Hierarchy of Polymorphic Variation and Desensitization
Permutations Relative to
1- and
2-Adrenergic Receptor Signaling*
Deborah A.
Rathz
,
Kimberly N.
Gregory
,
Ying
Fang
,
Kari M.
Brown§, and
Stephen B.
Liggett
§¶
From the Departments of
Pharmacology and
§ Medicine, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45267-0564
Received for publication, June 18, 2002, and in revised form, January 3, 2003
 |
ABSTRACT |
Agonist-promoted desensitization of
G-protein-coupled receptors results in partial uncoupling of receptor
from cognate G-protein, a process that provides for rapid adaptation to
the signaling environment. This property plays important roles in
physiologic and pathologic processes as well as therapeutic efficacy.
However, coupling is also influenced by polymorphic variation, but the relative impact of these two mechanisms on signal
transduction is not known. To determine this we utilized recombinant
cells expressing the human
1-adrenergic receptor
(
1AR) or a gain-of-function polymorphic variant
(
1AR-Arg389), and the
2-adrenergic receptor (
2AR) or a
loss-of-function polymorphic receptor
(
2AR-Ile164). Adenylyl cyclase activities
were determined with multiple permutations of the possible states of
the receptor: genotype, basal, or agonist stimulated and with or
without agonist pre-exposure. For the
1AR, the enhanced
function of the Arg389 receptor underwent less
agonist-promoted desensitization compared with its allelic counterpart.
Indeed, the effect of polymorphic variation on absolute adenylyl
cyclase activities was such that desensitized
1AR-Arg389 signaling was equivalent to
non-desensitized wild-type
1AR; that is, the genetic
component had as much impact as desensitization on receptor coupling.
In contrast, the enhanced signaling of wild-type
2AR
underwent less desensitization compared with
2AR-Ile164, thus the heterogeneity in
absolute signaling was markedly broadened by this polymorphism. Inverse
agonist function was not affected by polymorphisms of either subtype. A
general model is proposed whereby up to 10 levels of signaling by
G-protein-coupled receptors can be present based on the influences of
desensitization and genetic variation on coupling.
 |
INTRODUCTION |
Like a number of other G-protein-coupled receptors, the
1- and
2-adrenergic receptors
(
1AR and
2AR,1
respectively) undergo desensitization during continuous exposure to
agonist. Such desensitization occurs maximally after several minutes of
agonist exposure and is due to decreased interaction with
Gs, which is evoked by receptor phosphorylation (1). Thus the signal transduction of these receptors can be characterized as one
of two potential conditions or states, defined here as "control"
(no recent exposure to agonist) and "desensitized." However, we
have recently shown that an alteration in receptor-Gs coupling can also be imposed by genetic mechanisms. A single nucleotide polymorphism found in the
1AR gene in the human
population (2, 3) results in either Gly or Arg being encoded at amino
acid position 389 of the proximal portion of the cytoplasmic tail. In
studies using transfected cells with equivalent expression of the two
receptors, the
1AR-Arg389 displays an
increase in Gs coupling compared with
1AR-Gly389 (2). So, one can consider that
the human
1AR can exist in four agonist-stimulated
states: Gly389 control and desensitized, and
Arg389 control and desensitized. And, since basal
(non-agonist) activity is also affected by these genetic and
desensitization processes, eight states can be considered. For the
2AR, a polymorphism that results in a substitution of
Ile for Thr at amino acid 164 in the fourth transmembrane-spanning
domain results in a decrease in Gs coupling (4). Thus,
eight such states can be considered for the
2ARs as
well, again based on genotype and desensitization status. As opposed to
their allelic variants, only the
1AR-Gly389
and the
2AR-Thr164 (the receptors often
designated as "wild-type") have been studied in regards to
desensitization in recombinantly expressing cells (5, 6). Based on the
significant impact that both desensitization and polymorphic variation
have on coupling, we considered that control signaling with one variant
might even be equivalent to the desensitized signaling of the other.
