The
3-Adrenergic Receptor Activates
Mitogen-activated Protein Kinase in Adipocytes through a
Gi-dependent Mechanism*
Kurt J.
Soeder
,
Sheridan K.
Snedden
,
Wenhong
Cao§,
Gregory
J.
Della Rocca¶,
Kiefer W.
Daniel§,
Louis M.
Luttrell
, and
Sheila
Collins
§**
From the Departments of § Psychiatry and Behavioral
Sciences,
Pharmacology, ¶ Biochemistry, and
Medicine, Duke University Medical Center, Durham, North Carolina
27710
 |
ABSTRACT |
Promiscuous coupling between G
protein-coupled receptors and multiple species of heterotrimeric G
proteins provides a potential mechanism for expanding the diversity of
G protein-coupled receptor signaling. We have examined the mechanism
and functional consequences of dual
Gs/Gi protein coupling of the
3-adrenergic receptor (
3AR) in 3T3-F442A
adipocytes. The
3AR selective agonist disodium
(R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in
cAMP production in adipocyte plasma membrane preparations, and
pretreatment of cells with pertussis toxin resulted in a further 2-fold
increase in cAMP production by CL316,243. CL316,243 (5 µM) stimulated the incorporation of
8-azido-[32P]GTP into G
s (1.57 ± 0.12; n = 3) and G
i (1.68 ± 0.13;
n = 4) in adipocyte plasma membranes, directly
demonstrating that
3AR stimulation results in
Gi-GTP exchange. The
3AR-stimulated increase
in 8-azido-[32P]GTP labeling of G
i was
equivalent to that obtained with the A1-adenosine receptor
agonist N6-cyclopentyladenosine (1.56 ± 0.07; n = 4), whereas inclusion of unlabeled GTP (100 µM) eliminated all binding. Stimulation of the
3AR in 3T3-F442A adipocytes led to a 2-3-fold
activation of mitogen-activated protein (MAP) kinase, as measured by
extracellular signal-regulated kinase-1 and -2 (ERK1/2)
phosphorylation. Pretreatment of cells with pertussis toxin (PTX)
eliminated MAP kinase activation by
3AR, demonstrating
that this response required receptor coupling to Gi.
Expression of the human
3AR in HEK-293 cells
reconstituted the PTX-sensitive stimulation of MAP kinase,
demonstrating that this phenomenon is not exclusive to adipocytes or to
the rodent
3AR. ERK1/2 activation by the
3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive
3AR
coupling to Gi proteins serves both to restrain
Gs-mediated activation of adenylyl cyclase and to initiate
additional signal transduction pathways, including the ERK1/2 MAP
kinase cascade.
 |
INTRODUCTION |
Long before the discovery of the
3AR and its
recognition as a unique, adipocyte-specific receptor controlling
lipolysis and thermogenesis, Rodbell and colleagues (1) made the
observation that there was an unusual, biphasic stimulation of cAMP
production in adipocytes in response to the
-adrenergic receptor
agonist isoproterenol. Depending upon the concentration of GTP in the assay, isoproterenol could either stimulate or inhibit adenylyl cyclase
activity in adipocyte plasma membranes. Murayama and Ui (2) showed that
this inhibitory phase could be relieved by pretreatment of adipocytes
with pertussis toxin (PTX).1
This curious observation lay fallow until studied later in greater detail by Bégin-Heick (3-5). However, it was not until the
cloning and characterization of the
3AR gene and the
development of selective
3AR agonists (6, 7) that it was
postulated that this novel adipocyte-specific
AR may be responsible
for the biphasic adenylyl cyclase response in adipocytes (8). We have
previously noted that despite the relatively high level of expression
of the
3AR in adipocytes, the efficiency of coupling of
the
3AR to stimulation of adenylyl cyclase is low (9).
However, there has been no clear biochemical demonstration of physical
coupling of the
3AR to Gi, other than
comparative functional experiments in the presence or absence of PTX
(10), nor has there been any indication of what additional second
messenger pathway may be activated as a consequence of this putative
coupling of
3AR to Gi.
