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Vol. 58, Issue 5, 1162-1173, November 2000
2-Adrenergic Receptor by the GRK Pathway
Department of Integrative Biology and Pharmacology (A.S., B.W., Z.-F.H., J.F., R.B.C.), University of Texas-Houston Medical School, Houston, Texas; and Departments of Pediatrics (R.H.M.) and Molecular Physiology and Biophysics (B.J.K.), Baylor College of Medicine, Houston, Texas
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Abstract |
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The human
2-adrenergic receptor (
AR) is rapidly
desensitized in response to saturating concentrations of agonist by G
protein-coupled receptor kinases (GRKs) and cAMP-dependent protein
kinase A (PKA) phosphorylation of the
AR, followed by
-arrestin
binding and receptor internalization.
AR sites phosphorylated by GRK
in vivo have not yet been identified. In this study, we examined the
role of the carboxyl terminal serines, 355, 356, and 364, in the
GRK-mediated desensitization of the
AR. Substitution mutants of
these serine residues were constructed in which either all three
(S355,356,364A), two (S355,356A and S356,364A), or one of the serines
(S356A and S364A) were modified. These mutants were constructed in a
AR in which the serines of the PKA consensus site were substituted with alanines (designated PKA
) to eliminate any PKA
contribution to desensitization, and they were stably transfected into
human embryonic kidney 293 cells. Treatment of the PKA
mutant with 10 µM epinephrine for 5 min caused a 3.5-fold increase in
the EC50 value and a 42% decrease in the
Vmax value for epinephrine stimulation of
adenylyl cyclase. Substitution of all three serines completely
inhibited the epinephrine-induced shift in the EC50. Both
double mutants, S355,356A and S356,364A, showed a nearly complete loss
of the EC50 shift, whereas the single substitutions, S356A
and S364A, caused only a slight decrease in desensitization. None of
the mutations altered the epinephrine-induced decrease in
Vmax, which seems to be downstream of the
receptor. The triple mutation caused a 45% decrease in
epinephrine-induced internalization and a 90 to 95% reduction in
phosphorylation of the
AR relative to the PKA
(1.9 ± 0.2- and 16.6 ± 3.8-fold phosphorylation over basal,
respectively). The double mutants caused an intermediate reduction in
internalization (20-21%) and phosphorylation (43-52%). None of the
serine mutations altered the rate of
AR recycling. Our data
demonstrate that the cluster of serines within the 355 to 364
AR
domain confer the rapid, GRK-mediated, receptor-level desensitization
of the
AR.
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Introduction |
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The
2-adrenergic receptor (
AR) is rapidly
inactivated after exposure to epinephrine. Rapid
AR desensitization
at high concentrations of epinephrine results from phosphorylation of
the receptor by cAMP-dependent protein kinase (PKA) and one or more
members of the G protein-coupled receptor kinase (GRK) family (Clark et
al., 1989
, 1999
; Kunkel et al., 1989
; Krupnick and Benovic, 1998
;
Lefkowitz et al., 1998
). Considerable recent evidence supports the
proposal that GRK-mediated phosphorylation of receptors greatly
promotes their binding to
-arrestin, leading to receptor
internalization through a clathrin-mediated mechanism (Tsuga et al.,
1994
; Ferguson et al., 1995
, 1996
; Goodman et al., 1996
). At high
occupancy with strong agonists, it is clear that these events cause the
majority of the desensitization of the
AR (Clark et al., 1999
).
The current model of G protein-coupled receptor desensitization was
first developed through studies of rhodopsin. Phosphorylation of the
light-activated rhodopsin increased its affinity for visual arrestin
(Kuhn et al., 1984
). Arrestin binding is thought to be the most
critical step in desensitization, serving to uncouple rhodopsin from
the G protein transducin (Wilden et al., 1986
; Bennett and Sitaramayya,
1988
). Desensitization of the
AR is hypothesized to occur similarly,
requiring receptor phosphorylation followed by
-arrestin binding
(Lohse et al., 1990
; Palczewski and Benovic, 1991
). Unlike rhodopsin,
AR interaction with
-arrestin leads to receptor internalization
through a clathrin-coated pit-dependent pathway (Ferguson et al., 1996
;
Goodman et al., 1996
). Mutagenesis studies have suggested that multiple
domains within visual arrestin (Gurevich and Benovic, 1993
; Gurevich,
1998
; Vishnivetskiy et al., 1999
) and the
-arrestins (Kovoor et al.,
1999
) interact with receptor sites. The complementary domains within
either rhodopsin or the
AR have not been identified, although they
are thought to include regions that undergo agonist-induced
conformational changes and phosphorylation (Kovoor et al., 1999
).
Overwhelming evidence indicates that
AR phosphorylation and
subsequent
-arrestin binding are important for desensitization. Despite this, identification of the receptor sites at which GRK phosphorylation occurs and that are required for desensitization has
proven difficult. In the first study of putative GRK sites, it was
shown that substitution of all 11 carboxyl-terminal serines and
threonines reduced GRK-mediated desensitization without affecting regulation by PKA (Bouvier et al., 1988
). Relative to the wild-type (WT)
AR, phosphorylation of this mutant in response to a high concentration of agonist was reduced by half but its internalization was unaffected. Sequence analysis of
AR phosphorylated in vitro by
GRK suggested that the critical residues were in the distal portion of
the carboxyl tail (Fredericks et al., 1996
). However, our recent
mutagenesis studies of these sites demonstrated that they were not
required for in vivo desensitization (Seibold et al., 1998
), suggesting
that other regions of the receptor carboxyl tail, namely the 355-364
domain, may be important for GRK regulation. Interestingly, Hausdorff
et al. (1991)
found that substitution of four residues, S355, S356,
T360, and S364, in the proximal portion of the
AR carboxyl tail (a
subset of the 11 carboxyl tail serines and threonines previously
described), eliminated rapid desensitization mechanisms, both PKA- and
GRK-mediated. The effect on desensitization was not specific, because
phosphorylation and internalization of the mutant were also completely
blocked. The discrepancy between this study and the previous work in
which all 11 serines and threonines were mutated led to the conclusion that the four amino acid substitutions caused an altered receptor conformation that prevented normal regulation. Adding further complexity, Yu et al. (1993)
showed that substitution of serines 356 and 364 did not alter desensitization, but eliminated
AR internalization and resensitization. The inability of this mutant to
resensitize after agonist removal led to the proposal that receptor
internalization was required for the reversal of desensitization.
The inconsistencies in these reports coupled with our demonstration of
the lack of effect of mutating the more distal six serines and
threonines in the carboxyl tail prompted the studies presented in this
article on the potential role of the S355-S364 domain in GRK-mediated
desensitization. Mutants in this domain were constructed in which one
(or more) of the three serine residues was substituted with alanine.