Since inverse agonists act to lower the frequency of spontaneous
activation of
ARs, there is the potential for coupling polymorphisms
to influence this response as well. Knowing the hierarchy of these
states facilitates understanding the molecular basis of receptor
response to therapeutic agents and receptor dysfunction that can occur
in pathologic states, where both genetic and post-translational
modifications occur together. Such studies also provide for a general
model that depicts the interaction of genetic and desensitization
mechanisms in G-protein-coupled receptor signaling. To investigate
this, we expressed these four receptors in Chinese hamster fibroblasts
at equivalent levels and studied the relative effects of these genetic
modifications and those of short term agonist-promoted desensitization
on receptor function.
 |
EXPERIMENTAL PROCEDURES |
Constructs and Transfections--
Site-directed mutagenesis was
performed on the wild-type cDNA templates as described previously
so as to mimic the human Arg389
1AR and
Ile164
2AR variants (2, 4). Wild-type and
polymorphic cDNAs were cloned into the mammalian expression vector
pBC12B1. CHW-1102 cells were stably transfected by calcium phosphate
precipitation. Positive clones were selected based on resistance to 300 µg/ml G418. Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C, 5%
CO2, in 100 µg/ml streptomycin, 100 units/ml penicillin,
and 80 µg/ml G418. COS-7 cells were transiently transfected and
maintained as described (7).
Radioligand Binding--
Confluent layers of CHW cells were
washed three times with cold phosphate-buffered saline, lysed in
hypotonic buffer (5 mM Tris, 2 mM EDTA, pH
7.4), and mechanically detached with a rubber policeman in a small
volume. The particulates were homogenized with a polytron and then
centrifuged at 42,000 × g for 10 min. Pellets were
then resuspended in 75 mM Tris, 5 mM
MgCl2, 2 mM EDTA, pH 7.4. Expression levels
were determined in saturation binding assays. Membranes were incubated
with 400 pM 125I-cyanopindolol and 100 µM GTP for 2 h at room temperature with nonspecific
binding determined in the presence of 1 µM propranolol. Reactions were stopped by dilution and vacuum filtration over Whatmann
glass fiber filters. The percentage of the receptor pool that is
expressed at the cell surface was determined exactly as described
previously (8). Briefly, cells were homogenized as above, centrifuged
at 400 × g for 10 min, and the supernatant layered
over a 35% sucrose cushion and centrifuged at 150,000 × g for 1.5 h. The 0-35% interface (light vesicular
membranes) and the pellet (plasma membranes) were collected, diluted in
5 mM Tris, 2 mM EDTA, pH 7.4, and centrifuged
at 200,000 × g for 1 h. Radioligand binding with
125I-cyanopindolol was then carried out with each fraction
as described above.
Adenylyl Cyclase Activities, cAMP Measurements, and
Desensitization Protocol--
Confluent monolayers of cells were
washed twice with Hanks' balanced salt solution and allowed to
equilibrate in fresh DMEM for 30 min at 37 °C, 5% CO2.
Cells were then incubated with either a 10 µM
concentration of the indicated agonist with 100 µM
ascorbic acid or with ascorbic acid alone (control) for 20 min, washed five times with cold phosphate-buffered saline, detached, and membranes
prepared as above. Membranes were incubated with 30 mM
Tris, pH 7.4, 2 mM MgCl2, 0.8 mM
EDTA, 120 µM ATP, 60 µM GTP, 100 µM cAMP, 2.8 mM phosphoenolpyruvate, 2.2 µg
myokinase, the indicated concentrations of agonist, and 1 µCi of
[
-32P]ATP for 30 min at 37 °C as described
previously (2). The stop buffer contained a [3H]cAMP
standard, which accounted for column recovery. [32P]cAMP
was separated from [
-32P]ATP chromatographically using
alumina columns. Untreated (control) cells bearing the two
1ARs are designated Arg389C and
Gly389C, while those studied after treatment
with a 10 µM concentration of the agonist epinephrine
(desensitized) are designated Arg389D and
Gly389D. Similarly, control
2AR
are designated Thr164C and
Ile164C, while the desensitized state has the
subscript "D." An additional qualifier, based on whether the
adenylyl cyclase response is in the absence of agonist (basal, B) or in
response to isoproterenol (I), provides for eight different
permutations. To ascertain the effects of inverse agonists, COS-7 cells
were transfected with the indicated receptors and G
s. At
confluence, cells in 24-well plates were washed and incubated with 100 µM isobutylmethylxanthine with or without varying
concentrations of inverse agonists for 45 min. cAMP produced over this
time was quantitated by a competitive immunoassay (Amersham Biosciences).
