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INTRODUCTION |
Activation of 
1-adrenergic receptors
(AR)1 influences both the
contractile activity and the growth potential of cardiac myocytes. However, despite intense investigation, the signaling pathways linking
activation of specific 1-AR subtypes to these particular physiological responses remain controversial (1). The situation is
complicated by the diversity of 1-AR subtypes. Three
distinct 1-AR subtypes have been identified by molecular
cloning (2-4). Recently, the relationships between the cloned and
native 1-AR subtypes have been established by comparison
of their affinity constants for a wide variety of 1-AR
subtype-selective antagonists (5, 6). From this comparison, it has been
suggested that the cloned 1b-AR represents the native
1B-AR subtype; the cloned 1a/c-AR2
corresponds to the native 1A-AR subtype; and the cloned
1d-AR is considered to represent a novel
1D-AR subtype. With the recognition that multiple
1-AR subtypes exist, the roles of the individual 1-AR subtypes in mediating specific physiological
effects need to be investigated further.
The assignment of particular physiological responses and signaling
pathways first requires the elucidation of the specific 1-ARs subtypes present in cardiac myocytes. In a
previous study, we showed that all three 1-AR subtypes
are expressed at the mRNA level, but only the 1A-
and 1B-AR subtypes are detectable at the protein level
in neonatal rat cardiac myocytes (7). Based on a wide range of
subtype-selective receptor antagonists, this study suggested that the
1A-AR subtype appeared to be largely responsible for the
stimulation of the phosphatidylinositol hydrolysis pathway in response
to agonist treatment of these cells, whereas activation of the
mitogen-activated protein kinase (MAPK) pathway appeared to occur
through a subtype whose pharmacological profile most closely resembled
that of the 1B-AR subtype. Although intriguing, the
limited selectivities of the currently available 1-AR
antagonists do not allow the definitive assignments of functional
responses and signaling pathways to be made. Thus, for a given
1-AR antagonist, the binding constants for inhibition of
signaling responses show a range of differences among published reports
that is almost as great as the range of putative differences among the
cloned 1-AR subtypes (8). Therefore, to circumvent the
limitations of a pharmacologic approach, we sought to develop a
molecular approach that could be used to identify conclusively which
signaling pathways, and ultimately which functional responses, are
activated in response to the individual 1A- and
1B-AR subtypes in cardiac myocytes. To this end, we
constructed a constitutively active mutant of the 1a-AR
subtype by analogy to a previously described constitutively active
mutant of the 1b-AR subtype (9). Such constitutively
active receptors have the advantage that their signaling properties can
be examined in the absence of agonist. Following introduction of the
individual constitutively active 1-AR subtypes into the
normal cellular context of cardiac myocytes in which the wild type
receptor subtypes are expressed, we determined which signaling pathways
are activated in response to each mutant receptor subtype in the
absence of agonist.
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EXPERIMENTAL PROCEDURES |
Materials--
Materials were obtained from the following
sources: BMY 7378, prazosin, 5-methylurapidil, oxymetazoline, and
WB-4101 (Research Biochemicals International); phenylephrine,
propranolol, and phentolamine (Sigma); [3H]prazosin,
[125I]HEAT (NEN Life Science Products);
myo-[3H]inositol (Amersham Pharmacia Biotech);
AG1-X8 resin (Bio-Rad); and pREP4 and pREP8 expression vectors and
Sindbis Virus Expression Kit (Invitrogen).
Construction and Subcloning of the Mutant
1a-S290/293-AR cDNA--
The
1a-S290/293-AR cDNA was constructed by
site-directed mutagenesis of the bovine 1a-AR cDNA,
which was generously provided by Dr. Jon Lomasney, Northwestern
University Medical School. The oligonucleotides
5'-CGCGAGCATAAAGCGCTCAAAACGCTG-3' and 5'-CGTTTTGAGCGCTTTATGCTCGCGGGA-3' were used to generate the appropriate mutations at Lys290
His and Ala293 Leu. The
mutant 1a-S290/293-AR cDNA was verified
by DNA sequencing.
For expression studies in COS-m6 cells, the cDNA encoding the
mutant 1a-S290/293-AR subtype was subcloned
into the BamHI site of the pBC12B1 expression vector. The
cDNA encoding the mutant 1b-S288-294-AR
subtype in the pBC12B1 expression vector was kindly provided by Dr.
Susanna Cotecchia, Lausanne, Switzerland. The
1b-S288-294-AR should be phenotypically
identical to the 1a-S290/293-AR, since only
substitutions at Lys290 His and
Ala293 Leu were shown to be responsible for
constitutive activity (9). The cDNAs encoding the wild type
1a-AR and 1b-AR subtypes in the pREP4 and
pREP8 expression vectors, respectively, were generously provided by Dr.
Kenneth Minneman (Emory University Medical School).
For expression studies in cardiac myocytes, the cDNA fragments
encoding the mutant 1a-S290/293-AR and
1b-S288-294-AR subtypes were subcloned into
the pSinRep5 vector (Invitrogen). The 1.5-kb cDNA fragment encoding
the mutant 1a-S290/293-AR was released from
the pBC12B1 vector by digestion with BamHI. After filling in
the sticky ends with dNTPs and Klenow polymerase, this cDNA
fragment was ligated into the StuI site of the pSinRep5 vector. Similarly, the 2-kb cDNA fragment encoding the mutant 1b-S288-294-AR was released from the
pBC12BI vector by digestion with EcoRI and Pml1.
The 2-kb cDNA fragment was isolated and further digested with
ApaLI to generate a 1.9-kb cDNA fragment. After filling
in the sticky ends with dNTPs and Klenow polymerase, this cDNA
fragment was ligated into the StuI site of the pSinRep5 vector.
Expression of the Wild Type and Mutant
1a-S290/293 and
1b-S288/294-AR Subtypes in COS-m6
Cells--
Expression of the wild type and mutant receptors in COS-m6
cells was carried out by the DEAE-dextran transfection procedure (10).
