From the Departments of Medicine and Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Received for publication, September 5, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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A polymorphic variant of the human
Like the Polymorphism Detection--
The sequence encoding the third
intracellular loop of the human Constructs and Cell Transfection--
To create the polymorphic
Adenylyl Cyclase Activities--
Radioligand Binding--
Expression of mutant and wild-type
Intact Cell Receptor Phosphorylation--
Transiently
transfected COS-7 cells expressing equivalent levels of each receptor
were grown to confluence and incubated with [32P]orthophosphate (~4 mCi/100-mm plate) for 2 h
at 37 °C in 5% CO2 atmosphere. Cells were then
incubated in the presence or absence of 100 µM
norepinephrine for 15 min, washed five times with ice-cold phosphate-buffered saline, and solubilized in 1 ml of a buffer containing 1% Triton X-100, 0.05% SDS, 1 mM EDTA, and 1 mM EGTA in phosphate-buffered saline, by rotation in a
microcentrifuge tube for 2 h at 4 °C. This and all subsequent
steps included the protease inhibitors benzamidine (10 µg/ml),
soybean trypsin inhibitor (10 µg/ml), aprotinin (10 mg/ml), and
leupeptin (5 µg/ml) and the phosphatase inhibitors sodium fluoride
(10 mM) and sodium pyrophosphate (10 mM). (A
separate flask was scraped in 5 mM Tris/2 mM
EDTA, and the membranes were prepared for radioligand binding as
described above.) Unsolubilized material was removed by centrifugation at 40,000 × g at 4 °C for 10 min. The hemagglutinin
epitope tagged Miscellaneous--
Protein determinations were by the copper
bicinchoninic acid method (28). Data from adenylyl cyclase and
radioligand binding assays were analyzed by iterative least square
techniques using Prizm software (GraphPad, San Diego, CA). Agreement
between genotypes observed and those predicted by the Hardy-Weinberg
equilibrium was assessed by a Chi-squared test with one degree of
freedom. Genotype comparisons were by Fisher's exact test. Comparisons of results from biochemical studies were by t tests, and
significance was considered when p < 0.05. Data are
provided as the means ± S.E.
Sequence analysis of the third intracellular loop of the
2B-adrenergic receptor (
2BAR),
which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in
Caucasians (31%) and to a lesser extent in African-Americans (12%).
The consequences of this deletion were assessed by expressing wild-type
and the Del301-303 receptors in Chinese hamster ovary and COS cells.
Ligand binding was not affected, although a small decrease in coupling
efficiency to the inhibition of adenylyl cyclase was observed with the
mutant. The deletion occurs within a stretch of acidic residues that is
thought to establish the milieu for agonist-promoted phosphorylation
and desensitization of the receptor by G protein-coupled receptor
kinases (GRKs). Agonist-promoted phosphorylation studies carried out in
cells coexpressing the
2BARs and GRK2 revealed that the
Del301-303 receptor displayed ~56% of wild-type phosphorylation.
Furthermore, the depressed phosphorylation imposed by the deletion was
found to result in a complete loss of short term
agonist-promoted receptor desensitization. Thus the major phenotype of
the Del301-303
2BAR is one of impaired phosphorylation
and desensitization by GRKs, and thus the polymorphisms renders the
receptor incapable of modulation by this key mechanism of dynamic regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
2-Adrenergic receptors
(
2AR)1 are
cell surface receptors for catecholamines that bind to the
Gi/Go family of G proteins, coupling to
multiple effector systems including inhibition of adenylyl cyclase
activity (1).
2AR are widely expressed within the
central and peripheral nervous system (2-4) and participate in a broad
spectrum of physiologic functions such as regulation of blood pressure
both centrally and within the vasculature, sedation, analgesia,
regulation of insulin release, renal function, and cognitive and
behavioral functions (5-12). Three human
2AR subtypes have been cloned and characterized (
2A,
2B, and
2C). The
2BAR has
a distinct pattern of expression within the brain, liver, lung, and
kidney, and recent studies using gene knockouts in mice have shown that
disruption of this receptor effects mouse viability (13), blood
pressure responses to
2AR agonists (13), and the
hypertensive response to salt loading (14).