Miscellaneous--
Protein concentrations were determined by the
copper bicinchoninic acid method (9). Curve fitting was carried out
with PRISM software (GraphPad, San Diego, CA). Dose-response curves were compared by ANOVA with post-hoc t tests when the
p value was <0.05. Other results were compared with paired
t tests as indicated. Data are presented as means ± S.E.
 |
RESULTS |
Expression levels of the two
1AR variants in the
membrane preparations utilized for the adenylyl cyclase assays were
206 ± 16 for Gly389 and 170 ± 22 fmol/mg for
Arg389. Likewise, the two
2AR cell lines had
similar levels of expression (Thr164 = 783 ± 88, Ile164 = 1104 ± 111 fmol/mg). Of note, signaling
characteristics were compared between the two
1AR
variants or between the two
2AR variants, but not
between subtypes. There was no evidence for relevant intracellular
accumulation of either polymorphic variant as determined by radioligand
binding of light vesicular membrane and plasma membrane fractions
(Table I). Our initial goal was to assess
the degree of agonist-promoted desensitization for the wild-type
1AR (Gly389) and the Arg389
polymorphic receptor, and the wild-type
2AR
(Thr164) and its variant, Ile164. Concerning
the
1ARs, we knew from previous studies that basal and
agonist-stimulated adenylyl cyclase activities (in absolute values)
were higher for the Arg389
1AR compared with
the Gly389 receptor. For the current work, cells in culture
were exposed to vehicle or agonist for 20 min, washed, membranes
prepared, and adenylyl cyclase activities determined. When
desensitization is quantitated as the percent decrease of the response
relative to that in the absence of agonist pretreatment, the
1AR-Gly389 underwent 21 ± 6.7%
agonist-promoted desensitization (Fig.
1a, Table I). No change in the
EC50 was observed. The hyperfunctional
1AR-Arg389 underwent a greater degree of
desensitization compared with its allelic variant, amounting to 34 ± 4.1% desensitization (p < 0.01 versus
1AR-Gly389, Fig. 1b). For the
2ARs, we also found that the two polymorphic variants
differed in the extent of agonist-promoted desensitization (Fig. 1,
c and d). However, in contrast to what was
observed with the
1AR, the hyperfunctional
2AR-Thr164 actually underwent decreased
desensitization.
2AR-Thr164 displayed
26 ± 4.0% desensitization versus 37 ± 4.6%
found for Ile164-
2AR (p < 0.05).
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Table I
Adenylyl cyclase activation under control and desensitized conditions
for the polymorphic 1- and 2-adrenergic receptors
Results are from five to seven independent experiments. There was a
significant relationship between genotype and basal and maximal
isoproterenol stimulated adenylyl cyclase activities (p < 0.001 by ANOVA). The subscripts C and D denote activities under
control (no agonist pretreatment) and desensitized (with agonist
pretreatment) conditions, respectively. Iso = maximal
isoproterenol stimulated values.
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Fig. 1.
Agonist-promoted desensitization of
polymorphic 1AR and
2AR. Adenylyl cyclase activities
were determined in membranes from CHW cells expressing the indicated
receptors. Cells were exposed to vehicle (control) or 10 µM epinephrine (desensitized) for 20 min as described
under "Materials and Methods." Data are normalized to the control
maximal response after subtraction of basal levels. Shown are results
from four experiments. For the 1AR, the desensitization
was greater for the Arg389 compared with the
Gly389 variant (34 ± 4.1% versus 21 ± 6.7%, p < 0.01). For the 2AR, the
Ile164 receptor displayed greater desensitization than the
Thr164 allelic counterpart (37 ± 4.0%
versus 26 ± 4.0%, p < 0.05).
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|
Although the above data examine the extent of desensitization as a
percentage of the control response, the absolute levels of adenylyl
cyclase activities (pmol/min/mg) establish a hierarchy of signal
transduction based on genotype and desensitization. For the
1AR, these data are shown in Fig.