On day 0, COS-m6 cells were plated at a density of 2 × 106 cells/100-mm dish in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin. On day 1, cells were
transfected with varying concentrations of plasmid DNA and
DEAE-dextran. The cells were harvested 48 h later for measurement
of total or cell surface receptor binding. For total receptor binding,
the cells were lysed with a Polytron homogenizer in ice-cold buffer
containing 5 mM Tris-HCl, pH 7.4, and 5 mM
EDTA, and the cell lysates were centrifuged at 50,000 × g for 20 min at 4 °C. The resulting membrane pellets were
resuspended in buffer containing 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA at a protein concentration of 1-3 mg/ml.
Receptor expression was monitored by selective displacement of the
1-AR antagonist, [3H]prazosin, from these
membranes. Briefly, 0.03 mg of membrane protein was incubated in a
total volume of 90 µl of assay buffer consisting of 50 mM
Tris-HCl, pH 7.4, 1 mM EDTA, 1% bovine serum albumin, and
[3H]prazosin for 45 min at room temperature. In the
competition binding experiments, membranes were incubated with 1 nM of the [3H]prazosin in the presence or
absence of varying concentrations of competing ligands. In the
saturation binding experiments, membranes were incubated with 0.2-15
nM [3H]prazosin in the presence or absence of
10 µM phentolamine to determine nonspecific and total
binding, respectively. The incubation was terminated by the addition of
1 ml of ice-cold assay buffer, followed by rapid filtration over
Whatman GF/C glass fiber filters. After washing the tubes and filters
three times with 1 ml of ice-cold assay buffer, membrane-bound
[3H]prazosin retained on the filters was counted by
liquid scintillation spectrometry.
For cell surface receptor binding, the cells were washed 8 times with
Dulbecco's phosphate-buffered saline to remove antagonists that were
present in the growth media (see "Results" for details) and then
gently released from the dishes by incubation in 1 ml of Dulbecco's
phosphate-buffered saline containing 0.05% trypsin and 0.5 mM EDTA for 2 min at 37 °C. The cell suspensions were treated with 5 ml of Dulbecco's modified Eagle's medium containing 10% serum to inactivate the trypsin and centrifuged at 1,500 × rpm. The cell pellets were resuspended in assay buffer to give approximately 2.5 × 106 cells/ml. The binding of the
1-AR antagonist, [3H]prazosin, to the
resuspended cells was performed as described above in a final volume of
500 µl containing approximately 1 × 106 cells. An
aliquot of cells was counted by trypan blue staining to ensure the
intactness of the cells.
Expression of Mutant 1a-S290/293 and
1b-S288/294-AR Subtypes in Rat Cardiac
Myocytes--
Cardiac myocytes were prepared from hearts of
1-2-day-old Harlan Sprague-Dawley rats. Briefly, the ventricles were
removed, digested with a mixture of trypsin, chymotrypsin, and elastase in a Celstir apparatus at 37 °C, and subjected to Percoll step gradients to obtain an enriched fraction of greater than 94% myocytes, as described previously (11). Myocytes were suspended in modified Eagle's medium (MEM) containing 5% newborn calf serum, 100 µM 5-bromo-2'-deoxyuridine, 50 units/ml penicillin, and
50 µg/ml streptomycin and plated at a density of 5 × 105/18-mm well, 1 × 106/35-mm well, and
2 × 106/60-mm dish. Following overnight incubation,
the serum-containing medium was removed and replaced with a defined
serum-free medium, as detailed previously (11). Myocytes were
maintained in the defined serum-free medium for 24 h before being
used for viral infection.
By standard gene transfer methods, cardiac myocytes are not easily
transfected. Therefore, it was necessary to develop a viral infection
procedure in order to be able to measure activation of signaling
pathways in response to the introduction of a specific mutant receptor
subtype in the whole cell population. In another study, we demonstrated
the ability of a recombinant Sindbis virus to infect greater than 90%
of the cardiac myocytes, as measured by positive 
-galactosidase
staining.3 Therefore, in the
present study, recombinant Sindbis viruses encoding the mutant
1a-S290/293-AR and
1b-S288-294-AR subtypes were used to infect
cardiac myocytes. For construction of recombinant Sindbis viruses, the
pSinRep5 vector containing the cDNA for either the mutant
1a-S290/293-AR or mutant
1b-S288-294-AR was linearized with
NotI. The pSinRep5 vector containing the cDNA for the
LacZ was linearized with XhoI. The capped RNA transcripts were generated by in vitro transcription of the pSinRep
cDNAs as well as the DH-BB helper virus DNA, as described by the
manufacturer (Invitrogen). The recombinant Sindbis viruses were
harvested from the medium of baby hamster kidney cells that had been
electroporated 28 h earlier with the capped RNA transcripts.
For measurement of receptor expression, neonatal cardiac myocytes
growing on 60-mm dishes were infected with recombinant Sindbis virus
encoding either LacZ, mutant 1a-S290/293-AR,
or mutant 1b-S288-294-AR. The appropriate
dilutions of recombinant Sindbis virus were determined empirically to
yield comparable levels of mutant
1a-S290/293-AR and mutant
1b-S288-294-AR expression. After the
initial 1-h infection period, the medium was supplemented with the
1-AR antagonist, WB-4101, to a final concentration of 1 µM. The inclusion of WB-4101 in the medium was found to
increase significantly the number of mutant receptors expressed on the
cell surface. At 48 h post-infection, the cardiac myocytes were
lysed in homogenization buffer consisting of 20 mM HEPES,
pH 8.0, 2 mM MgCl2, 1 mM EDTA, 1 mM benzamidine, 1 mM AEBSF, and 10 µg/ml
pepstatin A by 12 passages through a 25-gauge needle. The cell lysates
were centrifuged at 265,000 × g for 30 min at 4 °C
to pellet the membrane fractions. The membrane pellets were resuspended
in 100 µl of 50 mM Tris-HCl, pH 7.4, and 1 mM EDTA, assayed for protein concentration, and used for measurement of
receptor expression. Briefly, 0.01 mg of membranes were incubated with
a saturating concentration of [125I]HEAT (4 nM) in a total volume of 90 µl of incubation buffer consisting of 50 mM Tris-HCl, pH 7.4, 1 mM
EDTA, and 1% bovine serum albumin for 45 min at 25 °C (7). The
reactions were stopped by addition of 1 ml of ice-cold incubation
buffer and rapid filtration of the mixture over Whatman GF/C filters.