2AAR subtype (15, 16), the
2BAR
undergoes short term agonist promoted desensitization (17). This
desensitization is due to phosphorylation of the receptor, which evokes
a partial uncoupling of the receptor from functional interaction with
Gi/Go (18, 19). Such phosphorylation appears to
be due to G protein-coupled receptor kinases (GRKs), a family of
serine/threonine kinases that phosphorylate the agonist-occupied
conformations of many G protein-coupled receptors (20). The process
serves to finely regulate receptor function providing for rapid
adaptation of the cell to its environment. Desensitization may also
limit the therapeutic effectiveness of administered agonists. For the
2BAR, phosphorylation of serines/threonines in the third
intracellular loop of the receptor is dependent on the presence of a
stretch of acidic residues in the loop that appears to establish the
milieu for GRK function (18). In this report we delineate the phenotype
of a common polymorphism of the
2BAR (21, 22), which
consists of a deletion of three glutamic acid residues in this region;
such a variation has a pronounced effect on receptor phosphorylation
leading to a loss of agonist-promoted desensitization.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
2BAR
(GenBankTM accession number AF005900) was examined for
polymorphic variation by performing polymerase chain reactions (PCRs)
to amplify this portion of the cDNA from genomic DNA derived from
blood samples. In this paper the adenine of the initiator ATG codon of
the open reading frame of the receptor is designated as nucleotide 1, and amino acid 1 is the encoded methionine. The human receptor consists of 450 amino acids. For initial examination, DNA from 39 normal individuals was utilized. Two overlapping fragments encompassing the
third intracellular loop region were generated using the following primer pairs: fragment 1 (534 bp), 5'-GCTCATCATCCCTTTCTCGCT (sense) and
5'-AAAGCCCCACCATGGTCGGGT (antisense) and fragment 2 (588 bp), 5'-CTGATCGCCAAACGAGCAAC (sense) and 5'-AAAAACGCCAATGACCACAG
(antisense). The 5' end of each sense and antisense primer also
contained sequences corresponding to the M13 forward
(5'-TGTAAAACGACGGCCAGT) and M13 reverse (5'-CAGGAAACAGCTATGACC)
universal sequencing primers, respectively. The PCR reactions consisted
of ~100 ng of genomic DNA, 5 pmol of each primer, 0.8 mM
dNTPs, 10% Me2SO, 2.5 units of Platinum Taq DNA
polymerase (Life Technologies, Inc.), 20 µl of 5× buffer J
(Invitrogen) in a 100-µl reaction volume. Reactions were started by
an initial incubation at 94 °C for 4 min, followed by 35 cycles of
94 °C for 30 s, 58 °C (fragment 1) or 60 °C (fragment 2)
for 30 s, and 72 °C for 1 min, followed by a final extension at
72 °C for 7 min. PCR reactions were purified using the QIAquick PCR
purification system (Qiagen), and automated sequencing of both strands
of each PCR product was performed using Applied Biosystems 370 sequencer using dye primer methods. As discussed below, a 9 bp in-frame
deletion beginning at nucleotide 901 was detected that resulted in a
loss of three glutamic acid residues at amino acid positions 301-303.
Thus, this polymorphism was denoted Del301-303. No other nonsynonymous
or synonymous polymorphisms were identified. PCR amplification of 209- and 200-bp fragments encompassing this polymorphic region allowed
screening of additional DNA samples whose genotypes were distinguished
by size when run on 4% Nuseive agarose gels. PCR conditions were the
same as described above except that buffer F was used with the
following primers: 5'-AGAAGGAGGGTGTTTGTGGGG (sense) and
5'-ACCTATAGCACCCACGCCCCT (antisense).