Recently, many G protein-coupled receptors have been shown to mediate
cellular growth or differentiation responses through the activation of
MAP kinase cascades (11). Receptors signaling via PTX-sensitive
Gi/o proteins, as well as PTX-insensitive Gq/11 proteins may activate the ERK1/2 MAP kinase cascade through a mechanism
involving tyrosine protein phosphorylation and the activation of the
low molecular weight G protein p21ras (12-14). Little is known
about the potential role of
ARs in the regulation of the MAP kinase
pathway. Recently, we have found that in fibroblasts the
2AR mediates Ras-dependent ERK1/2 activation through its ability to couple to a PTX-sensitive Gi protein
(15).
2AR coupling to Gi occurs as a result
of PKA-dependent phosphorylation of the receptor, which
effectively "switches" receptor coupling from Gs to
Gi proteins. In contrast,
2AR-mediated
ERK1/2 activation in S49 lymphoma cells is an entirely
Gs-dependent process (16). In this system,
PKA-mediated phosphorylation of the low molecular weight GTPase, Rap1,
promotes Ras-independent ERK1/2 activation; this process was shown to
be independent of
2AR interaction with Gi/o
proteins. Therefore, it is not yet clear whether there is a common
mechanism by which
ARs activate MAP kinase.
Here, we demonstrate that stimulation of the
3AR in
adipocytes induces the direct activation of both Gs and
Gi. In these cells, Gi activation results in
both the attenuation of
3AR-mediated stimulation of
adenylyl cyclase and the activation of the ERK1/2 MAP kinase pathway.
Unlike the
2AR signal in fibroblasts,
3AR activation of the ERK1/2 pathway is independent of cAMP and PKA. These
data suggest that the promiscuous coupling of the
3AR in adipocytes permits the simultaneous transduction of two independent signaling pathways. This property of the
3AR may be
responsible, in part, for the unique physiological effects of selective
3AR agonists in vivo, such as their potency
for stimulating lipolysis (17, 18) and their ability to prevent or
reverse obesity (6, 19-22).
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MATERIALS AND METHODS |
Chemicals--
CL316,243 was a gift from American Cyanamid Co.
(Pearl River, NY). CGP20712A was a gift from CibaGeigy. Forskolin,
bovine serum albumin (fraction V), insulin,
(S)-(
)-propranolol,
N6-2'-O-dibutyryladenosine
3':5'-cyclic monophosphate, and salbutamol were purchased from Sigma.
EGF was obtained from Upstate Biotechnology (Lake Placid, NY).
8-Azido-[32P]GTP was purchased from Andotek Life Sciences
(Irvine, CA). Pertussis toxin and genistein were purchased from
Calbiochem. N6-Cyclopentyladenosine was obtained
from RBI (Natick, MA). ICI 118551 was purchased from Cambridge Research
Biochemicals (Wilmington, DE). Precast polyacrylamide gels were
obtained from Novex (San Diego, CA).
Cell Culture and Transfections--
3T3-F442A preadipocytes were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin
(Life Technologies, Inc.) at 37 °C in a humidified 5%
CO2 atmosphere. Upon reaching 90% confluence, the cells
were stimulated to differentiate into adipocytes by culturing in a
differentiation media composed of Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5 µg/ml insulin (23). HEK-293 cells were
grown in six-well dishes and transfected with 1.2 µg of pBC
3 or
pBC
2 DNA (24) by calcium phosphate co-precipitation (25). Ten h
later, cells were washed in 1 mM EGTA in phosphate-buffered
saline and incubated in growth medium for 24 h. The cells were
then serum-starved for 24 h and treated with PTX (100 ng/ml),
propranolol (10 µM), SR59230A (25 µM),
CL316,243 (10 µM), or isoproterenol (100 µM), and MAP kinase assays were performed as described
below. Cells that were pretreated with PTX were incubated with the
toxin for 16-20 h at a concentration of 100 ng/ml.