The tubes and filters were washed three times with ice-cold incubation
buffer, and the filters were counted in a Beckman gamma counter.
Nonspecific binding was determined by the inclusion of 1 µM prazosin.
Measurement of Phosphatidylinositol Hydrolysis--
In the
studies employing COS-m6 cells, cells growing on 18-mm wells were
transfected and then labeled for 24 h with medium containing
myo-[3H]inositol (2 µCi/ml). After 24 h
labeling, the medium was replaced with medium containing 20 mM LiCl followed by addition of agonist or vehicle for
another 45 min. The cells were stopped by the addition of ice-cold
trichloroacetic acid (final concentration of 6%). The precipitated
proteins were removed by centrifugation, solubilized in 0.25 M NaOH, 0.2% SDS, and protein concentrations were
determined. The supernatants were extracted three times with 3 volumes
of water-saturated diethyl ether and incubated at 55 °C for 60 min, and the neutralized aqueous extracts were applied to 1-ml columns of
Dowex AG-1-X8, formate form. Total inositol phosphates were eluted
using a standard procedure (13) and counted by liquid scintillation spectrometry.
For the neonatal cardiac myocyte studies, cells growing on 18-mm wells
were infected with the various recombinant Sindbis viruses. Twenty-four
hours later, the cells were labeled with media supplemented with
myo-[3H]inositol (2 µCi/ml). At 48 h
post-Sindbis virus infection, the cells were treated with 10 mM LiCl in the presence or absence of 100 µM
phenylephrine and 1 µM propranolol for 30 min at
37 °C. The cells were stopped by two rapid 1-ml washes of ice-cold phosphate-buffered saline followed by the addition of 1 ml of 6%
ice-cold trichloroacetic acid. After scraping, the cells were centrifuged at 14,000 × g for 10 min to remove the
precipitated proteins. The supernatants were extracted three times with
diethyl ether, and the extracts were applied to 0.5-ml columns of Dowex AG-1-X8, formate form. Inositol phosphates were eluted by a standard procedure (13) and counted by liquid scintillation spectrometry.
Measurement of MAPK Activation--
Neonatal rat cardiac
myocytes growing on 35-mm wells were treated in the presence or absence
of 100 µM phenylephrine in the presence of 1 µM propranolol for 5 min at 37 °C. Reactions were stopped by two washes with ice-cold phosphate-buffered saline followed
by addition of 75 µl of MAPK extraction buffer, consisting of 20 mM -glycerophosphate, 20 mM NaF, 2 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mM vanadate, 1 mM DTT, 10 mM
benzamidine, 1 mM AEBSF, 25 µg/ml leupeptin, and 5 µg/ml pepstatin A. After scraping, the cell extracts were incubated
on ice for 15 min and centrifuged at 14,000 × g for 10 min at 4 °C. The supernatant fractions were assayed for protein
concentration using the Coomassie Plus Protein Assay (Pierce). Equal
amounts of protein were electrophoresed on 11% SDS-polyacrylamide
gels, and the proteins were transferred to Immobilon-P membrane
(Millipore). MAPK activation was determined by immunoblotting with a
polyclonal antisera that recognizes only the doubly phosphorylated
forms of ERK1 and ERK2 (Anti-active MAPK, Promega, 1:10,000), which is
the form of MAPK that possesses myelin basic protein phosphorylating
activity. Western blots stripped and reprobed with a monoclonal
pan-ERK1/ERK2 antibody (Promega) showed equivalent amounts of ERK1/ERK2
protein across the lanes. The amounts of active and total ERK1 and ERK2
were visualized by incubating the immunoblot with a goat anti-rabbit
horseradish peroxidase conjugate (1:5000) followed by enhanced
chemiluminescent detection using the SuperSignal Chemiluminescent
Substrate Kit (Pierce).
Luciferase Gene Expression--
Cultured cardiac myocytes were
transfected with 1 µg of plasmid DNA using either the SRE-luciferase
reporter plasmid (Stratagene, Inc.) or the ANF promoter-luciferase
reporter plasmid, which was the generous gift of Dr. J. H. Brown.
An aliquot of 1 µg of luciferase reporter plasmids (SRE or ANF) was
mixed with 7 µl of the "Plus" component of LipofectAmine Plus
reagent (Life Technologies, Inc.) and 100 µl of Opti-MEM medium (Life
Technologies, Inc.) and incubated for 15 min at room temperature. In a
separate tube, 7 µl of the lipid component of LipofectAMINE Plus
reagent was added to 100 µl of Opti-MEM medium and incubated at room
temperature for 15 min. The contents of the two tubes were then mixed
together and incubated an additional 30 min at room temperature. The
cardiac myocyte tissue culture plates (60 mm) were washed with Opti-MEM medium and then 1 ml of fresh Opti-MEM medium without antibiotics was
added to each cell plate. The contents of the DNA/LipofectAMINE Plus
mixture was then added to each dish of myocytes. After 5 h, the
transfection media were removed and the myocytes maintained in Opti-MEM
medium without antibiotics.
For luciferase gene expression assays, the cardiac myocytes were lysed
in 0.5 ml of 1× lysis buffer (Promega Corp.). The cell lysates were
centrifuged at 15,000 × g for 1 min at 4 °C, and the supernatants were transferred to a new tube. Approximately 50 µl
of the supernatant was assayed in duplicate for SRE- and/or ANF-luciferase gene expression using a Luciferase Assay System with
Reporter Lysis Buffer (Promega Corp.). Relative light units were
measured utilizing a Lumat LB9507 EG&G Berthold luminometer with
myocyte cell lysates. Following luciferase assays, -galactosidase enzyme assays were performed in duplicate samples. The luciferase relative light unit numbers were divided by the -galactosidase enzyme assay numbers to correct for the transfection efficiency in the
myocytes. The cardiac cell lysate from the luciferase assays was
assayed spectrophotometrically for the -galactosidase enzyme activity in cardiac myocytes using the -Galactosidase Enzyme Assay
System with Reporter Lysis Buffer (Promega).