2BAR construct, a 1585-bp PCR product encompassing the
2BAR gene was amplified from a homozygous deletion
individual using the following primers: 5'-GGCCGACGCTCTTGTCTAGCC (sense) and 5'-CAAGGGGTTCCTAAGATGAG. This fragment was
digested and subcloned into the XcmI and BamHI
sites of the wild-type
2BAR sequence in the expression
vector pBC12BI (17). The integrity of the construct was verified by
sequencing. Chinese hamster ovary cells (CHO-K1) were stably
transfected by a calcium phosphate precipitation technique as described
previously using 30 µg of each receptor construct and 0.5 µg of
pSV2neo to provide for G418 resistance (23). Selection of
positive clones was carried out in 1.0 mg/ml G418, and expression of
the
2BAR from individual clonal lines was determined by
radioligand binding as described below. Several clonal lines with
matched expression levels between 500 and 1000 fmol/mg were utilized as
indicated. Cells were grown in monolayers in Ham's F-12 medium
supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 80 µg/ml G418 (to maintain selection
pressure) at 37 °C in a 5% CO2 atmosphere. For
phosphorylation experiments, receptors were epitope tagged with the
influenza hemagglutinin nonopeptide (YPYDVPDYA) at the amino terminus.
This was accomplished by constructing vectors using the
above constructs with insertions of in-frame sequence encoding the
peptide using PCRs essentially as described previously (24). Tagged
receptors were expressed at ~15 pmol/mg, along with GRK2 (
ARK1),
in COS-7 cells using a DEAE Dextran technique as described (16).
2AR inhibition
of adenylyl cyclase was determined in membrane preparations from CHO
cells stably expressing the two receptors using methods similar to
those described previously (25). Briefly, cell membranes (~20 µg)
were incubated with 27 µM phosphoenolpyruvate, 0.6 µM GTP, 0.1 mM cAMP, 0.12 mM ATP,
50 µg/ml myokinase, 0.05 mM ascorbic acid, and 2 µCi of
[
-32P]ATP in a buffer containing 40 mM
HEPES, pH 7.4, 1.6 mM MgCl2, and 0.8 mM EDTA for 30 min at 37 °C. Activities were measured in
the presence of water (basal), 5 µM forskolin, and 5 µM forskolin with the indicated concentrations of
agonists. Reactions were terminated by the addition of a stop solution
containing excess ATP and cAMP and ~100,000 dpm of
[3H]cAMP. Labeled cAMP was isolated by gravity
chromatography over alumina columns with [3H]cAMP used to
quantitate column recovery. Results are expressed as percentages of
inhibition of forskolin-stimulated activity. For desensitization
experiments, cells were pretreated for 30 min at 37 °C with medium
alone or with medium containing 10 µM norepinephrine,
placed on ice, and washed five times with cold phosphate-buffered
saline prior to membrane preparation. Desensitization of
2BAR is manifested by a shift to the right in the
dose-response curve for the inhibition of adenylyl cyclase
(i.e. increase in EC50) without a significant
change in the maximal response (17-19). To quantitate the magnitude of
this desensitization, the inhibitory response under control conditions
at a submaximal concentration of agonist (the EC50) in the
assay was determined from the curve and compared with the response to
this same concentration from membranes derived from cells exposed to
norepinephrine. This method has been previously validated (26) in
several G protein-coupled receptor systems.
2BAR was determined using saturation binding assays as
described (25, 27) with [3H]yohimbine or
[125I]aminoclonidine with 10 µM
phentolamine or 10 µM yohimbine, respectively, used to
define nonspecific binding. For competition studies, membranes were
incubated in 50 mM Tris-HCl, pH 7.4, 10 mM
MgSO4, 0.5 mM EDTA with 2.0 nM
[3H]yohimbine and 16 concentrations of the indicated
competitor in the absence or presence of guanine nucleotide for 30 min
at 25 °C. Reactions for the above radioligand binding studies were terminated by dilution with 4 volumes of ice-cold 10 mM
Tris-HCl pH 7.4 buffer and vacuum filtration over Whatman GF/C glass
fiber filters.
2BARs were immunoprecipitated using an
anti-hemagglutinin high affinity monoclonal antibody (Roche Molecular
Biochemicals) as described previously (24). Briefly, solubilized
material was preincubated for 2 h at 4 °C with protein
G-Sepharose beads to remove nonspecific binding. The supernatant was
then incubated with protein G-Sepharose beads and a 1:200 dilution of
antibody for 18 h at 4 °C. Following immunoprecipitation, the
beads were washed three times by centrifugation and resuspension and
then incubated at 37 °C for 1 h in SDS sample buffer. Proteins
in the supernatant were then fractionated on a 10% SDS-polyacrylamide gel with equal amounts of receptor (based on radioligand binding) loaded in each lane. Signals were visualized and quantitated using a
Molecular Dynamics PhosphorImager with ImageQuant Software.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
2BAR gene from 78 chromosomes revealed a single sequence
variant. This consisted of an in-frame 9-bp deletion (GAAGAGGAG)
beginning at nucleotide 901 (Fig.