Adenylyl Cyclase Assays--
Plasma membranes were prepared from
3T3-F442A adipocytes or Sprague-Dawley epididymal white adipose tissue
as follows. For cultured cells, the monolayers were gently rinsed with
ice-cold phosphate-buffered saline. All subsequent steps were performed at 4 °C. Lysis buffer (5 mM Tris/2 mM EDTA,
pH 7.2, containing 10 µg/ml soybean trypsin inhibitor, 10 µg/ml
benzamidine, 2 µg/ml aprotinin, and 0.1 mM
phenylmethylsulfonyl fluoride) was added, and the cells were released
from the dish with a rubber policeman. Following gentle disruption with
a glass/Teflon homogenizer, nuclei were removed by centrifuging for 5 min at 800 × g. The supernatant was collected and
centrifuged at 40,000 × g for 20 min. The pellet was
resuspended in fresh lysis buffer with a Teflon pestle, centrifuged again, and then resuspended in a small volume of lysis buffer. For rat
adipocytes obtained from epididymal adipose tissue, the same procedure
was followed except that first the tissue was finely minced, and
adipocytes were isolated by incubating the tissue pieces with
collagenase (Worthington) (26). Adenylyl cyclase activity in plasma
membranes was measured as described previously (9). Control incubations
in the absence or presence of isobutylmethylxanthine (0.25 mM) found no evidence of particulate cyclic nucleotide
phosphodiesterase activity in these plasma membranes. In some
experiments, cAMP production was measured in intact 3T3-F442A
adipocytes. For these whole cell assays, growth media was replaced with
serum-free medium 3 h prior to stimulation. The serum-free medium
consisted of Dulbecco's modified Eagle's medium supplemented with 1 g/liter fraction V bovine serum albumin, 10 mM HEPES, pH
7.4, 100 units/ml penicillin, and 100 µg/ml streptomycin. Then cells
were incubated with the indicated concentrations of
-agonist in
fresh serum-free medium for 10 min at 37 °C in the presence of 0.25 mM isobutylmethylxanthine. Reactions were stopped by the
addition of ice-cold 5% trichloroacetic acid, and particulate material
was removed by centrifugation. The cAMP concentrations from both assay
methods were determined by radioimmunoassay using a polyclonal
antiserum to iodinated cAMP (27). Protein concentrations were
determined by the method of Bradford (28).
Western Blotting and Photolabeling of Heterotrimeric G
Proteins--
Western blotting was performed with antibodies specific
for individual G
subunits. Adipocyte membranes (30 µg of protein) were solubilized in SDS-polyacrylamide gel electrophoresis sample buffer and resolved on 4-20% Novex gradient gels (San Diego, CA). A
recombinant G protein standard mix containing G
i1,
G
i2, G
i3, and G
o was
included as a positive control (gift of Dr. Pat Casey). The proteins
were then transferred to nitrocellulose membranes. The membranes were
incubated for 16 h at 4 °C with rabbit
anti-G
i1+2, anti-G
i3, or
anti-G
s, 1:1000 dilution, followed by alkaline phosphatase-conjugated goat anti-rabbit antisera (1:10,000 dilution, Amersham Pharmacia Biotech) as secondary antibody. Immunoreactive bands
were visualized by Storm PhosphorImager (Molecular Dynamics).
Synthesis and purification of the 8-azido-[32P]GTP was
performed as described (29, 30) with minor modifications (31). For some
experiments, 8-azido-[32P]GTP was purchased from Andotek
Life Sciences. The photoaffinity labeling procedure of Offermanns (32)
was followed with modifications (31). For labeling of the
G
i subunits, adipocyte membranes (50 µg of protein)
were incubated with 8-azido-[32P]GTP (0.5 µCi) for 10 min at 30 °C in a volume of 60 µl containing 50 mM
HEPES (pH 7.6), 1 mM EDTA, 50 mM NaCl, 500 µM MgCl2, 50 µM GDP, 100 µM ATP, and 1 µg/ml adenosine deaminase in the presence or absence of the adenosine A1 receptor agonist
N6-cyclopentyladenosine or the
3AR-selective agonist CL316,243. Labeling of the
G
s subunit was performed as above at a free
Mg2+ concentration of 2-5 mM. The binding
reactions were stopped by placing tubes on ice, and all subsequent
steps were performed at 4 °C. The membranes were collected by
centrifugation for 10 min at 14,000 × g and
resuspended in a buffer containing 50 mM HEPES (pH 7.6), 1 mM EDTA, 50 mM NaCl, 500 µM
MgCl2, and 2 mM dithiothreitol. Following
irradiation of the samples with a UV lamp (4 watts, 254 nm) at a
distance of 3 cm for 5 min, the samples were collected by
centrifugation as before and solubilized in Laemmli sample buffer.