Data Analysis--
Values represent the mean ± S.E.
Saturation binding and competition binding curves were analyzed using
the nonlinear, least squares regression analysis program GraphPad Prizm
(GraphPad Software). Estimates of ligand binding affinity
(KD) and receptor density
(Bmax) were obtained from saturation isotherms
by fitting the data to a rectangular hyperbola. IC50 values
from competition binding experiments were converted to
Ki values using the Cheng and Prusoff equation (14):
Ki = IC50/(1 + [A]/KD) (14), where [A] is
the concentration of ligand used and KD was
determined from the saturation binding studies.
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RESULTS |
Construction and Characterization of the Constitutively Active
1a-S290/293-AR--
To address whether
constitutively active mutants of the 1a-AR and the
1b-AR subtypes could be used to probe their
subtype-specific coupling to signaling pathways, a constitutively
active mutant of the 1a-AR needed to be constructed. A
constitutively active mutant 1b-AR subtype has already
been constructed and characterized by Dr. Cotecchia (9). Accordingly,
the 1a-AR subtype was mutated at Lys290 and
Ala293 (1a-S290/293-AR) to
mirror the substitutions previously found to result in maximal
constitutive activation of the mutant 1b-AR subtype (9). Properties that have been shown to be characteristic of constitutive activation include the following: 1) an increased affinity of a
receptor for agonist but not for antagonist; 2) an elevated basal level
of signaling; and 3) an increased agonist-stimulated level of
signaling. To determine whether the mutant
1a-S290/293-AR subtype exhibited any or all
of these properties, the mutant 1a-S290/293-AR subtype was expressed and
then characterized in COS-m6 cells. Since COS-m6 cells do not express
wild type 1-ARs, these cells provide a convenient
background in which to study the properties of the mutant
1a-S290/293-AR subtype.
Increased Affinity for Agonist--
COS-m6 cells transfected with
either the wild type 1a-AR or the mutant
1a-S290/293-AR were characterized in terms
of their affinity for various agonists and antagonists by monitoring
the displacement of [3H]prazosin through agonist or
antagonist competition binding assays. Phenylephrine and
norepinephrine, both full 1-AR agonists (15), displaced
[3H]prazosin from membranes expressing the
1a-S290/293 mutant with approximately
40-fold higher affinities than those expressing the wild type receptor
(Fig. 1; Table
I). The partial imidazoline agonist,
oxymetazoline (15), also demonstrated a higher affinity for the
1a-S290/293-AR (Fig. 1), although the
difference in affinity, in this case, was only 6-fold (Table I). The
antagonist, 5-methylurapidil, demonstrated similar affinity for both
the wild type and mutant receptors (Fig. 1; Table I). The agonist and
antagonist competition curves fitted best to a single-site model, where
Hill coefficients were between 0.7 and 1.0. These data demonstrate that
the mutant 1a-S290/293-AR showed an increase
in binding affinity for agonist but not for antagonist compared with
its wild type counterpart. These data provide the first confirmation
that the mutant 1a-S290/293-AR possesses
properties reminiscent of other constitutively active receptors (9).
Furthermore, these data suggest that the difference in agonist binding
affinity observed between the wild type and mutant
1a-ARs may be related to the intrinsic activity of the agonist (i.e. a larger difference is observed for the full
agonist, phenylephrine, compared with the partial agonist,
oxymetazoline), and this observation is in agreement with that
previously described for a constitutively active mutant of the
2-AR (16).
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Fig. 1.
Competition binding relationships of agonists
and antagonists to the wild type
1a-AR and
1a-S290/293 mutant
receptors expressed in COS-m6 cells. The ability of
1-AR agonists and antagonists to displace specific
[3H]prazosin binding from membranes prepared from COS-m6
cells transiently expressing homogeneous populations of either wild
type 1a-AR or 1a-S290/293
mutant receptor was performed and analyzed as described under
"Experimental Procedures." Each value represents the mean ± S.E. of three to four individual experiments performed in duplicate.
The corresponding Ki values are reported in Table I.
Mean receptor expression levels were 1400 (1a-AR) and
300 (1a-S290/293) fmol/mg membrane protein
obtained using 0.2 and 2.6 µg/ml of cDNA/transfection,
respectively.
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Table I
Agonist and antagonist binding affinities of the wild type
1a-AR and 1a-S290/293 mutant receptor
Values are Ki (nM) calculated from the
data shown in Fig. 1. The ratio between wild type 1a-AR and
1a-S290/293 Ki values are shown
in parentheses. Each value represents the mean ± S.E. of at least
three individual experiments performed in duplicate.
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Enhanced Basal and Agonist-stimulated Phosphatidylinositol
Hydrolysis--
Previously, it was shown that all 1-AR
subtypes couple to the phosphatidylinositol hydrolysis pathway when
overexpressed at high, "non-physiological" levels (e.g.
>1500 fmol of receptor/mg of protein) (17-19). In the present report,
we decided to study the signaling properties of both the constitutively
active and wild type 1-ARs when they were expressed at
more physiological levels in order to get a better idea of how these
receptors operate in vivo. By altering the amount of plasmid
DNA used in the transfections, COS-m6 cells expressing various levels
of receptor were obtained. In particular, cells expressing receptor in
the 300 fmol/mg protein range were studied, since this density seems
comparable with many intact cellular systems that normally express
1-ARs. Examples of Bmax values
for various tissues are as follows: 256 fmol/mg protein in rat cardiac
myocytes (20); 158 fmol/mg protein in rat cerebral cortex (21); and
233, 313, and 690 fmol/mg protein in rat hippocampus, vas deferens, and
liver, respectively (22).
The second confirmation that the mutant
1a-S290/293-AR exhibits properties
characteristic of other constitutively active receptors was a
reproducible and marked increase in the basal level of
phosphatidylinositol hydrolysis (i.e. in the absence of
agonist). As shown in Fig. 2, COS-m6
cells expressing the mutant 1a-S290/293-AR
displayed a high basal level of inositol phosphate accumulation at the
lowest receptor density examined of 199 fmol/mg protein. By contrast,
cells expressing the wild type 1a-AR showed no
detectable basal level of inositol phosphate accumulation over a wide
range of receptor densities from 308 to 2098 fmol/mg protein. These data demonstrate that the mutant
1a-S290/293-AR had an increased ability to
interact with G protein to stimulate inositol phosphate accumulation in
the absence of agonist, which is indicative of constitutive activity.