1A) that results in loss of
three glutamic acid residues at amino acid positions 301-303 of the
third intracellular loop of the receptor (Fig.
2). Using the rapid detection method (Fig. 1B), allele frequencies were determined in a larger
population. The frequencies of the wild-type and the Del301-303
polymorphic
2BAR are shown in Table
I. The deletion polymorphism is more common in Caucasians than African-Americans, with allele frequencies of
0.31 and 0.12, respectively. The distribution of homozygous and
heterozygous alleles in either population was not different than that
predicted from the Hardy-Weinberg equilibrium (p > 0.8).
View larger version (17K):
[in a new window]
Fig. 1.
Identification of the human
2BAR variant. Shown in
A and B are representative automated sequence
chromatograms identifying a deletion of the nucleotides
GAAGAGGAG. In C, a rapid screening technique
identifies homozygous and heterozygous PCR products by size.
View larger version (55K):
[in a new window]
Fig. 2.
Localization of the
2BAR polymorphism. Shown are the
fifth and sixth transmembrane spanning domains (TMD) and the
third intracellular loop of the receptor.
Frequency of the 2BAR Del301-303 polymorphism
The consequences of this polymorphism on ligand binding and receptor
function were evaluated by stably expressing the human wild-type
2BAR and the Del301-303 receptor in CHO cells (Table II). Saturation radioligand binding
studies revealed a small but statistically significant lower affinity
for the
2AR antagonist [3H]yohimbine for
Del301-303 compared with the wild-type receptor (Kd = 5.1 ± 0.2 versus 3.8 ± 0.3 nM,
respectively, n = 5, p < 0.05).
Agonist (epinephrine) competition binding experiments carried out in
the presence of quanosine 5'(
-
-imido)triphosphate revealed a small increase in the Ki for the
polymorphic receptor (285 ± 8.7 versus 376 ± 66 nM, n = 5, p < 0.05). In
similar studies carried out in the absence of guanine nucleotide,
two-site fits were obtained for both receptors with no differences in
the KL or the percentage of receptors in the
high affinity state (RH; Table II). However, a
trend toward an increased KH was observed with
the Del301-303 mutant. These results prompted additional studies with
the partial agonist radioligand [125I]aminoclonidine.
Saturation binding studies (in the absence of quanosine
5'(
-
-imido)triphosphate) with concentrations of the ligand from
0.2 to 4 nM revealed a single site with a
Kd of ~1 nM as reported by others
(27). Comparison of the wild-type
2BAR and the
Del301-303 receptor revealed essentially identical Kd
values for [125I]aminoclonidine (1.33 ± 0.12 versus 1.22 ± 0.07 nM, respectively). Taken together, the data suggest that there is little, if any, effect
of the deletion in the third intracellular loop on the conformation of
the ligand binding pocket within the transmembrane spanning
domains.
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To address the functional consequences of the mutation, studies
examining agonist-promoted inhibition of forskolin-stimulated adenylyl
cyclase activities were carried out in lines expressing the wild-type
2BAR and the Del301-303 receptor at densities of 626 ± 54 and 520 ± 82 fmol/mg (n = 7, p > 0.05). The results of these studies are shown in
Table III. As can be seen, the
Del301-303 receptor displayed less inhibition of adenylyl cyclase
(23.4 ± 2.2%) compared with wild-type
2BAR
(28.5 ± 1.6%, p < 0.05). Furthermore, the
polymorphic receptor had a greater EC50 (19.6 ± 5.5 versus 7.9 ± 2.1 µM, p < 0.01). Thus, the loss of the three glutamic acids in the third
intracellular loop, which is known to contain regions important for G
protein coupling, results in a modest decrease in agonist-mediated
receptor function.