After stable transfection into human embryonic kidney (HEK) 293 cells,
the mutant receptors were examined for coupling efficiency,
epinephrine-induced desensitization, internalization, recycling, and
phosphorylation. To focus specifically on the role of GRK-mediated
desensitization, the mutants were constructed in a
AR in which the
PKA consensus sites were ablated by substitution of serines 261, 262, 345, and 346 with alanine (designated PKA
).
Ablation of the serines of the PKA consensus sites aided analysis because previous studies have shown that PKA effects contribute to the
level of overall desensitization but do not affect the component
attributed to GRK-mediated homologous desensitization (Green et al.,
1981
; Clark et al. 1988
; Hausdorff et al., 1989
; Yuan et al., 1994
).
The data reported here show that mutation of all three serines
(S355,356,364) in the C-terminal domain was required for complete
elimination of homologous receptor-level desensitization and for a 90 to 95% reduction in phosphorylation of the
AR, although mutation of
only two amino acids in this cluster resulted in a dramatic reduction
of desensitization. We conclude that these serines are the likely sites
for GRK-meditated desensitization.
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Materials and Methods |
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Description of Mutant
ARs.
Mutations were introduced into
the
AR in amino acid region 355 to 364, as shown in Table
1 and in Fig.
1. All of the mutant
ARs contain
alanine substitutions for the serines of the two consensus PKA sites.
Mutagenesis was performed using the polymerase chain reaction as
described previously (Seibold et al., 1998
). The mutants were sequenced
through the entire
AR coding region and epitope tags to ensure
accuracy of the mutagenesis procedure. All of the
ARs in Table 1
include the hemagglutinin (HA) epitope at the amino terminus and the
6HIS tag at the carboxyl tail, as described previously (January et al.,
1997
). Recycling data also were obtained for the untagged WT
AR, and
desensitization data were obtained for both the untagged WT
AR and
for the amino-terminally HA-tagged WT
AR. All of the plasmids were
stably transfected into HEK 293 cells, as described in Table 1.
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Transfection of HEK 293 Cells.
The HEK 293 cells were
cultured at 37°C in 5% CO2 in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The
plasmids were linearized by PvuI digestion and transfected into subconfluent HEK 293 cells using the CaPO4
method. Sixteen hours later, the cells were shocked with 25% glycerol
in DMEM and placed in media containing 0.4 mg/ml G418. Stable
transfectants expressing
AR were identified using an intact cell
[125I]iodocyanopindolol
(125ICYP) binding assay described below.
Membrane Preparation.
Cells were plated into 100-mm dishes
that had been precoated with poly-L-lysine. Pretreatment
with epinephrine or carrier was performed in 5%
CO2 at 37°C and was stopped by removal of media
followed by six 5-ml washes with ice-cold HME buffer (20 mM HEPES, pH
8.0, 2 mM MgCl2, 1 mM EDTA, 1 mM benzamidine, 10 µg/ml trypsin inhibitor, 0.1 mg/ml BSA). The final concentration of
the carrier components were 0.1 mM ascorbate and 1 mM thiourea (AT), pH
7.0. The cells were scraped into HME buffer containing 10 µg/ml
leupeptin, 2 mM tetrasodium pyrophosphate and 0.1 µM okadaic acid and
homogenized with seven strokes in a type B Dounce homogenizer. The
homogenates were layered onto sucrose step gradients (23 and 43%)
prepared in HE buffer (20 mM HEPES, pH 8.0, 1 mM EDTA) and centrifuged
at 25,000 rpm in a Beckman SW28.1 rotor for 35 min. The fraction at the
23/43% sucrose interface was taken, frozen in liquid nitrogen, and
stored at
80°C.
Measurement of Receptor Levels.
Cells were cultured in
12-well dishes for measurement of intact cell receptor number by
125ICYP binding. The
125ICYP was prepared as described previously
(Barovsky and Brooker, 1980
; Hoyer et al., 1984
). The cells were rinsed
in serum-free DMEM and then removed from the plates by pipetting up and
down with 0.5 ml of serum-free DMEM. Aliquots (25-50 µl) of the
resuspended cells were used in triplicate binding reactions, each
containing about 200 pM 125ICYP. Nonspecific
binding was measured in triplicate reactions containing 1 µM
alprenolol. The reactions were performed on ice for 50 min and were
terminated by the addition of 2.5 ml of ice-cold 50 mM Tris·HCl, pH
7.5, and 10 mM MgCl2. The
125ICYP-bound
AR was collected by filtration
through Whatman GF/C filters. The filters were rinsed three times with
2.5 ml of the cold Tris/MgCl2 buffer and then
counted using a Beckman 4000 gamma counter (Beckman Instruments,
Columbia, MD). Protein was measured using a Bio-Rad dye reagent.
Measurement of Equilibrium Binding Constants for ICYP and
Epinephrine
The Kd
values for 125ICYP and epinephrine were determined for each
of the mutant
ARs using methods described previously (January et
al., 1997
, 1998
). The range of 125ICYP concentrations used
for the Kd measurement was 1 to 150 pM. Reactions were performed in triplicate with 1 µg of membrane protein, and nonspecific binding was measured with the inclusion of alprenolol at 1 µM. The Kd value was estimated by fit
of the data to a rectangular hyperbola. The assays to measure
epinephrine Kd included 40 to 50 pM
125ICYP, 10 µM guanosine
5'-3-O-(thio)triphosphate (GTP
S), and concentrations of epinephrine ranging from 0.1 to 100 µM. Reactions were performed with triplicate points using 1 µg of membrane protein. Graph-Pad (San
Diego, CA) analysis was used to fit the data to a one-component sigmoidal curve with a Hill coefficient of
1. Because the mutant
ARs had 125ICYP Kd values
similar to one another, an average value of 7.7 pM was used in the
Cheng-Prusoff calculation of the epinephrine Kd.
Adenylyl Cyclase Assay.
Adenylyl cyclase activity was
assayed using a modification of the method described by Salomon et al.
(1974)
. Membranes were diluted to a final protein concentration of 0.1 to 0.2 mg/ml, to achieve 5 to 10 µg per reaction. Incubation was
carried out at 30°C for 10 min with 40 mM HEPES, pH 7.7, 6 mM
MgCl2, 1 mM EDTA, 100 µM ATP, 1 µM GTP, 0.1 mM 1-methyl-3-isobutylxanthine, 8 mM creatine phosphate, 16 U/ml
creatine kinase, and 2 µCi of [
-32P]ATP
(30 Ci/mmol; NEN Life Science Products, Boston, MA) in a total volume
of 100 µl. Each point was assayed in triplicate, with six to eight
concentrations of epinephrine bracketing the EC50. The [32P]cAMP
produced in the assay was collected using Dowex and Alumina columns as
described previously (Clark et al., 1988
). Graph-Pad software was used
to estimate the EC50 and the
Vmax values.