2a. As is seen, the influence
of genetic variation was such that even after desensitization, the
maximal Arg389 receptor function (ArgDI) was
equivalent to the maximal non-desensitized Gly389 variant
(GlyCI). The rank order of activities for the various states for the
1AR are: ArgCI > GlyCI = ArgDI > GlyDI > ArgCB > ArgDB > GlyCB
GlyDB. For the
2AR, since the genetically
uncoupled Ile164 receptor underwent an even greater degree
of desensitization than the wild-type (Fig. 1, c and
d), the heterogeneity in adenylyl cyclase activities due to
the various permutations was substantial. The eight states are shown in
Fig. 2b. The rank order was thus: Thr164CI > Thr164DI > Ile164CI > Ile164DI > Thr164CB > Thr164DB > Ile164CB = Ile164DB.

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Fig. 2.
Effects of desensitization on absolute
activities of adenylyl cyclase stimulation by polymorphic
1AR and
2AR. Shown are data from
experiments described in the legend to Fig. 1 plotted as absolute
adenylyl cyclase activities (pmol/min/mg). Subscripts indicate control
(C) or desensitization (D) conditions. For both
1AR and 2AR there was a relationship
between genotype, desensitization status, and activities
(p < 0.005 by ANOVA). Basal and maximal stimulated
activities were all different from their allelic or desensitization
counterparts except for 1AR-GlyC and
ArgD maximal stimulations, which were the same.
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We also explored whether the polymorphic variations affected the
response to inverse agonists. We considered that the conformational effects of these substitutions might constrain the receptor so that it
could not be fully "inactivated" (i.e. decreased
spontaneous activation) by the binding of inverse agonists. We were
unable to obtain consistent results in CHW cells, likely due to the low levels of basal adenylyl cyclase activity and the relatively low expression levels in the stable lines. Thus COS-7 cells were
transfected with the various receptors along with G
s,
exposed to the phosphodiesterase inhibitor isobutylmethylxanthine, and
whole cell cAMP production over the ensuing 30 min determined in the
absence or presence of various concentrations of the inverse agonists
CGP-20712 (
1AR) or ICI-118551 (
2AR).
Here, it is the absolute levels of cAMP that are relevant rather than a
percent change. Results are shown in Fig.
3. The basal levels of cAMP were greater
for
2AR Thr164 compared with
2AR Ile164, as might have been predicted
from the membrane studies. Interestingly, a similar difference between
the two
1ARs, which also might be expected, was not
observed. This may be because spontaneous (i.e. non-agonist)
activation is less apparent with
1AR (10), and thus,
differences in the two variants may not be as readily discerned. Regarding the responses to inverse agonists for the
1ARs, exposure to CGP-20712 resulted in a
dose-dependent decrease in cAMP production for both
receptors. The response was identical for both, with minimal cAMP
levels of 7.1 ± 0.8 and 7.0 ± 0.9 pmol/mg for
Arg389 and Gly389 (n = 5).
Similarly, for the
2ARs the inverse agonist ICI-118551 lowered cAMP to the same levels for both receptors (Thr164 = 7.4 ± 0.1, Ile164 = 6.5 ± 1.0 pmol/mg,
n = 4). These results indicate that the responses to
inverse agonists are not influenced by these genetic variations.

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Fig. 3.
Efficacy of inverse agonists acting at
polymorphic 1AR and
2AR. Accumulation of whole cell
cAMP was determined over a 45-min period after addition of 100 µM isobutylmethylxanthine in the absence or presence of
varying concentrations of the inverse agonist CGP-20712
( 1AR) or ICI-118551 ( 2AR). Shown are the
maximal responses from four to five experiments. The absolute levels of
cAMP accumulation in the presence of inverse agonist were not different
between genotypic variants. Expressions were 1AR
Arg389 = 4.8 ± 0.6, 1AR
Gly389 = 3.7 ± 0.7, 2AR
Thr164 = 2.1 ± 0.34, 2AR
Ile164 2.2 ± 0.39 pmol/mg.