Moreover, since the mutant 1a-S290/293-AR
was expressed at a lower level than the wild type 1a-AR, these data likely underestimate the greater constitutive activity of
the mutant 1a-S290/293-AR.
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Fig. 2.
Effect of expressing the wild type
1a-AR or the
1a-S290/293 mutant receptor
on phosphatidylinositol hydrolysis in COS-m6 cells. Basal
(unstimulated) and phenylephrine-stimulated phosphatidylinositol
hydrolysis were measured in
myo-[3H]inositol-labeled COS-m6 cells
transiently expressing either the
1a-S290/293 mutant receptor or an increasing
density of the wild type 1a-AR. Cells were stimulated
with either 100 µM phenylephrine or vehicle
(unstimulated) in the presence of 20 mM LiCl for 45 min,
and the accumulated inositol phosphates were extracted and measured as
described under "Experimental Procedures." The mean receptor
expression levels (Bmax) shown were measured
simultaneously using a whole cell binding assay and were obtained by
using 2.6 µg/ml cDNA for the
1a-S290/293 mutant and 0.04, 0.2, and 5.0 µg/ml cDNA/transfection for the 308, 963, and 2098 fmol/mg
membrane protein receptor densities, respectively, of the wild type
1a-AR. Each value represents the mean ± S.E. of at
least four individual experiments performed in duplicate. Data shown
have had subtracted the total inositol phosphate accumulation measured
in cells transfected in the presence of vector alone (28,854 ± 6160 dpm/mg protein; n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the corresponding 1a-S290/293
value.
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Also, as shown in Fig. 2, COS-m6 cells expressing the mutant
1a-S290/293-AR displayed a higher
agonist-stimulated level of inositol phosphate accumulation compared
with cells expressing the wild type 1a-AR. In fact, the
wild type 1a-AR had to be expressed at a 10-fold higher
level than the mutant 1a-S290/293-AR before
their maximal responses were comparable. These data are consistent with
previous studies of wild type and mutant 2-ARs, which
showed that greatly elevated expression of the wild type receptor was
able to increase agonist-stimulated cAMP accumulation to a level
comparable to that achieved with the constitutively active mutant
receptor (19).
Increased Potency of Agonist-stimulated Phosphatidylinositol
Hydrolysis--
Previously, it was shown that an increased agonist
potency is yet another feature of a constitutively active receptor (9, 16). Fig. 3 compares the dose-response
curves for the wild type 1a-AR and mutant
1a-S290/293-AR to agonist. As can be seen,
phenylephrine exhibits a 13-fold greater potency for stimulating
phosphatidylinositol hydrolysis via the mutant
1a-S290/293-AR (EC50 = 15 ± 8 nM, n = 3) compared with the wild type
1a-AR (EC50 = 192 ± 64 nM,
n = 3, p < 0.01). These results
demonstrate that the mutant 1a-S290/293-AR
also fulfilled this criterion of a constitutively active receptor.
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Fig. 3.
Concentration response relationships for
phenylephrine-stimulated total inositol phosphate accumulation in
COS-m6 cells transiently expressing either the wild type
1a-AR or the
1a-S290/293 mutant
receptor. Receptor expression densities of 1400 and 300 fmol/mg
membrane protein were obtained by using 0.2 and 2.6 µg/ml of
cDNA/transfection of the wild type 1a-AR or
1a- S290/293 mutant receptor,
respectively. Phosphatidylinositol hydrolysis was measured as described
under Fig. 2 and under "Experimental Procedures." Each value
represents the mean ± S.E. of at least three individual
experiments performed in duplicate. Calculated EC50 values
are 15 ± 8 nM, n = 3, for the mutant
1a-S290/293-AR and 192 ± 64 nM, n = 3, for the wild type
1a-AR.
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Increased Receptor Expression in the Presence of
Antagonist--
We observed that for any given plasmid DNA
concentration used for transfection, the level of expression of the
mutant 1a-S290/293-AR was considerably lower
than that of the wild type receptor. A similar finding was recently
reported for a constitutively active mutant of the 2-AR
(23). Moreover, inclusion of antagonist in the post-transfection period
was shown to increase the level of expression of the constitutively
active mutant of the 2-AR. To determine whether this
property was shared by the constitutively active mutant of the
1a-S290/293-AR, inclusion of a variety of
antagonists in the post-transfection period was studied. These
antagonists were removed just prior to collection of the cells by
exhaustive washing (8 times) with Dulbecco's phosphate-buffered
saline. As shown in Fig. 4, the 1a-AR-selective antagonists, 5-methylurapidil and
WB-4101, in the post-transfection period led to a significant increase
in the number of mutant 1a-S290/293-ARs on
the cell surface. The 1d-AR selective antagonist, BMY 7378, resulted in a similar increase. Interestingly, the non-selective 1-AR antagonist, prazosin, was unique in that it did not
elevate the density of mutant
1a-S290/293-AR. These data suggest that the
ability of antagonists to enhance receptor expression is a property
shared by constitutively active mutants of the 2-AR and
1a-S290/293-AR. The mechanism by which
antagonists result in higher receptor expression is not yet clear but
may relate to stabilization of receptor conformation (23). In this
regard, the finding that not all antagonists display this property is
interesting in that it might support the existence of multiple receptor
conformations of the mutant
1a-S290/293-AR.
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Fig. 4.
Effect of
1a-AR antagonists on the level of cell
surface expression of the
1a-S290/293-AR in COS-m6
cells. Following transfection, cell were grown for 48 h in
medium supplemented with either prazosin (1 µM),
5-methylurapidil (1 µM), WB-4101 (1 µM),
BMY 7378 (100 µM), or vehicle (control).