Quantification of Coupling Efficiency and Desensitization.
We have previously described the equation for coupling efficiency,
given below (Whaley et al., 1994
)
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(1) |
1 is the first-order rate
constant for inactivation. The coupling efficiency (k1/k
1) is
calculated from three experimentally determined values: the
Kd is the low-affinity binding constant for
the agonist, the EC50 is obtained from the
activation of adenylyl cyclase by agonist, and r represents
Bmax. Eq. 1 can be combined with the equation for Vmax to give eq. 2, as
described previously (January et al., 1997
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(2) |
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(3) |
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(4) |
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(5) |
Kd, a
decrease in k1r resulted in a large change in
EC50 and almost no decrease in
Vmax, a finding predicted from eqs. 4 and
5. Therefore at the high receptor densities used here, receptor-level
desensitization will be represented primarily through
EC50 shifts. Thus, any significant decrease in
Vmax for epinephrine stimulation after
agonist-induced desensitization can be attributed to effects on
components downstream of
AR/Gs coupling.
Measurement of Receptor Internalization by
[3H]CGP-12177 Binding.
Epinephrine-stimulated
AR
internalization was measured as described previously (January et al.,
1997
; Seibold et al., 1998
). Cells were plated into 12-well dishes that
had been precoated with poly-L-lysine to aid cell adhesion.
The cells were pretreated with either carrier or epinephrine from 100×
stocks. Pretreatment was performed at 37°C in 5%
CO2 for various times and was stopped by removal
of media followed by 6 ice-cold DMEM rinses. To each well, 1 ml of
serum-free DMEM was added containing 10 nM
[3H]CGP-12177 (CGP) to measure surface receptor
number. Incubations were performed on ice for 1 h. The assays
included triplicate points, and nonspecific binding was determined by
inclusion of 1 µM alprenolol. Some assays included 0.2% digitonin
during the CGP incubation to measure total receptor number, including
the internalized pool. After incubation, the CGP mixture was removed and the wells were washed twice with ice-cold PBS. The cells were scraped into 0.5 ml of trypsin and liquid scintillation counting was
performed. Internalization data are plotted as the percentage of
surface receptor number measured in carrier (AT)-treated samples. The
data were fit to the curve for monoexponential decay and Graph Pad
software was used to estimate the apparent rate of internalization.
AR Recycling Assay.
Cells were seeded in 12-well dishes
coated with poly-L-lysine and grown to confluence. The
cells were pretreated with either carrier or 1 µM epinephrine for 20 min. The concentration of 1 µM epinephrine permitted more complete
washout compared with 10 µM and still provided about 70% receptor
occupancy. At 20 min, the medium was removed and the cells were rinsed
three times with 2 ml of warm (37°C) DMEM plus 10% fetal bovine
serum and then refed with the same. The cells were incubated at 37°C
for 0 to 60 min to allow recycling. Recycling was stopped by removal of media and two rinses with ice-cold PBS. Serum-free DMEM containing about 10 nM CGP was then added with and without 1 µM alprenolol and
the cells incubated on ice for 1 h. The CGP mixture was removed and the cells rinsed twice with ice-cold PBS. The cells were scraped into 0.5 ml of trypsin and liquid scintillation counting was performed. Surface receptor number is reported as a percentage of that found in
the carrier-treated control. The return of receptors to the cell
surface was fit to the curve for monoexponential decay and the rate of
recycling determined. The rate of endocytosis was calculated according
to eq. 6, described by Koenig and Edwardson (1994)
.
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(6) |
Observation of
AR Internalization by Immunofluorescence.
Stably transfected cells were plated on
poly-D-lysine-coated #1 glass cover slips in 35-mm culture
dishes, grown to 50 to 80% confluence, then chilled on ice. Monoclonal
antibody mHA.11 (Berkeley Antibody Co., Berkeley, CA) was added to 2 µg/ml and the incubation on ice continued for 60 min. The monolayers
were washed three times with ice-cold medium, then warmed to 37°C for 5 min or 30 min in the presence or absence of 10 µM isoproterenol. The monolayers were then rapidly chilled, washed once with PBS containing 1.2% sucrose (PBSS) and fixed at 4°C for 10 min with PBSS
containing 4% paraformaldehyde (Electron Microscopy Sciences, Ft.
Washington, PA). The fixed cells were incubated in 0.34%
L-lysine, 0.05% Na-m-periodate in PBSS for 20 min, washed, and permeabilized with 0.2% Triton X-100 for 5 min, then
blocked for 15 min with 10% heat-inactivated goat serum. Goat
anti-mouse IgG conjugated with Alexa Fluor 488 (Molecular Probes,
Eugene, OR), diluted to 5 µg/ml in PBSS with 0.2% heat-inactivated
goat serum and 0.05% Triton X-100, was added to the cells and left
overnight in the dark. The cover slips were mounted in Mowiol
(Calbiochem, La Jolla, CA) and imaged with a DeltaVision Restoration
Microscopy System (Applied Precision, Issaquah, WA). The digitized
images were then deconvoluted with a DeltaVision workstation running
DVSoftWoRx and assembled using Adobe Photoshop 5.5 (Adobe Systems,
Mountain View, CA).
Determination of
AR Phosphorylation.
The methods used are
modifications of those described previously (January et al., 1997
).
Cells were plated into 100-mm dishes precoated with
poly-L-lysine and grown to confluence. Cells were rinsed
once with phosphate-free DMEM and then incubated with 0.5-mCi [32P]orthophosphate in phosphate-free DMEM
containing 1% fetal bovine serum for 3 h at 37°C in 5%
CO2. The labeling medium was removed and replaced
with 5 ml of bicarbonate- and phosphate-free DMEM containing 10% FBS.
After 30 min equilibration the cells were treated with 10 µM
epinephrine or AT carrier for the indicated times. The medium was
removed and the cells rinsed with 5 ml ice-cold PBS. The dishes were
placed on ice and scraped into 3 ml of PBS containing 10 µg/ml
leupeptin and 100 nM okadaic acid. The cells were collected by
centrifugation at 2000 rpm in an IEC DPR 6000 centrifuge. The cell
pellet was solubilized by vortexing in buffer containing 20 mM HEPES,
pH 7.4, 300 mM NaCl, 0.8%
n-dodecyl-
-D-maltoside (DBM), 5 mM
EDTA, 3 mM EGTA, 20 mM sodium pyrophosphate, 10 mM sodium fluoride, 25 mM imidazole, 10 µg/ml benzamidine, 10 µg/ml trypsin inhibitor, 100 nM okadaic acid, 10 µg/ml leupeptin, and 14 mM
-mercaptoethanol.