Proteins labeled with 8-azido-[32P]GTP were resolved by
SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel
and visualized by a Storm PhosphorImager (Molecular Dynamics).
MAP Kinase Assays--
Activation of MAP kinase was determined
by measuring the phosphorylation state of ERK 1 and ERK 2 (33). For
this assay, 3T3-F442A cells were grown and differentiated in six-well
culture plates and serum-starved for 24 h prior to stimulation
with
-adrenergic agonists or growth factors. The serum-free medium
consisted of Dulbecco's modified Eagle's medium supplemented with 1 g/liter fraction V bovine serum albumin, 10 mM HEPES, pH
7.4, 100 units/ml penicillin, and 100 µg/ml streptomycin. Five min
after the addition of
-agonists or growth factors, the medium was
removed, and the cells were solubilized by the direct addition of
Laemmli sample buffer (34). These cell lysates were sonicated for
5 s, and aliquots (30 µg protein/lane) were resolved by
SDS-polyacrylamide gel electrophoresis. ERK1/2 phosphorylation was
detected by protein immunoblotting using a 1:3000 dilution of rabbit
polyclonal phospho-MAP kinase-specific antisera (NEBiolabs) with a
1:10,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit
antisera (Amersham Pharmacia Biotech) as secondary antibody.
Quantitation of ERK1/2 phosphorylation was performed using a Storm
PhosphorImager (Molecular Dynamics). After quantitation of ERK1/2
phosphorylation, nitrocellulose membranes were stripped of
immunoglobulin and reprobed using rabbit polyclonal anti-ERK 2 IgG
(Upstate Biotechnology, Inc.) to confirm equal amounts of ERK 2 protein.
 |
RESULTS AND DISCUSSION |
Stimulation of 3T3-F442A adipocyte plasma membranes with the
selective
3AR agonist CL316,243 resulted in a 3-4-fold
increase in adenylyl cyclase activity Fig.
1A). Membranes prepared from cells pretreated with PTX exhibited a more robust increase in cAMP
production, which was at least 2-fold above that observed in membranes
from naive cells. Whereas basal levels of cAMP generation were elevated
following PTX treatment, the effect of PTX on the -fold activation of
adenylyl cyclase by forskolin was minimal (Fig. 1B). We
obtained similar results from Chinese hamster ovary cells, which
heterologously express the mouse
3AR (data not shown) (35). These results suggest that the
3AR is
constitutively coupled to both Gs and Gi,
because inhibition of Gi function leads to increased tonic
production of cAMP.

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Fig. 1.
3AR agonist
stimulation of cAMP production in 3T3-F442A adipocytes.
A, dose-response curve for agonist stimulation of cAMP
production in response to CL316,243. Membranes of untreated or
PTX-pretreated adipocytes were prepared and incubated with increasing
agonist concentrations. Cyclic AMP production was measured by
radioimmunoassay using polyclonal antisera to iodinated cAMP. The data
shown are representative of two dose-response experiments.
B, basal (Bas) and forskolin
(FSK)-stimulated cAMP production in untreated and
PTX-pretreated membranes. The data shown are the average of four
experiments.
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Rat white adipocytes have been shown to contain the two splice variants
of G
s and the three isoforms of G
i
(
i1,
i2,
i3) (5, 36). As
shown in Fig. 2, 3T3-F442A adipocytes
express the same repertoire of G proteins as found in rat white
adipocytes: two splice variants of G
s,
G
i3, and G
i1/G
i2. Others
have previously shown that adipocytes do not contain the PTX-sensitive
G
o protein (36-39). To determine whether the PTX
effects on cAMP production resulted from constitutive coupling of the
3AR to both Gs and Gi, the
photolabile GTP analog 8-azido-[32P]GTP was used to
measure
3AR agonist-dependent GTP loading of G
s and G
i. This method has been used by
several investigators to demonstrate physical coupling between G
protein-coupled receptors and individual G protein
subunits, based
upon several well defined criteria for receptor-G protein interaction
(40-42). Because the binding of GTP analogs to G
subunits is highly
dependent upon free magnesium concentrations (31), we conducted our
experiments in adipocyte plasma membranes under separate conditions
favorable for 8-azido-[32P]GTP labeling of each G
family. Because the
3AR is coupled to G
s
and activation of adenylyl cyclase, Fig.