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The deletion polymorphism occurs in a highly acidic stretch of amino
acids (EDEAEEEEEEEEEEEE) within the third intracellular loop of
2BAR (Fig. 2). The structural importance of this region has been previously assessed and shown to be critical for short term
agonist-promoted receptor phosphorylation leading to desensitization (18). These data and reports by others (29) suggest that this acidic
environment is necessary for receptor phosphorylation by GRKs.
Therefore, to investigate the consequences of this deletion polymorphism on receptor desensitization, agonist-promoted inhibition of adenylyl cyclase activity was determined in membranes from CHO cells
expressing the wild-type and Del301-303 receptor after pretreatment
with norepinephrine. In these experiments, cells were incubated with
medium alone or medium containing agonist (10 µM
norepinephrine) for 30 min at 37 °C and subsequently extensively washed at 4 °C; membranes were prepared, and agonist-mediated inhibition of forskolin stimulated adenylyl cyclase activity was determined. As described previously (17) and shown in Fig.
3 and Table II, desensitization of
wild-type
2BAR expressed in CHO cells is manifested by
an increase in the EC50 for agonist-mediated inhibition of
adenylyl cyclase. Analysis of composite curves derived from four
independent experiments shows an increase from 7.4 to 29.4 µM for the wild-type
2BAR. In
contrast, there was no change in the EC50 for the deletion
receptor following agonist pretreatment (29.5 µM
versus 31.2 µM). Desensitization was
quantitated by examining adenylyl cyclase activities at a submaximal
concentration of agonist (the EC50 for the control
condition). At this concentration, wild-type
2BAR
inhibited adenylyl cyclase activity by 16.5 ± 3.9%; with agonist
pre-exposure, inhibition at this same concentration of agonist was
7.6 ± 2.3% (n = 4, p < 0.05, Fig. 3C), amounting to ~54% desensitization of receptor
function. Submaximal inhibition of adenylyl cyclase for the Del301-303
receptor, however, was not different between control and
agonist-treated cells (17.1 ± 3.0% versus 15.9 ± 1.7%, n = 4, p = ns). In another
two cell lines with matched expression of ~600 fmol/mg, the same
desensitization phenotypes for wild-type and the Del301-303
polymorphic receptor were observed (data not shown).
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We next performed whole cell phosphorylation studies of the wild-type
and polymorphic 2BAR under the same conditions used for
desensitization. We hypothesized that agonist-promoted phosphorylation would be decreased in the polymorphic receptor. However, given that
this receptor displays rightward-shifted dose-response curves for
inhibition of adenylyl cyclase at base line, we also considered the
possibility that the receptor is significantly phosphorylated in the
basal state. Studies were carried out in cells cotransfected with the
receptor and GRK2 (
ARK1), a strategy that we have previously shown
to be useful in identifying receptor-GRK interactions (30). The results
of a representative study are shown in Fig.
4. The wild-type
2BAR underwent a 5.84 ± 0.49-fold increase in
phosphorylation with agonist exposure. In contrast, although the
Del301-303 receptor displayed some degree of agonist-promoted
phosphorylation, the extent was clearly less (3.28 ± 0.24-fold,
p < 0.05 compared with wild type). Basal
phosphorylation was equivalent between the two receptors.
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It is interesting to note that this partial loss of phosphorylation
results in a receptor that fails to undergo any degree of functional
desensitization. Although it might seem reasonable to assume that such
phosphorylation would be associated with some degree of
desensitization, several previous studies with the 2AAR and
2BAR subtypes indicate that full (i.e.
wild type) phosphorylation is necessary for the desensitization process
(16, 18, 24). For the
2AAR, we have shown that four
serines in the third intracellular loop are phosphorylated after
agonist exposure (16). Removal of serines by alanine substitution
mutagenesis results in a proportional decrease in phosphorylation. Such
partial phosphorylation (compared with wild type), however, was found
to be insufficient to cause any detectable desensitization. In a
previous study of the
2BAR, we deleted and substituted
the entire aforementioned acidic region (18). Agonist-promoted
phosphorylation was reduced by ~50% in this mutant, and
desensitization was ablated. These results are entirely consistent with
the current work, where a restricted deletion resulted in a decrease in
phosphorylation and a complete loss of desensitization. Finally, we
have also recently shown that a chimeric
2A/
2CAR, which undergoes agonist-promoted
phosphorylation, fails to exhibit desensitization (24). Taken together
with our current work, these results indicate that the conformation of the third loop evoked by GRK mediated phosphorylation which provides for the binding of arrestins (which is the ultimate step that imparts
uncoupling) is highly specific. Thus, a precise
phosphorylation-dependent conformation is apparently
required for arrestin binding to
2AR and subsequent
functional desensitization. Perturbations of the milieu can thus have
significant functional consequences, as occurs with the Del301-303
polymorphic
2BAR.