After 30 min rocking at 4°C, the solubilized cells were centrifuged
for 30 min at 45,000 rpm in a Beckman 50 Ti rotor.
AR was purified using either of two
procedures. Procedure 1 used for the majority of experiments consisted
of a Ni-NTA affinity step followed by either wheat germ agglutinin-agarose (WGA) chromatography or immunoprecipitation. The
solubilized supernatant was applied to Ni-NTA superflow resin (Qiagen,
Valencia, CA) packed into disposable columns (Bio-Rad), 0.8 ml
of 2× slurry per column. The eluate was collected and recycled onto
the columns. The columns were washed once with 5 ml of buffer containing 0.05% DBM, 20 mM HEPES, pH 7.4, 300 mM NaCl, 25 mM imidazole, 4 M guanidine HCl, and 1 M LiCl. After a rinse with 5 ml of
Ni2+ column buffer (0.05% DBM, 20 mM HEPES, pH
7.4, 300 mM NaCl, 25 mM imidazole), the
AR was eluted in a single
step with 4 ml of buffer containing 0.05% DBM, 20 mM HEPES, pH 7.4, 300 mM NaCl, and 100 mM imidazole. The
AR was further purified using
either WGA or immunoprecipitation. Similar results were obtained with both procedures. The WGA step was performed as described previously (January et al., 1997
AR antibody added (antibody SC-569 directed against the
C-terminal 20 amino acids; Santa Cruz Biotechnology, Santa Cruz, CA).
The samples were incubated for 90 min at 4°C with rocking, after
which 50 µl of Protein A Sepharose was added and the samples further
incubated for 1 h at 4°C with rocking. The immune complexes were
centrifuged for 5 min in the IEC DPR 6000, the supernatants aspirated,
and the pellets washed twice with 2 ml of Ni column buffer. To each
pellet was added 125 µl of SDS sample buffer (50 mM Tris, pH 6.8, 2%
SDS, 0.025% bromphenol blue, 6 M urea, and 14 mM
-mercaptoethanol).
The samples were incubated at 60°C for 15 min with frequent
vortexing. The samples were transferred to Eppendorf tubes, briefly
spun in a Microfuge and loaded onto 7.5% SDS-polyacrylamide
gels along with prestained molecular mass markers. After
electrophoresis, the proteins were transferred from the gel to
0.22-µm polyvinylidene difluoride (PVDF) membranes.
In the course of these studies, a substantially modified procedure was
developed that allowed better recovery and considerably improved
quantification and will be referred to as procedure 2. In outline, this
procedure consisted of a C-tail antibody affinity column,
N-glycosidase F treatment of the antibody column eluate, and
a Talon affinity resin step
(Co2+-carboxymethylaspartate-agarose; Clontech,
Palo Alto, CA). Solubilized
AR (equal amounts from control and
epinephrine-treated based on
AR levels in the extracts) was applied
to a 100-µl packed volume of antibody resin in a column (SC-569
C-tail antibody from Santa Cruz linked to agarose) that had been
prewashed with PBS, pH 7.0. After recycling the extract three times
through the column, it was washed once with 3 ml of 10 mM phosphate
buffer, pH 6.8, containing 0.05% DBM. The
AR was eluted with 1 ml
of 100 mM glycine buffer, pH 2.5, plus 0.05% DBM and the eluate
collected in 0.3 ml 1 M phosphate buffer, pH 8.0 for neutralization.
The eluate was digested with 1500 units of N-glycosidase F
(New England Biolabs, Beverly, MA) for 2 h at 37°C, and applied
to the Talon Co2+-carboxymethylaspartate-agarose
column (0.5 ml packed resin) that had been prewashed with Talon buffer
(0.05% DBM, 20 mM HEPES, pH 7.4, 150 mM NaCl) and recycled through the
resin two times. The column was washed twice with Talon buffer, once
with 4 ml of 10 mM imidazole, and finally with 0.25 ml of 20 mM
imidazole, all in the same buffer. The
AR was eluted with 0.75 ml of
Talon buffer containing 100 mM imidazole and concentrated to 50 µl in a centricon (Amicon; 30-kDa cutoff). SDS-sample buffer was added to the 50 µl of eluate and heated at 60°C for 15 min. Samples were
run on SDS-polyacrylamide gel electrophoresis (PAGE; 12% gel) and
transferred to nitrocellulose.
PhosphorImager analysis was performed on the PVDF or nitrocellulose
membranes from either procedure using a Molecular Dynamics Storm
PhosphorImager model 860 and ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). Western blotting was performed using the anti-HA
antibody or the anti-carboxyl terminal
AR antibody as the primary
antibody as described previously (Seibold et al. 1998| |
Results |
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Determination of the Coupling Efficiency for Epinephrine Activation
of Adenylyl Cyclase for the HA-
AR-6HIS and Mutant
ARs.
To
eliminate the possibility that the mutations we introduced into the
AR caused nonspecific effects on coupling, we determined the
coupling efficiency value for each mutant using eq. 1. Calculation of
coupling efficiency requires measurement of receptor number, the
low-affinity Kd value for agonist binding,
and the EC50 value for adenylyl cyclase
activation (Whaley et al., 1994
). We calculated the coupling efficiency
for at least two clones of each mutant. The coupling efficiencies and
the experimentally determined values used in its calculation are
summarized in Table 2. None of the mutant
receptors showed a significant alteration of the
Kd value for epinephrine binding relative
to the HA-
AR-6HIS or the PKA
. The variation
we observed in coupling efficiencies in this study are typical and
reflect the variation in the three parameters used in the calculation
of coupling efficiency.
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Desensitization of the HA-
AR-6HIS and Mutant
ARs.
To
measure desensitization, cells expressing the mutant
ARs were
pretreated with 10 µM epinephrine or carrier for various times.
Membranes were prepared and assayed for activation of adenylyl cyclase
with a range of epinephrine concentrations. Figure
2 shows typical data from epinephrine
dose-response curves in which adenylyl cyclase activity was measured in
membranes prepared from cells pretreated with carrier (control) or 10 µM epinephrine for 2 or 5 min. The increase in
EC50 value and the decrease in the
Vmax value with 10 µM epinephrine
pretreatment were measured in many experiments, such as those in Fig.
2, and used to calculate desensitization as fraction activity remaining
for the various pretreatment times (1 to 30 min), as summarized in Fig.