3A shows that under high
magnesium conditions, which favor receptor-stimulated GTP loading of
G
s, CL316,243 could stimulate specific
8-azido-[32P]GTP labeling of G
s (1.57 ± 0.12; n = 3; p < 0.001). Under low magnesium conditions, which are optimal for determining
receptor-stimulated GTP loading to G
i, there was a
similar 1.68 ± 0.13-fold increase (n = 4;
p < 0.001) in specific 8-azido-[32P]GT G
protein labeling of the 40-42-kDa G
i species (Fig.
3B). We obtained similar data from 3T3-F442A membranes (not
shown). Fig. 3B also shows that the ability of CL316,243 to
stimulate Gi-GTP exchange in adipocyte membranes was
equivalent to that obtained with the A1-adenosine receptor
agonist N6-cyclopentyladenosine (1.56 ± 0.07; n = 4; p < 0.001).
N6-Cyclopentyladenosine served as a positive
control because the A1-adenosine receptor is expressed in
both primary adipocytes (43, 44) and differentiated 3T3-F442A
adipocytes (45) and couples to all three G
i species (46,
47).

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Fig. 2.
G protein subunit
determination. Plasma membranes from 3T3-F442A adipocytes and rat
epididymal white adipose tissue (EWAT) were resolved by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The complement of G protein subunits
present was assessed by Western blotting as described under
"Materials and Methods." Std, G protein standard.
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Fig. 3.
Labeling of G
subunits by 8-azido-[32P]GTP in adipocyte membranes
in response to 3AR
stimulation. A, 8-azido-[32P]GTP labeling
of G s in response to no stimulation (NS) or
labeling with CL316,243 (CL) (5 µM) in rat
white adipocyte membranes. B, 8-azido-[32P]GTP
labeling of the 40-42-kDa G i/o species in response to
no stimulation, N6-cyclopentyladenosine
(CPA) (50 nM), and CL316,243 (5 µM) in rat white adipocyte membranes. The effect of
excess GTP is also shown. Data shown are the average of three
experiments (for G s) or four experiments (for
G i) and are expressed as -fold change over nonstimulated
control. *, significantly different from unstimulated control,
p < 0.001.
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A number of Gi-coupled receptors have been shown capable of
activating MAP kinase (11). Recently, Daaka and colleagues (15) reported that the
2AR could couple to both
G
s and G
i, with consequent activation of
MAP kinase. We therefore determined whether one of the functional
consequences of the coupling of the
3AR to
G
i in 3T3-F442A adipocytes was the stimulation of MAP
kinase. Intact 3T3-F442A adipocytes treated with CL316,243 (5 µM) exhibited approximately a 2.5-fold stimulation of
ERK1/2 phosphorylation (Fig. 4).
Activation of the endogenous
2AR in 3T3-F442A adipocytes with the
2AR-selective agonist salbutamol also yielded a
similar 2-fold stimulation of ERK1/2 phosphorylation. Both
AR
responses were less robust than that observed following activation of
endogenous receptors for EGF, consistent with previous findings (13).
Also shown in Fig. 4, pretreatment of 3T3-F442A adipocytes with PTX completely blocked ERK1/2 phosphorylation in response to either
2AR or
3AR stimulation, but it did not
affect stimulation by EGF. The addition of propranolol (0.1 µM) eliminated ERK1/2 phosphorylation in response to
salbutamol but had no effect on the activation of MAP kinase induced by
CL316,243 (not shown), consistent with a CL-mediated effect exclusively
through the
3AR (7).

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Fig. 4.
Effect of -agonists
and PTX on ERK1/2 phosphorylation in 3T3-F442A adipocytes.
Quantitation of ERK1/2 phosphorylation subsequent to agonist
stimulation was determined by Western blotting with phosphospecific
anti-ERK1/2 rabbit antisera. Quantitation was performed using a Storm
PhosphorImager (Molecular Dynamics). Data shown are the average of four
experiments and are expressed as the -fold change over nonstimulated
control for salbutamol (1 µM), CL316,243 (5 µM), and EGF (10 ng/ml). Pretreatment with PTX was as
detailed under "Materials and Methods." *, significantly
different from unstimulated control by one-way analysis of variance,
p < 0.001.