Following extensive washing to remove the incubation antagonists, whole
cell binding was performed as described under "Experimental
Procedures." In each case, 2.6 µg/ml of the
1a-S290/293 mutant cDNA/transfection was
used. Each value represents the mean ± S.E. of at least three
individual experiments performed in duplicate. *, p < 0.05; **, p < 0.01, compared with corresponding
1a-S290/293 control value.
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Comparison of the Signaling Properties of the Constitutively Active
1a-S290/293 and
1b-S288/294-AR Subtypes in COS-m6
Cells--
By having shown that the mutant
1a-S290/293-AR possesses all of the
characteristics of a constitutively active receptor, the second aim of
this study was to compare the signaling properties of the constitutively active mutant 1a-S290/293-AR
subtype with that of the analogous constitutively active mutant 1b-S288-294-AR subtype developed by Dr.
Cotecchia (9), since it is their ability to activate signaling pathways
in the absence of agonist that makes them useful tools for linking a
specific receptor subtype to a particular signaling pathway. Activation
of both the phospholipase C and MAPK signaling pathways has been
demonstrated upon addition of 1-AR agonists to cardiac
myocytes (1, 7, 27, 30). Fig. 5 shows a
comparison of basal and agonist-stimulated phosphatidylinositol hydrolysis in COS-m6 cells expressing either the mutant
1a-S290/293-AR subtype or the
1b-S288-294-AR subtype. As can be seen, the
mutant 1a-S290/293-AR subtype displayed an
elevated basal activity and a higher agonist-stimulated
phosphatidylinositol hydrolysis activity compared with the mutant
1b-S288-294-AR subtype. In fact, the mutant
1b-S288-294-AR subtype had to be expressed
at a five times greater receptor density in order to observe similar
levels of basal and agonist-stimulated activity as the mutant
1a-S290/293-AR subtype, suggesting that the
mutant 1b-S288-294-AR subtype had a lower
potency in activating the phosphatidylinositol hydrolysis signaling
pathway. These data indicate that, even though they are closely related
structurally, the mutant 1a-S290/293-AR and
1b-S288-294-AR subtypes differ functionally
in terms of their relative abilities to stimulate the
phosphatidylinositol hydrolysis signaling pathway. Although
interesting, meaningful interpretation of these results is difficult
since COS-m6 cells do not normally express the 1a- and
1b-ARs and, thus, are unlikely to possess the
physiologically appropriate G proteins and downstream signaling
components. To circumvent this problem, we decided to study the
signaling properties of the mutant
1a-S290/293- and
1b-S288-294-AR subtypes in another type of
cells in which these receptor subtypes are normally expressed. For this
purpose, we selected neonatal rat cardiac myocytes.
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Fig. 5.
Effect of expressing the
1a-S290/293-AR and
1b-S288/294-AR mutant
on basal and phenylephrine-stimulated phosphatidylinositol hydrolysis
in COS-m6 cells. Cells were stimulated with either 100 µM phenylephrine or vehicle (unstimulated), and the
accumulation of inositol phosphates was measured as described under
Fig. 2 or under "Experimental Procedures." The mean receptor
expression levels (Bmax) shown were measured
simultaneously using a whole cell binding assay. Receptor expression
levels were obtained using 2.6 µg/ml cDNA/transfection for the
1a-S290/293-AR and 0.008 and 0.2 µg/ml
DNA/transfection for the 369 and 1306 fmol/mg membrane protein
densities of the 1b-S288-294 mutant. Each
value represents the mean ± S.E. of at least four individual
experiments performed in duplicate. Data shown have had subtracted the
total inositol phosphate accumulation measured in cells transfected in
the presence of vector alone (28,854 ± 6160 dpm/mg protein;
n = 4). ##, p < 0.01 compared with
corresponding 1a-S290/293 value.
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Comparison of the Signaling Properties of the Constitutively Active
1a-S290/293 and
1b-S288/294-AR Subtypes in Rat Cardiac
Myocytes--
In a previous study, we showed that both the
1A- and 1B-AR subtypes are expressed in
neonatal rat cardiac myocytes (7). Activation of both the phospholipase
C and MAPK signaling pathways has been demonstrated upon addition of
1 agonists to cardiac myocytes (1, 7, 27, 30). Moreover,
several studies have demonstrated that the combined activation of these
receptor subtypes is associated with stimulation of several signaling
pathways, including stimulation of phosphatidylinositol hydrolysis (19) and activation of ERK (24), JNK (25), and p38 kinase (26). However, the
definitive assignment of these signaling pathways to the activation of
the individual 1A- and 1B-AR subtypes has been hampered by the lack of pharmacological tools with the requisite subtype specificity. With the development of constitutively activated 1a-AR and 1b-AR subtypes, we now have the
molecular tools to examine their respective signaling properties in the
appropriate cellular context in which the wild type 1-AR
receptors are normally expressed, but in the absence of agonist to
stimulate the various wild type 1-AR subtypes.
Whereas standard gene transfer methods were effective for introducing
luciferase reporter plasmids into cardiac myocytes, they were
ineffective for expression of the constitutively active 1-AR subtypes in myocytes. However, we recently
demonstrated the first successful use of recombinant Sindbis viruses to
express G protein subunits in cardiac myocytes.3 This
viral infection procedure was found to provide a rapid and efficient
method to introduce genes into cardiac myocytes, with greater than 90%
of cardiac myocytes being successfully targeted. Thus, activation of
signaling pathways in response to the introduction of the individual
constitutively activated receptor subtypes can be measured on the whole
cell population. Therefore, to utilize this method, we
constructed recombinant Sindbis viruses encoding the
constitutively activated 1a-S290/293-AR
and 1b-S288-294-AR subtypes. To control for
any nonspecific effects of Sindbis virus infection, a recombinant
Sindbis virus containing the bacterial lacZ gene or pSinRep5
vector was utilized. The expression of the constitutively activated
1a-S290/293-AR and
1b-S288-294-AR subtypes was quantitated by
radioligand binding. As shown in Fig. 6,
uninfected cardiac myocytes and myocytes infected with the LacZ Sindbis
virus expressed similar numbers of 1-ARs
(Bmax = 270 and 178 ± 16 fmol/mg protein
for control and LacZ virus-infected cells, respectively). By contrast,
cardiac myocytes infected with either the
1a-S290/293-AR Sindbis virus or the
1b-S288-294-AR Sindbis virus expressed
approximately 10-fold higher receptor densities than myocytes infected
with the LacZ Sindbis virus (Bmax = 2120 ± 219 and 1518 ± 350 fmol/mg protein for
1a-S290/293-AR virus-infected and
1b-S288-294-AR virus-infected cells,
respectively).