3. Compared with the HA-
AR-6HIS and
the PKA
, the triple and double mutants showed
greatly reduced desensitization. Desensitization was measured in at
least two clones for each mutant. For the 30-min pretreatment with 10 µM epinephrine, the percentage of activities remaining for the
HA-
AR-6HIS, PKA
, S355,356,364A, S355,356A,
and S356,364A, respectively, were 3% ± 0.3, 8% ± 1.0, 72% ± 8, 28% ± 4, and 64% ± 15. Relative to PKA
,
these values are 9 (S355,356,364A), 3 (S355,356A), and 8 (S356,364A) times greater and highly significant (P < .001).
Compared with HA-
AR-6HIS, these values are 24, 9, and 21 times
greater for the triple- and double-serine mutants. The
PKA
mutant provides the most appropriate
comparison because all of the mutant
ARs described here contain
alanine substitutions for the serines of consensus PKA sites. The two
single-serine substitutions, S356A and S364A, were not as effective as
the double mutants in impairing desensitization. However, the
percentage of activities remaining after 30-min desensitization
were 16% ± 3 and 20% ± 1, which differed significantly from the
PKA
(P < .05).
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and HA-
AR-6HIS increased 3.4-fold
and 10.8-fold, respectively. For the S355,356A and S356,364A mutants
the EC50 after desensitization increased 1.5 and
1.2-fold, respectively. In contrast to the EC50 changes, the extent of the epinephrine-induced decrease in
Vmax was similar for the mutant
ARs,
PKA
, and HA-
AR-6HIS (Table 3), ranging
between 31.0 and 47.7%. The data show that the reduced desensitization
measured for the 355-364 domain mutants resulted from inhibition of
EC50 shifts rather than
Vmax effects.
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S, and forskolin stimulation. We found this treatment of the
PKA
cells caused decreases in both
PGE1 (10 µM) and GTP
S (10 µM) stimulation
of adenylyl cyclase (54 ± 5.4% and 25.4 ± 6.7%
respectively). The effects on PGE1 and GTP
S
stimulation support our suggestion that there is probably downstream
desensitization. Similar effects of epinephrine-induced desensitization
on these two activities were observed in the HA-
AR-6HIS. Forskolin
(20 µM) stimulation was reduced only 8 to 14% in these cell lines,
which is perhaps not surprising given that forskolin activates all
adenylyl cyclases except type 9 and is unlikely to activate only that
adenylyl cyclase coupled to the
AR. In fact, maximal forskolin
activation is about double that of the maximal epinephrine stimulation.
Another possibility we evaluated was whether stimulation of
Gi contributed to the epinephrine-induced
decrease in Vmax. To test this,
HA-
AR-6HIS and the PKA
cells were treated
overnight in the presence or absence of pertussis toxin (100 ng/ml) and
then desensitized by a 5-min treatment with epinephrine. Although
pertussis toxin predictably increased the Vmax value for epinephrine stimulation of
adenylyl cyclase in both controls and epinephrine-treated cells, it did
not alter either the extent of the desensitization-induced decrease in
the Vmax values for epinephrine stimulation
of adenylyl cyclase or the overall extent of the desensitization.
Internalization and Recycling of the Mutant
ARs
Cells expressing the various
ARs were
exposed to 10 µM epinephrine for various times, and receptor
internalization was measured by [3H]CGP-12177 binding to
intact cells. The results are shown in Fig.
5 as the loss of surface receptor with
time of epinephrine treatment. After 30 min of 10 µM epinephrine
pretreatment, the percentage of surface receptors internalized was 79%
(±2, n = 8), 66% (±2, n = 4), and 72% (±2, n = 4), for the HA-
AR-6HIS, PKA
, and S356A, respectively. The extent of
internalization measured for the double mutants S355,356A and S356,364A
and for the S364A mutant was reduced, with values of 52% (±3,
n = 6), 53% (±3, n = 5), and
51% (±4, n = 5), respectively, a decrease of
about 20% compared with PKA
. The triple-serine mutation
caused the greatest decrease in the extent of internalization, giving a
value of 36% (±3, n = 3). Fit of the averaged
data shown in Fig. 5 to the curve for monoexponential decay showed that
the observed rate of internalization was also reduced for the double,
triple, and S364A mutants. The kobs determined for the HA-
AR-6HIS, PKA
, and S356A was 0.19 min
1, whereas that determined for the triple and double
mutants and S364A was 0.11 min
1, a reduction of 42%. The
correlation coefficients for the first-order decay curves were 0.996 or
better for the different mutants. [3H]CGP-12177 binding
performed in the presence of digitonin showed no loss of total
AR
number during the internalization time course.
|
AR-6HIS, the
PKA
, or the double and triple mutant
ARs
were pretreated with 1 µM epinephrine for 20 min, then rinsed to
remove the hormone, and incubated for various times to allow the
recycling of the
ARs to the cell surface. The return of the
ARs
to the cell surface was measured by
[3H]CGP-12177 binding (Fig.
6). The HA-
AR-6HIS and mutant
ARs all recycled to the cell surface after washout of epinephrine with
similar rate constants, estimated from fit of the data to a
monoexponential decay curve. From the average value of the rate constants, a recycling t1/2 of 10 min
(krecycle= 0.07) was determined. Because the
HA-
AR-6HIS and mutant
ARs were found to recycle at the same rate,
the differences in the rate and extent of internalization indicate
different rates of endocytosis. The rate of endocytosis calculated for
the double mutants was 0.08 min
1, a value 38%
less than the rate of 0.13 min
1 calculated for
PKA
. We were not able to obtain recycling rates
for the triple mutant because the internalization was so small.
However, it seems that its rate of recycling is not altered relative to
the PKA
or the double mutants (0.07 min
1). Using this value, it can be calculated
that the rate of endocytosis of the triple mutant is reduced about 70%
relative to PKA
.
|
AR carboxyl terminus inhibited receptor recycling to the plasma
membrane (Cao et al., 1999
AR and found the data to be similar to those
obtained for the HA-
AR-6HIS. In response to a 20-min pretreatment
with 1 µM epinephrine, 66.2% (±2.77, n = 5) of the HA-
AR-6HIS, and 68.5% (±1.85, n = 2) of the
untagged WT
AR were internalized. After removal of epinephrine and a
60 min recycling incubation, all but 16.7% (±3.09, n = 5) of the HA-
AR-6HIS, and 19.9% (±1.70, n = 2)
of the untagged WT
AR had returned to the plasma membrane.
Observation of
AR Internalization Using Deconvolution
Microscopy.