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The relative sensitivity of the
3AR to activate MAP
kinase versus cAMP accumulation in adipocytes was assessed
by treating intact cells with increasing concentrations of CL316,243
(Fig. 5). Because the MAP kinase assays
involve the stimulation of intact cells, we included cAMP dose-response
data also from intact cells. The EC50 for cAMP production
in these experiments was 12 nM, whereas parallel
measurements of MAP kinase activation (EC50 = 280 nM) indicated that the PTX-sensitive coupling of the
3AR to MAP kinase activation is less potent under these
conditions. Nevertheless, the dose-response curves for both
measurements yielded a unit slope, indicative of the high selectivity
of CL316,243 for the
3AR (7, 48). Note that the
EC50 for cAMP production in these whole cell experiments is
significantly less than that found in plasma membranes (Fig. 1).
However, it is generally recognized that dose-response curves as well
as ligand binding data from intact cells are shifted "to the left,"
when compared with data obtained using isolated membrane preparations
(49, 50). These comparative studies shown in Fig. 5 suggest that cAMP
production is the favored pathway in response to activation of the
3AR in adipocytes. It must be remembered, however, that
the catalytic activity of the subsequent kinases and their
juxtaposition to substrate targets will ultimately determine the
relative importance of these two pathways. Future studies will address
these issues.

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Fig. 5.
Comparative dose-dependent
stimulation of cAMP production ( ) and MAP kinase activation ( ) in
3T3-F442A adipocytes. Differentiated 3T3-F442A adipocytes were
incubated with increasing concentrations of CL316,243 and processed for
measurement of cAMP or ERK1/2 phosphorylation as detailed under
"Materials and Methods." The data were analyzed by nonlinear
curve-fitting routines (GraphPad Prism) (9). The results shown are from
two independent experiments.
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To determine whether the dual Gs/Gi coupling of
the
3AR and activation of MAP kinase was an
adipocyte-specific event or a unique feature of the rodent
3AR, we attempted to reconstruct these effects by
transiently transfecting the human
3AR into HEK-293
cells. As shown in Fig. 6A,
transient transfection of the h
3AR resulted in a low
basal level of ERK1/2 phosphorylation (lane 2), and
stimulation with the
3AR agonist increased ERK1/2 phosphorylation ~4-fold above basal level (lane 3), as
compared with the 7-8-fold activation achieved by EGF (lane
7). There was no effect of the
3AR agonist on
mock-transfected cells (lane 1). This activation of MAP
kinase by the h
3AR was completely blocked by the
selective
3AR antagonist, SR59230A (lane 4)
(51). In addition, the nonselective
AR agonist isoproterenol at a
concentration capable of activating the
3AR (100 µM), in the presence of propranolol, also led to an
~4-fold stimulation of ERK1/2 phosphorylation (lane 5).
Finally, PTX completely eliminated MAP kinase activation (lane 6), as observed in 3T3-F442A adipocytes (Fig. 4). Together these data suggest that the dual coupling of the
3AR to
Gs/Gi, along with its activation of MAP kinase,
is a general property of this receptor.

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Fig. 6.
HEK-293 cells transiently expressing
the human 3AR activate MAP
kinase. A, HEK-293 cells were transiently transfected
with the h 3AR as described under "Materials and
Methods." Cells were treated as shown, and ERK1/2 phosphorylation was
determined. B, HEK-293 cells transiently expressing human
3AR or human 2AR were pretreated with
H-89 (20 µM), genistein (5 µM), or AG1478
(3 µM) for 40 min before agonist stimulation.
The results shown represent one of three experiments. CL,
CL316,243; ISO, isoproterenol.
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Mechanistically,
2AR-mediated MAP kinase activation in
fibroblasts requires sequential receptor coupling to Gs and
Gi, because cAMP-dependent, PKA-mediated
phosphorylation of the
2AR is a prerequisite for
receptor-Gi coupling (15). To compare the role of cAMP and
PKA in
AR-mediated MAP kinase activation in adipocytes, we compared
the effects of the PKA inhibitor H-89 on
2AR- and
3AR-stimulated MAP kinase activation. As shown in Fig.