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Fig. 6.
Expression of endogenous and transfected
mutant 1-AR subtypes in cardiac
myocytes. The level of expression of endogenous and mutant
1-AR subtypes was measured by radioligand binding of
[125I]HEAT to cardiac myocyte membranes as described
under "Experimental Procedures." [125I]HEAT binding
was performed on 0.01 mg of membranes derived from untransfected
cardiac myocytes (control, n = 2) or myocytes
transfected with either LacZ (n = 3), or the
1a-S290/293 mutant (n = 3),
or the 1b-S288-294 mutant
(n = 4) for 48 h. Each value represents mean ± S.E.
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By having confirmed receptor expression, we examined which signaling
pathways were activated in cardiac myocytes expressing either the
constitutively active 1a-S290/293-AR subtype
or the 1b-S288-294-AR subtype. Fig.
7 shows a comparison of basal and
phenylephrine-stimulated phosphatidylinositol hydrolysis in cardiac
myocytes expressing similar levels of constitutively activated
1a-S290/293-AR and
1b-S288-294-AR subtypes. Under the basal
condition (30 min with 10 mM LiCl in the absence of
agonist), cells expressing the constitutively activated
1a-S290/293-AR subtype displayed a 2.4-fold
increase in inositol phosphate accumulation compared with the LacZ
cells, which showed no enhancement of inositol phosphates accumulation
during the 30 min of incubation with 10 mM LiCl. On the
other hand, cells expressing the constitutively activated
1b-S288-294-AR subtype showed no increase
in inositol phosphates accumulation during the 30 min of incubation
with 10 mM LiCl, which makes them indistinguishable from
the LacZ cells (Fig. 7A). Under the agonist-stimulated condition, phenylephrine produced a 6-fold increase in inositol phosphates accumulation in the LacZ cells. Cells expressing the constitutively active 1a-S290/293-AR subtype
showed a further 2-fold increase in phenylephrine-stimulated inositol
phosphate accumulation compared with the LacZ cells, whereas the
phenylephrine-stimulated activity in the cells expressing the
constitutively activated 1b-S288-294-AR
subtype was indistinguishable from that observed in the LacZ cells
(Fig. 7B). These data clearly demonstrate that the
constitutively activated 1a-S290/293-AR
subtype couples to the phosphatidylinositol hydrolysis signaling pathway in both the absence and presence of agonist in cardiac myocytes, which confirms and extends previous pharmacological data from
our own laboratory (7). By contrast, the constitutively activated
1b-S288-294-AR subtype was not able to
couple to the phosphatidylinositol hydrolysis signaling pathway in
cardiac myocytes.
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Fig. 7.
Effect of expressing the
1a-S290/293-AR or the
1b-S288/294-AR mutant on
basal and phenylephrine-stimulated phosphatidylinositol hydrolysis
in cardiac myocytes. Myocytes were transfected for 48 h with
Sindbis virus capable of expressing the LacZ, the
1a-S290/293-AR mutant, or the
1b-S288-294-AR mutant and labeled with
myo-[3H]inositol for 24 h as described
under "Experimental Procedures." Cells were treated with either 10 mM LiCl (basal) (A) or 100 µM
phenylephrine, 1 µM propranolol, and 10 mM
LiCl (B) for 30 min, rapidly quenched, and total inositol
phosphates measured as described under "Experimental Procedures."
Each value represents mean ± S.E. of 6-12 experiments. Data
shown have had subtracted the total inositol phosphate accumulation in
un-stimulated (basal) LacZ-transfected cells incubated in the presence
of 10 mM LiCl for 30 min (1403 ± 144 dpm).
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Comparison of the MAPK Signaling Pathways of the Constitutively
Active 1a-S290/293 and
1b-S288/294-AR Subtypes in Rat Cardiac
Myocytes--
Next, we examined the MAPK signaling pathway. Fig.
8 shows a comparison of basal and
phenylephrine-induced activation of MAPK activity in cardiac myocytes
expressing similar levels of the activated
1a-S290/293-AR and
1b-S288-294-AR subtypes. Under the basal
condition (in the absence of agonist), cardiac myocytes expressing the
constitutively activated 1a-S290/293-AR
subtype showed a slight activation (1.5-fold), with the level of active
MAPK being similar to that observed in LacZ cells (Fig. 8A).
By contrast, cells expressing the constitutively activated 1b-S288-294-AR subtype displayed marked
activation, with the level of active MAPK being 5.8-fold above that
observed in LacZ cells. Under the agonist-stimulated condition,
phenylephrine produced a modest increase in the level of active MAPK in
the LacZ cells, which was less than the level of active MAPK observed
in the 1b-S288-294-AR subtype in the
absence of agonist. In cells expressing the constitutively activated
1a-S290/293-AR subtype, phenylephrine
stimulation increased the level of active MAPK only marginally
(1.3-fold) compared with that in the phenylephrine-stimulated LacZ
cells. In contrast, cells expressing the constitutively activated
1b-S288-294-AR subtype showed a further
3.3-fold increase in the agonist-stimulated level of active MAPK
compared with that in the phenylephrine-stimulated LacZ cells (Fig.
8B). These results clearly demonstrate that the constitutively activated 1b-S288-294-AR
subtype effectively couples to the MAPK signaling pathway both in the
absence and presence of agonist in cardiac myocytes, whereas the
constitutively activated 1a-S290/293-AR
subtype shows little or no ability to couple to the MAPK signaling pathway in cardiac myocytes. Not only was the strict and specific coupling of the 1b-S288-294-AR subtype to
the MAPK kinase signaling pathway, and the
1a-S290/293-AR subtype to the
phosphatidylinositol hydrolysis pathway unexpected, this specificity
was observed even though the mutant receptor subtypes were being
expressed at 10-fold higher levels than the wild type receptor
subtypes.