Cells expressing the PKA- or the triple and double
mutants were incubated with antibody directed against the
amino-terminal HA epitope tag, followed by treatment with either
carrier or 10 µM isoproterenol for 5 or 30 min. The cellular location
of the receptors in response to isoproterenol treatment was determined using immunofluorescent deconvolution microscopy. In the absence of
agonist, all of the antibody-labeled mutant receptors remained at the
cell surface. In the presence of agonist, there was rapid and
substantial receptor internalization of the double mutants into
peripheral endocytic vesicles (Fig. 7).
The triple mutant seemed to internalize somewhat more slowly. Similar
results were obtained using 10 µM epinephrine (data not shown).
Although the immunofluorescent studies are not easily quantified, they
are consistent with the results of CGP binding and indicate that
internalization of the double and triple mutants and the
PKA
results in a similar localization to
endocytic vesicles.
|
Phosphorylation of the Mutant
ARs in Response to 10 µM
Epinephrine.
Cells expressing the PKA
,
S355,356A or the S356,364A mutant
ARs, were
labeled with
[32P]orthophosphate and
then treated with either 10 µM epinephrine or carrier for 2 min. The
cells were solubilized and the
AR protein purified by procedure 1 as
described under Materials and Methods. The purified
AR
was subjected to SDS-PAGE and transferred to a PVDF membrane. The
PhosphorImager analysis of a representative experiment is shown in Fig.
8A. To estimate relative loading, a
Western blot was performed on the same membrane, using antibody directed against the
AR carboxyl terminus (Fig. 8B). The data are
representative of 4 independent experiments and show that the double
mutants are rapidly phosphorylated in response to epinephrine pretreatment. Phosphorylation of the PKA
increased 7-fold in response to a 2-min pretreatment with 10 µM
epinephrine, whereas phosphorylation of the double mutants S355,356A
and S356,364A increased only 3.3- to 4-fold (Fig.
9). The time courses of phosphorylation
of the double mutants and PKA
were similar
(data not shown), showing a maximum level at 2 to 5 min, after which
phosphorylation declined.
|
|
,
[32P]labeled cells were pretreated with
epinephrine for 2 min and the
ARs purified by procedure 2 as
discussed under Materials and Methods. The PhosphorImager
scan and accompanying Western blots of a typical experiment (including
comparison with S356,364A) are shown in Fig.
10. In this purification procedure, the
glycosyl residues are removed by treatment with
N-glycosidase F (between the antibody affinity and the Talon
affinity steps); this results in the migration of the
AR to a
molecular mass of
48 to 50 kDa. The Western blot using the
anti-HA antibody shows that similar levels of
AR were purified from
the three cells lines (each antibody column was loaded with 375 fmol of
solubilized
AR). The 2-min epinephrine treatment of the triple
serine mutant and PKA
caused 2- and 15-fold
increases in phosphorylation over basal, respectively. Phosphorylation
of the double mutant was 6.4-fold over basal (i.e., 39% of
PKA
). From three independent experiments,
including the one in Fig. 10, we found the fold stimulation of the
triple mutant over basal was 1.86 ± 0.18-fold compared with
16.6 ± 3.8 for PKA
. Thus the
epinephrine-stimulated phosphorylation of the triple mutant is only
~5% that of the PKA
. Nevertheless it is
important to emphasize that phosphorylation was not eliminated.
|
AR to a tight band,
greatly improving our ability to quantify 32P
relative to the glycosylated
AR that spreads over a 15- to 20-kDa
region of the gels. Additionally we have found that the background is
reduced resulting in larger fold-stimulations relative to procedure 1.
| |
Discussion |
|---|
|
|
|---|
Our data show that mutations of either two or three of the serines
in the
AR 355-364 domain caused a striking reduction in epinephrine-induced receptor-level desensitization of the
AR (EC50-shift) without altering the decrease in
Vmax that seems to be downstream. These
mutations were made in a receptor in which both PKA consensus sites
were ablated to eliminate any contribution of PKA desensitization of
the
AR. The complete elimination of the EC50
shift in the S355,356,364A mutant coupled with the 90 to 95% loss of
phosphorylation and considerably reduced extent of internalization
(45%) relative to PKA
are consistent with the
proposal that the crucial sites for homologous GRK-mediated
desensitization of the
AR lie in the 355 to 364 amino acid region
with the caveat that phosphorylation was not eliminated. Our data are
consistent as well with the studies of rhodopsin phosphorylation by
GRK1 (rhodopsin kinase). Two serines in the carboxyl tail of mouse
rhodopsin are phosphorylated in vivo in response to light (Ohguro et
al., 1995
). These two serines lie in the region of rhodopsin most
homologous to the 355 to 364 domain of the
AR [see Collins et al.
(1991)
for amino acid alignment]. In addition, in vitro studies have
shown that rhodopsin kinase can phosphorylate the
AR (Benovic et
al., 1986
), and GRK2 (
ARK1) can phosphorylate rhodopsin (Benovic et
al., 1987
). The evidence suggests that the
AR and rhodopsin share
similar sites for recognition by their respective kinases.
Table 3 and Fig. 4 show further that the decreased desensitization of
the triple and double mutants is almost completely attributable to the
lack of change of the EC50 for epinephrine stimulation. What little desensitization is observed with S355,356,364A and S356,364A is caused for the most part by the decrease in the Vmax value. This is not significantly
different from the decrease in Vmax values
observed with the PKA
or the HA-
AR-6HIS
(Table 3), demonstrating that the disruption of desensitization found
in serine substitution mutants of the
AR 355-364 region results
from changes in values of EC50 rather than those
of Vmax. Loss of
AR/Gs coupling with receptor-level desensitization is predicted (by eqs. 4 and 5 under Materials and
Methods) to be represented primarily by changes in the value of
EC50 rather than changes in that of
Vmax at the high receptor density reported
here (Whaley et al., 1994
). The epinephrine-induced decreases in
PGE1 and GTP
S stimulation of adenylyl cyclase
support the idea that there is significant heterologous downstream
desensitization. Unfortunately, we were unable to detect significant
decreases in forskolin stimulation with desensitization; however, as
noted previously, this could be explained by the fact that forskolin activates all adenylyl cyclases except type 9, and this may obscure the
contribution of the subtype altered by the epinephrine pretreatment. The contribution of Gi to the 40% decrease in
Vmax was also explored through the use of
pertussis toxin and found not to contribute either to the
Vmax or to the overall desensitization. At
present therefore, the cause of the decrease in
Vmax in these cells remains unexplained;
however, the cells expressing the triple mutant will be ideal for
examining this phenomenon in future studies because the
Vmax effect is not complicated by
receptor-level changes.