6B, H-89 blocked
2AR-stimulated MAP kinase
activation (lane 8), consistent with the results of Daaka et
al. (15). In contrast, the
3AR signal was insensitive to
H-89 (lane 3). Fig. 6B also shows that MAP kinase
activation by the
3AR was sensitive to low micromolar concentrations of the tyrosine kinase inhibitor genistein (lane 4) and to the EGR receptor tyrphostin AG1478 (lane 5).
These results indicate that, like other G
i-coupled
receptors, the
3AR employs a tyrosine kinase receptor in
its mechanism of recruiting MAP kinase (52, 53). In other experiments,
treatment with dibutyryl cAMP enhanced
2AR-stimulated
ERK1/2 phosphorylation by ~70%, consistent with the model of
PKA-dependent
2AR-Gi coupling.
However, stimulation of MAP kinase by the
3AR was
unaffected by dibutyryl cAMP (data not shown). Together, these results
indicate that MAP kinase activation via the
3AR, unlike
the
2AR, is insensitive to modulation of the PKA
pathway. Thus
3AR-mediated MAP kinase activation can be
distinguished in several ways from the
2AR pathway.
First, the
3AR is not a substrate for PKA (24). Second, the
3AR appears to be constitutively coupled to both
Gs and Gi. Third,
3AR-stimulated
activation of cAMP is not required for activation of the MAP kinase
cascade in adipocytes. In contrast to the finding of
PKA-dependent
2AR-mediated MAP kinase
activation in S49 lymphoma cells (16), the
3AR pathway
in adipocytes is completely PTX-sensitive.
One of the remarkable features of the
3AR is that
treatment with
3AR-selective agonists in vivo
can prevent or reverse obesity due to either congenital or diet-induced
etiology (6, 19-22, 54). These agents are also efficacious over
prolonged periods of administration (21), which is quite distinct from
the rapid desensitization and down-regulation that is characteristic of chronic treatment with
1- and, particularly,
2AR-selective drugs (55, 56). These unique properties of
the
3AR have been suggested to be related to the fact
that
3AR is resistant to desensitization (57), because
it is not a substrate for either PKA or the G protein-coupled receptor
kinases (24). Instead, our finding that the
3AR is
capable of activating both the PKA pathway and the MAP kinase cascade
raises another possibility. The simultaneous recruitment of both
signaling networks may result in a more potent stimulation of lipolysis
and/or may promote the growth and differentiation of brown adipocytes,
which is observed in all
3AR agonist-treated animals
(20, 21, 58). It will now be important for us to test the consequence
of impairing one or the other of these signal transduction pathways to
determine their contribution to these
3AR-mediated responses.
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ACKNOWLEDGEMENTS |
We thank Drs. Patrick Casey and Timothy
Fields for advice and discussions on the synthesis and use of
8-azido-[32P]GTP and the gift of recombinant G protein
standard mix. We thank Dr. Suzie Mumby for the antisera to
G
i3, Dr. Tom Gettys for the cAMP antisera, Dr. Neil
Freedman for the gift of pBC
3 DNA, and Dr. Luciano
Manara of SANOFI MIDI Research for the gift of SR59230A. We also
acknowledge Dr. Robert J. Lefkowitz for helpful discussions during the
development of this project.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK46793 and DK53092 (to S. C.) and DK02352 (to L. M. L.), National Institutes of Health Training Program Grants T32ES0731 (to
K. J. S.) and T32GM07105 (to S. K. S.), and National Institutes of
Health Medical Scientist Training Program Grant T32GM07171 (to
G. J. D. R.).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.
**
To whom correspondence should be addressed: Duke University Medical
Center, P. O. Box 3557, Durham, NC 27710. Tel.: 919-684-8991; Fax:
919-684-3071; E-mail: colli008{at}mc.duke.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PTX, pertussis toxin;
AR,
-adrenergic receptor;
PKA, cAMP-dependent protein kinase;
EGF, epidermal growth
factor;
MAP, mitogen-activated protein;
ERK, extracellular
signal-regulated kinase;
CL316, 243, disodium
(R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate;
SR59230A, 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronapth-1-ylamino]-(2S)-2-propanol
oxalate;
AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline;
CGP20712A, 1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-O-methyl-4-trifluoromethyl-2-imidazoyl)phenoxy]-2-propanol
methane sulfonate.
 |
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