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 |
DISCUSSION |
The dynamic nature of signaling by G-protein-coupled receptors has
been considered indicative of the ability of these receptors to rapidly
adapt to changes in their signaling environment. Processes such as
receptor desensitization are critical for maintenance of homeostasis
during normal physiological circumstances, may be compensatory in
certain pathological states, or may aberrantly alter signaling and
contribute to characteristics of disease states (11). Desensitization
may also limit the effectiveness of therapeutically administered
agonists (tachyphylaxis). Using recombinantly expressed receptors, the
nature of rapid agonist-promoted desensitization of many
G-protein-coupled receptors has been explored. Some receptors, such as
the
2CAR and the
3AR, display little or
no rapid desensitization (12, 13). Others display a range of
desensitization attributed to various mechanisms including
phosphorylation by G-protein-coupled receptor kinases, or second
messenger-dependent kinases such as cAMP-dependent protein kinase or protein kinase C. Such comparative studies can be helpful in drug design or understanding
selected features of disease states. With the recent elucidation of
polymorphic variants of receptors such as the
1- and
2AR, which have significant functional impact on
receptor coupling, the potential interaction between desensitization
and genetic variation needs to be considered so as to establish how
receptor signaling is influenced by both processes.
Here we have carried out studies with polymorphic
1ARs
(2), which have either Gly or Arg at amino acid 389. This residue is
located in the cytoplasmic portion of the receptor, within a predicted
helix formed between the seventh transmembrane-spanning domain and
the membrane-anchoring palmitoylated cysteine(s) (14). Given the steric
properties of Gly within
helices, and the importance of this region
for G-protein binding, it is not surprising that functional coupling is
different between the Arg and Gly
1AR variants. The
2AR polymorphism (4) consists of a substitution of Ile
for Thr in the fourth transmembrane-spanning domain, and also displays
altered coupling to Gs, likely due to changes in the
agonist binding pocket that affects the conformation of the intracellular loops. A priori, it was not clear whether
these polymorphisms would enhance or depress agonist promoted
desensitization. For example, the robust signaling of
1AR-Arg389 might render it less likely to
desensitize; conversely, since its conformation is more favorable for
Gs coupling, it could be more sensitive to
G-protein-coupled receptor kinase-mediated phosphorylation, which is
dependent on the receptor being in the active conformation.
We show here that there is a significant impact of these polymorphisms
on agonist-promoted desensitization. In the case of
1AR
function, the desensitized hyperfunctional variant (Arg389)
is equivalent to that of the non-desensitized Gly389
receptor. Since the basal level of signaling, which represents spontaneous conversion to R*, is also relevant, the number of permutations, taking into account basal or agonist stimulation, non-desensitized or desensitized, and two polymorphic variants, for the
1AR amounts to eight. For the
1AR this is
graphically displayed in matrix format with the aforementioned states
in Fig. 4, which is useful for
considering a more generalized scheme of the role of genetic variation
in G-protein-coupled receptor signaling (see below). As is shown, there
is considerable variation in basal and agonist-stimulated activities
upon stratification by genotype and desensitization status. Such a
range of relative signaling efficacy, and the complex interactions
between desensitization and genotype, may explain the high degree of
interindividual variability in physiologic responses to agonists and
antagonists that has been observed (reviewed in Ref. 15). An additional
level of signaling can also be found when one considers the response to inverse agonists, which bind receptor and tend to stabilize the R
state, and thus minimize spontaneous conversion to R*. As such, Gs-coupled receptors display a decrease in basal adenylyl
cyclase activity/cAMP production. Depending on the nature of the
polymorphism, the response to inverse agonist could also be affected by
genetic variation. Interestingly, despite the other effects of these
polymorphisms, differences in inverse agonist efficacy were not
observed with either the
1AR or
2AR
variants, as cAMP levels were reduced by inverse agonists to the same
absolute levels regardless of genotype. (These values are not
incorporated into Fig. 4, since they were determined using a different
approach and are not directly comparable.)

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Fig. 4.
Matrix representation of relative adenylyl
cyclase activities of 1AR
stratified by genotype, desensitization status, and agonist
stimulation. Results are from the computer fit minimum and
maximal values from the mean curves from experiments in figure 2, normalized to agonist-stimulated adenylyl cyclase activities of
1AR-Arg389 in the non-desensitized (control)
state.