Although receptor-level desensitization of the double mutants was
greatly impaired relative to the HA-
AR-6HIS and
PKA
, internalization and phosphorylation were
not comparably reduced. We considered how our results could be
reconciled with the currently accepted scheme for the
AR
desensitization process which proposes that GRK-phosphorylation of the
AR is followed by
-arrestin binding,
-arrestin-promoted
endocytosis, and subsequent recycling (Krupnick and Benovic, 1998
;
Lefkowitz et al., 1998
; Clark et al., 1999
). Each step in this scheme
of
AR regulation, including GRK phosphorylation,
-arrestin
binding, and internalization, is dependent on the previous step.
However, as we have shown previously in HEK 293 cells, the
inter-relationships between these processes during the time of agonist
stimulation are complex (January et al., 1997
). Phosphorylation of the
AR shows a rapid rise, reaches a maximum by 2 to 5 min, and then
declines, whereas levels of endocytosed
AR continue to increase
until a steady state of endocytosis and recycling is achieved.
The most straightforward conclusion is that our double mutants have
ablated two of the three crucial GRK sites that are required for
-arrestin-mediated desensitization, but that the additional GRK site
must be blocked to achieve near complete blockade of
-arrestin
binding. The first evidence for multisite interaction between receptors
and arrestin came from studies of rhodopsin and visual arrestin.
Mutagenesis studies showed that at least three regions of visual
arrestin interact with phosphorylated, light-activated rhodopsin to
terminate signaling (Gurevich and Benovic, 1993
; Gurevich, 1998
;
Vishnivetskiy et al., 1999
). Similar work suggests that multisite
binding mediates
-arrestin-
AR interaction (Kovoor et al., 1999
).
Although domains of arrestin and
-arrestin important for receptor
interaction have been identified, the corresponding sites in the
respective receptors were not known previously. Regions of the
receptors likely to be important for arrestin interaction include
GRK-phosphorylated residues, the third intracellular loop (a region
important for contact with G proteins), and undefined domains that
result from agonist binding-induced conformational change (Gurevich and
Benovic, 1993
; Krupnick et al., 1994
; Gurevich, 1998
; Kovoor et al.,
1999
; Vishnivetskiy et al., 1999
). In support of this model, the
recently determined crystal structure of visual arrestin provided a
firm structural basis for the proposed conformational alterations of
arrestin and multisite binding to receptor (Granzin et al., 1998
;
Hirsch et al., 1999
).
Given the validity of the multisite model of
-arrestin binding to
the
AR, there are several schemes consistent with our desensitization, phosphorylation, and internalization data. When agonist-bound, the double or triple mutants may retain sites for low-affinity
-arrestin interaction sufficient to promote
internalization, albeit at a reduced rate, but not adequate to provide
a stable, uncoupled state of the
AR at the cell surface. A
mutagenesis study of the m2 muscarinic receptor
provides precedent for this scheme. Although two regions of the third
intracellular loop were phosphorylated in response to agonist,
mutagenesis of either region alone did not reduce receptor
phosphorylation or internalization, and were described as
"redundant" for these functions (Pals-Rylaarsdam and Hosey, 1997
).
In contrast, desensitization was eliminated by mutagenesis of the more
carboxyl terminal region. The m2 muscarinic receptor study and our results support the model of multisite receptor-
-arrestin interaction and demonstrate that selective mutagenesis of the receptor can identify a subset of GRK
phosphorylation sites important for high-affinity
-arrestin binding
and desensitization but possibly leave intact other weak
-arrestin
interaction sites that contribute to endocytosis.
As an alternative to the model of multisite
AR-
-arrestin
interaction, the data reported here may indicate the existence of a
separate but minor internalization pathway that is not dependent on
receptor phosphorylation by GRK or
-arrestin binding.
Arrestin-independent internalization pathways have been reported for
the m1, m3, and m4 muscarinic receptors (Lee et al., 1998
) and
the angiotensin II type 1A receptor (Zhang et al., 1996
). An
internalization pathway that is independent of
-arrestin and GRK
phosphorylation, but dependent on PKA, was reported for the secretin
receptor (Walker et al., 1999
). In addition, dominant negative
-arrestin does not completely block
AR internalization (Ferguson
et al., 1996
), perhaps because of the contribution of a
-arrestin-independent pathway. These studies suggest that the lack
of correlation between the extent of
AR desensitization and
internalization reported here may result from the use of an alternate
internalization pathway that depends on agonist binding but not GRK
phosphorylation or
-arrestin binding. Based on this model, the
absence of crucial GRK phosphorylation sites in the serine mutants of
the 355 to 364 domain would not block internalization through the
alternate pathway.
Still another possibility that must be considered is based on our
observation of a 2-fold phosphorylation in the triple mutant, even
while receptor-level desensitization was eliminated. This result leaves
open the possibility that there is an as-yet-unidentified phosphorylation site that could be involved in endocytosis of the
AR
because the inhibitory effect of the triple mutant on endocytosis was
far from complete. Further studies will be required to evaluate the
location of this site and its possible contribution to internalization.
Regardless of which model best explains our results, the data described
here resolve previous conflicting reports on the function of the
AR
carboxyl tail serines and threonines (Bouvier et al., 1988
; Hausdorff
et al., 1989
; Hausdorff et al., 1991
; Yu et al., 1993
). Although
Hausdorff et al. reported global, nonspecific effects by substitution
of four amino acids in the 355- to-364 region, we show that
substitution of only two residues in this domain significantly reduces
agonist-induced desensitization while only partially inhibiting
internalization and phosphorylation. The selective mutagenesis strategy
employed here more specifically impaired desensitization. The report
that glycine substitution of serines 356 and 364 blocked
internalization and resensitization without affecting desensitization
(Yu et al., 1993
) is very difficult to reconcile with our results. We
found that alanine substitution of these same sites (S356,364A)
resulted in only a partial inhibition of internalization, and recycling
was unaffected. It is possible to rationalize the conflicting results
if glycine and alanine substitution cause significantly different
effects. In addition, their work was carried out in Chinese hamster
ovary (CHO) cells, rather than the HEK 293 cells used here, and
AR
internalization is greatly reduced in CHO cells compared with HEK 293 (Menard et al., 1997
). Therefore a small effect of the mutations on the extent of internalization in HEK 293 cells may be a major effect in CHO cells.
