.
AR to activate and regulate specific G
proteins in a transfected CHO cell system. The A
AR is
capable of activating both G
-2 and
G
-3, the two pertussis toxin substrates expressed in
CHO cells, as well as G
-subunits. Moreover,
sustained exposure of A
AR-expressing cells to agonist
results in the selective down-regulation of G
-3 and
G-protein
-subunits but not of G
-2,
G
, or G
-subunits. However,
the extent to which both of these proteins are down-regulated and the
differing time courses exhibited suggest that distinct processes may be
involved in regulating the levels of these proteins in response to
receptor activation. Whatever these mechanisms are, we have clearly
shown that while the A
AR is capable of interacting with
multiple G-proteins in a given cell type, agonist treatment
specifically induces the down-regulation of G
-3,
thereby providing a potential mechanism for heterologous
desensitization of hormone signaling events mediated by this G-protein.
Table: Agonist regulation of G-protein expression in
CHO cells expressing the rat AAR
AR-transfected CHO cells were treated with or without
10 µM NECA for 24 hours at 37 °C prior to membrane
preparation and comparative immunoblotting using the antisera described
under ``Experimental Procedures.'' Under these conditions,
G
and G
co-migrate and therefore the
signal observed on immunoblots represents a composite signal for these
proteins. Data are presented as means ± standard error from the
number of experiments in parentheses, with the signal obtained for
untreated control membranes set at 100%.
P]GTP,
4-azidoanilido-[
-
P]guanosine
5`-triphosphate;
I-AB-MECA,
I-4-aminoben-zyl-5`-N-methylcarboxamidoadenosine;
NECA, 5`-N-ethylcarboxamidoadenosine; PVDF, polyvinylidene
difluoride; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel
electrophoresis; PTx, pertussis toxin.
AR-transfected cell line used in this study, Dr. John
Raymond for generously supplying AA-[
P]GTP, and
Linda Scherich for preparation of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.