Thus, the major signaling phenotype of the 2BAR
Del301-303 polymorphism is one of decreased agonist-promoted
phosphorylation that results in a complete loss of the ability for the
receptor to undergo agonist-promoted desensitization. In addition, the polymorphism imposes a small decrease in receptor coupling. The potential physiologic consequences of the polymorphism could be related
to either or both of the above phenotypes. A receptor that fails to
undergo desensitization would be manifested as static signaling despite
continued activation of the receptor by endogenous or exogenous
agonist. Such a lack of regulation by agonist may perturb the dynamic
relationship between incoming signals and receptor responsiveness that
maintains homeostasis under normal or pathologic conditions. Recently,
Gavras and colleagues (14) have shown that
2B
/+ mice
fail to display a hypertensive response to salt loading after subtotal
nephrectomy. Thus, a polymorphic
2BAR that fails to
desensitize (i.e. does not display regulatable function) may
predispose to salt-sensitive hypertension. Regarding the therapeutic
response to
2AR agonists, the phenotype of the Del301-303 receptor indicates that individuals with this polymorphism would display little tachyphylaxis to continued administration of
agonists. In addition, the initial response to agonist might also be
reduced based on the somewhat depressed coupling of the Del301-303 receptor.
Until recently, it has been difficult to differentiate
2BAR function from the other two subtypes in physiologic
studies. With the development of knockout mice lacking each
2AR subtype (5, 6, 13, 31), certain functions can now be
definitively attributed to specific subtypes. Characterization of the
2BAR knockout mouse has indicated that the
2BAR subtype is expressed on vascular smooth muscle and
is responsible for the hypertensive response to
2AR
agonists (13). This indicates that vascular
2BAR
contribute to overall vascular tone and thus participate in systemic
blood pressure regulation. This role may be more important, however,
during adaptive conditions, such as salt loading, because resting blood
pressure is normal in the heterozygous
2B
/+ mice (14). Whether the
2B
/
mice have altered resting
blood pressures has not been studied in detail because of high
perinatal lethality of the homozygous knockout (13). However, neither
the region of chromosome 2 near the
2BAR coding sequence
nor the deletion polymorphism has been linked or associated with
hypertension (21, 22, 32). However, no studies have assessed whether
the polymorphism is associated with salt-sensitive hypertension or
other phenotypes, or the response to
2AR agonist.
In summary, we have delineated the signaling phenotype of a
polymorphism of the 2BAR that results in a deletion of
three glutamic acids in the third intracellular loop of the receptor. The polymorphism is prevalent in the human population, with a frequency
that is ~2-fold greater in Caucasians as compared with African-Americans. The polymorphic receptor displays wild-type agonist
binding affinity but a small decrease in function in the resting state.
However, the major phenotype is a significant decrease in
agonist-promoted phosphorylation by GRKs, which results in a receptor
that fails to display agonist-promoted desensitization. To our
knowledge this is the first polymorphism of any G protein-coupled receptor to affect GRK-mediated phosphorylation.
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ACKNOWLEDGEMENT |
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We thank Esther Getz for manuscript preparation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL52318 and ES06096.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF316895.
To whom correspondence should be addressed: University of
Cincinnati College of Medicine, 231 Albert Sabin Way, Rm. G062, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax:
513-558-0835; E-mail: stephen.liggett@uc.edu.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008118200
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ABBREVIATIONS |
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The abbreviations used are: AR, adrenergic receptor(s); GRK, G protein-coupled receptor kinase; PCR, polymerase chain reaction; bp, base pair(s); CHO, Chinese hamster ovary.
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REFERENCES |
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