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For a general model (Fig. 5) of the
potential interaction of uncoupling events due to genetic variation and
to desensitization, we have made several assumptions to include
polymorphisms with various phenotypic effects on receptor signaling.
From prior characterization of agonist, receptor, and G-protein
interactions of the polymorphic
AR (2, 4), we have assumed that the
conformation of the receptor is altered by the polymorphisms under
study, thus the depiction of two active conformations
(RI*1, RII*3) based on
the two different receptors (RI and RII). It is
also assumed that when desensitized the conformation of the receptor is
altered by phosphorylation and
-arrestin interaction (16), and these
states are thus denoted as RI*2 and
RII*4. The "basal" (i.e.
non-agonist bound) signaling of a receptor, due to spontaneous toggling
to an active conformation, is also considered here as relevant, as is
the effect of desensitization on basal signaling. (Although not
affected with the
1AR or
2AR, the
potential for a coupling polymorphism to alter inverse agonist activity
is included in the general model.) The abundance of each of the above
species at equilibrium is indicated by the arrows and the subscripted
brackets. Within this general model 10 relevant levels of signaling,
due to the various states or abundance of a given species, are present
at steady state. Of note, other minor species of unknown signaling
significance, such as agonist-bound receptor that is not activated
(i.e. ARI), are not included.

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Fig. 5.
General model of the potential interaction of
coupling polymorphisms and desensitization. Shown are two
polymorphic receptors (RI, RII),
under conditions of agonist (A) or inverse agonist
(IA) occupancy. The desensitized state refers to recent
prior exposure to agonist in vivo. Different conformations
of the receptor are depicted as *1, *2,
*3, and *4, which depend on the effects of the
polymorphism and desensitization status. The italicized subscripts
outside the brackets indicate the relative abundance of a given
species. For example, at steady state, it would be expected that
k > j.
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From the standpoint of
AR subtypes in the heart, their functional
regulation has been linked to a variety of physiological states in
diseases such as heart failure. In human heart failure, myocardial
1AR and
2AR have been shown to be
desensitized. Along with receptor down-regulation, this response is
thought to be adaptive in that the pathologically altered heart with
limited physiologic and metabolic reserves is protected from constant stimulation by high circulating catecholamines. On the other hand, other studies in genetically altered mice have suggested that some
aspects of desensitization of
AR signaling may be maladaptive in
experimental heart failure (17). Recent studies have shown that
1AR or
2AR polymorphisms are associated
with certain physiologic or pathologic phenotypes in human heart
failure (18-21). However, prior to the current study it has not been
clear whether there was any potentiation, or attenuation, of
desensitization events by these polymorphisms. The in vitro
data presented here indicate that both desensitization and genetic
variation together can serve to set the ultimate level of signaling of
1AR and
2AR. Indeed, the signaling of
some receptors, even in the desensitized state, is equivalent to their
non-desensitized allelic variants. Stated another way, genetic
variation can have an effect of the same magnitude as that of
desensitization. Regarding
AR in heart failure, this may be
particularly important in defining patient subsets, tailoring
therapeutic regiments, or in the development of new agents (15, 22). As
a general paradigm, we present a model by which genetic variation and
desensitization of G-protein-coupled receptor signaling can be
considered as multiple states. Although the prevalence of functional
polymorphisms within the superfamily is not fully defined (23), many
G-protein-coupled receptors have been reported to be polymorphic in
their coding regions (24, 25), such that the model may be applicable to
multiple diverse signaling events by these receptors.
 |
ACKNOWLEDGEMENTS |
We thank Cheryl Theiss for cell culture and
Esther Getz for manuscript preparation.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants HL22619, HL52318, GM61376, and HD07463.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: University of
Cincinnati College of Medicine, 231 Albert Sabin Way, Rm. G-062, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: stephen.liggett@uc.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M206054200
 |
ABBREVIATIONS |
The abbreviations used are:
1AR
and
2AR,
1- and
2-adrenergic receptors, respectively;
Gs, stimulatory guanine nucleotide-binding protein;
C, control conditions;
D, desensitized conditions;
B, basal state of adenylyl cyclase
activation;
I, isoproterenol stimulated state of adenylyl cyclase
activation;
ANOVA, analysis of variance.
 |
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