We found that mutations of the
AR 355-364 domain did not
affect recycling, because all of the mutants recycled with kinetics indistinguishable from those of HA-
AR-6HIS. However, we considered whether the carboxyl terminal HIS6 tag, included
in the HA-
AR-6HIS and all the mutant
ARs described here, might
inhibit receptor recycling relative to the untagged WT
AR. It was
recently reported that the addition of various amino acids to the
AR
carboxyl terminus inhibited recycling to the cell surface (Cao et al.,
1999
). The study proposed that additions to the carboxyl terminus block
the receptor PDZ-binding domain, composed of the last three
amino acids, from mediating the protein-protein interactions required for recycling. We tested the effect of the HIS6
tag by comparing recycling of the HA-
AR-6HIS and the untagged
WT
AR. As we report here, the recycling of the HA-
AR-6HIS and the
untagged WT
AR were indistinguishable. Our data are similar to those
of Kallal et al. (1998)
, who found similar internalization and
recycling for both the untagged WT
AR and the
AR labeled at the
carboxyl terminus with green fluorescent protein. The kinetic data we
obtained were similar to those of Morrison et al. (1996)
, who described the internalization and recycling of the HA-
AR, which has an untagged carboxyl tail. In addition, we found that the
HIS6 tag did not affect
AR desensitization.
Comparison of the HA-
AR-6HIS with the untagged WT
AR showed that
their desensitization was similar, in agreement with previous work
(January et al., 1997
). After 30-min pretreatment with 10 µM
epinephrine, the fraction activity remaining for the HA-
AR-6HIS,
HA-
AR, and untagged WT
AR was 0.03 (±0.003, n = 9), 0.06 (±0.01, n = 6), and 0.07 (±0.01, n = 6), respectively. We show that agonist-induced
desensitization, internalization, and recycling are clearly not
impaired by addition of the HIS6 epitope tag to
the receptor carboxyl terminus. However, we must be open to the
possibility that there may be important cell-specific differences in
the effects of C-terminal epitope tags that clearly block interactions
with PDZ domain-containing proteins such as HNERF (Hall et al., 1998
).
Previous work showed that both PKA- and GRK-dependent mechanisms
contributed to receptor level desensitization of the
AR (Hausdorff
et al., 1989
; Yuan et al., 1994
). The functional importance of the PKA
consensus sites found in the third intracellular loop and carboxyl
terminus of the
AR has been supported by mutagenesis studies
(Hausdorff et al., 1989
; Yuan et al., 1994
) and by the use of S49
lymphoma cells lacking either Gs or PKA activity
(Green and Clark, 1981
; Green et al., 1981
; Clark et al., 1988
).
Identification of the sites required for GRK-dependent, homologous
desensitization has been more difficult. This is understandable in
retrospect given that it seems that GRK-mediated phosphorylation,
-arrestin binding, internalization, and recycling may be tightly
linked (Krupnick and Benovic, 1998
; Lefkowitz et al., 1998
; Clark et al., 1999
) and, in addition, further confounded by PKA-mediated desensitization when concentrations of epinephrine in the pretreatment are high. In this article, isolation of GRK-mediated receptor desensitization and thereby simplification of its analysis was achieved
by substitution of the serines in the two PKA consensus sites. Our work
provides strong evidence that the serines in the
AR 355 to 364 play
a key role in GRK-dependent desensitization.
| |
Footnotes |
|---|
Received January 21, 2000; Accepted August 14, 2000
This study was supported by National Institutes of Health Grants GM31208 (R.B.C.) and HL57445, HL50047 (B.J.K.) and HL03463 (R.H.M.)
Send reprint requests to: Dr. Richard B. Clark, University of Texas-Houston Medical School, Department of Integrative Biology and Pharmacology, P.O. Box 20708, Houston, Texas 77225. E-mail: richard.b.clark{at}uth.tmc.edu
| |
Abbreviations |
|---|
AR, human
2-adrenergic
receptor;
PKA, cAMP-dependent protein kinase;
GRK, G protein-coupled
receptor kinase;
WT, wild-type;
HEK, human embryonic kidney;
HA, hemagglutinin;
DMEM, Dulbecco's modified Eagle's medium;
125ICYP, [125I]iodocyanopindolol;
GTP
S, guanosine 5'-3-O-(thio)triphosphate;
AT, ascorbic
acid/thiourea;
CGP, [3H]CGP-12177;
PBSS, PBS containing
0.12% sucrose;
DBM, n-dodecyl-
-D-maltoside;
WGA, wheat germ
agglutinin;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide gel
electrophoresis;
PGE1, prostaglandin E1;
CHO, Chinese
hamster ovary.
| |
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T. M. Tran, J. Friedman, E. Qunaibi, F. Baameur, R. H. Moore, and R. B. Clark Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the {beta}2-Adrenergic Receptor Using Phosphoserine-Specific Antibodies Mol. Pharmacol., January 1, 2004; 65(1): 196 - 206. [Abstract] [Full Text] [PDF] |
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R. B. Clark and T. C. Rich Probing the Roles of Protein Kinases in G-Protein-Coupled Receptor Desensitization Mol. Pharmacol., November 1, 2003; 64(5): 1015 - 1017. [Full Text] [PDF] |
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J. G. Baker, I. P. Hall, and S. J. Hill Influence of Agonist Efficacy and Receptor Phosphorylation on Antagonist Affinity Measurements: Differences between Second Messenger and Reporter Gene Responses Mol. Pharmacol., September 1, 2003; 64(3): 679 - 688. [Abstract] [Full Text] [PDF] |
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V. Simon, M.-T. Robin, C. Legrand, and J. Cohen-Tannoudji Endogenous G Protein-Coupled Receptor Kinase 6 Triggers Homologous {beta}-Adrenergic Receptor Desensitization in Primary Uterine Smooth Muscle Cells Endocrinology, July 1, 2003; 144(7): 3058 - 3066. [Abstract] [Full Text] [PDF] |
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B. T. Nguyen, L. Yang, B. M. Sanborn, and C. W. Dessauer Phosphoinositide 3-Kinase Activity Is Required for Biphasic Stimulation of Cyclic Adenosine 3',5'-Monophosphate by Relaxin Mol. Endocrinol., June 1, 2003; 17(6): 1075 - 1084. [Abstract] [Full Text] [PDF] |
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J. Friedman, B. Babu, and R. B. Clark beta 2-Adrenergic Receptor Lacking the Cyclic AMP-Dependent Protein Kinase Consensus Sites Fully Activates Extracellular Signal-Regulated Kinase 1/2 in Human Embryonic Kidney 293 Cells: Lack of Evidence for Gs/Gi Switching. Mol. Pharmacol., November 1, 2002; 62(5): 1094 - 1102. [Abstract] [Full Text] [PDF] |
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M. Lamey, M. Thompson, G. Varghese, H. Chi, M. Sawzdargo, S. R. George, and B. F. O'Dowd Distinct Residues in the Carboxyl Tail Mediate Agonist-induced Desensitization and Internalization of the Human Dopamine D1 Receptor J. Biol. Chem., March 8, 2002; 277(11): 9415 - 9421. [Abstract] [Full Text] [PDF] |
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