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Fig. 8.
Effect of expressing the
1a-S290/293-AR or the
1b-S288/294-AR mutant on
basal and phenylephrine-stimulated MAPK activation in cardiac
myocytes. Myocytes plated on 35-mm wells were transfected for
48 h with viruses capable of expressing LacZ, the
1a-S290/293-AR, or the
1b-S288-294-AR mutant. Myocytes were
treated with vehicle or 100 µM phenylephrine for 5 min,
rapidly quenched, and analyzed for the presence of active MAPK as
described under "Experimental Procedures." A shows the
basal level of active MAPK, whereas B shows the effect of
phenylephrine on the level of active MAPK in LacZ, in the
1a-S290/293-AR mutant and/or in the
1b-S288-294-AR mutant expressing cardiac
myocytes. The lower section of each panel shows a
representative immunoblot of the detection of active MAPK (ERK1 and
ERK2) by the anti-active MAPK polyclonal antisera. Immunoblots stripped
and reprobed with pan-ERK1/2 monoclonal antibody for determination of
protein loading/lane showed equal amounts of ERK1/2 per lane (data not
shown). Bar graphs represent quantification of the amount of
active ERK2 (p42 kDa MAPK) by densitometric quantification and are the
mean ± S.E. for 5-9 experiments.
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Comparison of Gene Activation by the Signaling Pathways of the
Constitutively Active 1a-S290/293 and
1b-S288/294-AR Subtypes in Rat Cardiac
Myocytes--
We next examined the effect of the two signaling
pathways on downstream events at the level of gene regulation in
cardiac myocytes. In order to assess the impact of the two divergent
signaling pathways activated by the constitutively activated
1a-AR and 1b-AR subtypes, we chose to
measure the downstream effect on ANF and c-fos cardiac gene
transcription. Both of these genes are activated during
norephinephrine-induced cardiac hypertrophy in the heart, and as such
represent important targets for the 1A-AR and
1B-AR subtype signaling pathways. The ANF gene is activated by the 1-AR receptor agonist, phenylephrine,
through multiple promoter elements (AP-1, SP1, A/T, and SRE, see Refs. 27-31), and is linked to activation of PI hydrolysis (32-36).
Moreover, previous reports have suggested that the 1A-AR
subtype mediates the activation of ANF gene expression (33, 37). The
early response genes (c-fos, c-jun, and
c-myc) are activated by stimulation of 1-ARs
(36, 38-43). The c-fos gene is up-regulated during cardiac
hypertrophy of myocytes (44-45). Therefore, we investigated the affect
of the constitutively activated
1a-S290/293-AR and
1b-S288-294-AR subtypes on c-fos
gene regulation in cardiac myocytes.
As the c-fos gene is regulated by the (SRE) element in the
promoter, we utilized luciferase gene expression reporter constructs coupled to the SRE promoter element. To measure ANF and
c-fos gene activation, we measured the activation of the
SRE- and the ANF-luciferase gene reporter constructs in cardiac
myocytes co-transfected with the activated
1a-S290/293-AR and
1b-S288-294-AR subtypes. Fig.
9 shows a comparison of luciferase gene
activity in transfected cardiac myocytes expressing similar levels of
the activated 1a-S290/293-AR and
1b-S288-294-AR subtypes. In the absence of
agonist, cells expressing the constitutively activated
1a-S290/293-AR subtype and the
ANF-luciferase reporter construct showed a 2.4-fold increase in the
level of luciferase activity (Fig. 9). By contrast, cells expressing
the constitutively activated 1b-S288-294-AR
subtype and the ANF-luciferase reporter construct displayed no
significant increase in the level of luciferase activity compared with
control. In similar experiments in the absence of agonist, cells
expressing the constitutively activated
1b- S288-294-AR subtype and the
SRE-luciferase reporter plasmid displayed a marked 4.7-fold increase in
the level of luciferase activity. By contrast, the constitutively
activated 1a- S290/293-AR and the
SRE-luciferase construct showed no significant increase in the level of
luciferase activity compared with control. Taken together, these data
have several important ramifications. First, the
1a-S290/293-AR subtype preferentially
activates ANF-luciferase activity compared with
SRE/c-fos-luciferase activity. This is consistent
with the ability of 1a-S290/293-AR to
activate PI hydrolysis. Second, the lack of
1b-S288-294-AR subtype stimulation of
ANF-luciferase activity is consistent with the inability of the
1b-S288-294-AR subtype to activate PI
hydrolysis (Fig. 7). Third, the
1b-S288-294-AR subtype activation of
SRE/c-fos-luciferase activity compared with ANF-luciferase
correlates with its ability to activate MAPK activity. Taken together,
these results indicate that the
1a-S290/293-AR and
1b-S288-294-AR subtypes couple to distinct
effector pathways and physiological responses in rat cardiac myocytes.
These data confirm and extend previous pharmacological results that
reached a similar conclusion (7).
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Fig. 9.
Effect of expressing the
1a-S290/293-AR or the
1b-S288/294-AR mutant on
the activation of transfected SRE-luciferase and ANF-luciferase gene
reporter plasmids in cardiac myocytes. Myocytes plated on 60-mm
dishes were transfected for 24 h with luciferase reporter
constructs (SRE-luciferase or ANF-luciferase) using LipofectAMINE Plus
and then after 24 h with viruses capable of expressing LacZ or
pSindRep5 vector, the 1a-S290/293-AR mutant,
or the 1b-S288-294-AR mutant for an
additional 24 h. Cell lysates were isolated 48 h after the
initial transfection and assayed for luciferase gene expression and
-galactosidase expression. The fold induction is relative to cells
transfected with only the luciferase reporter construct, and the values
have been corrected for transfection efficiency with -galactosidase
enzyme values.
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DISCUSSION |
Constitutively Activated Receptors--
Constitutively activated
receptors demonstrate various properties that set them apart from their
wild type counterparts, but it is their ability to activate signaling
pathways in the absence of agonist that makes them valuable molecular
tools. Particularly in the case of the 1-ARs, where
pharmacological tools do not
1a- and the
§,
TOP
ABSTRACT
INTRODUCTION
