From the University of Texas at Houston Medical School, Department of Integrative Biology, Pharmacology, and Physiology, Houston, Texas 77225
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ABSTRACT |
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Tentative identification of the G protein-coupled
receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the 2-adrenergic receptor (
AR) was recently
reported based on in vitro phosphorylation of recombinant
receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz,
R. J. (1996) J. Biol. Chem. 271, 13796-13803).
Phosphorylated residues identified for GRK2 were threonine 384 and
serines 396, 401, and 407. GRK5 phosphorylated these four residues as
well as threonine 393 and serine 411. To determine if mutation of these
sites altered desensitization, we have constructed
ARs in which the
threonines and serines of the putative GRK2 and GRK5 sites were
substituted with alanines. These constructs were further modified to
eliminate the cAMP-dependent protein kinase (PKA) consensus
sites. Mutants
ARs were transfected into HEK 293 cells, and standard
kinetic parameters were measured following 10 µM
epinephrine treatment of cells. The mutant and wild type (WT) receptors
were all desensitized 89-94% after 5 min of 10 µM
epinephrine stimulation and 96-98% after a 30-min pretreatment. There
were no significant changes observed for any of the mutant
ARs
relative to the WT in the extent of 10 µM
epinephrine-induced internalization (77-82% after 30 min).
Epinephrine treatment for 1 min induced a rapid increase in the
phosphorylation of the GRK5 and PKA
mutant
ARs as well
as the WT. We conclude that sites other than the GRK2 and GRK5 sites
identified by in vitro phosphorylation are involved in
mediating the major effects of the in vivo
GRK-dependent desensitization of the
AR.
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INTRODUCTION |
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Epinephrine stimulation of the 2-adrenergic
receptor (
AR)1 in intact
cells activates the receptor and rapidly induces its desensitization.
The decreased responsiveness of the receptor after stimulation by
near-saturating concentrations of epinephrine appears to be caused by
rapid cAMP-dependent protein kinase (PKA) and G
protein-coupled receptor kinase (GRK) phosphorylation. GRK phosphorylation in turn promotes
-arrestin binding and receptor internalization (1, 2). Identification of the specific amino acids
phosphorylated by these protein kinases has been the focus of numerous
studies. Through the use of several deletion and substitution mutants,
the sites for PKA-mediated desensitization of the
AR in intact cells
were shown to be serines 261 and 262 in the third intracellular loop
PKA consensus site (3-5). For the GRKs, mutagenesis studies indicate
the involvement of 11 serines and threonines in the carboxyl terminus
(5, 6). By utilizing in vitro GRK phosphorylation of
recombinant
AR reconstituted into liposomes followed by sequencing
of proteolytic fragments of the carboxyl tail, it was found that four
sites were phosphorylated by GRK2 (
AR kinase 1), serines 396, 401, and 407, and threonine 384, and six by GRK5 that included the same four
phosphorylated by GRK2 and additionally threonine 393 and serine 411 (7). On the basis of this study it was proposed that these amino acids were the sites of GRK-mediated phosphorylation in intact cells; however, the effects of mutating these sites on the desensitization of
the
AR in vivo was not addressed.
In the studies presented here, we have determined the effects of
substitutions of the putative GRK phosphorylation sites identified by
the in vitro approach of Fredericks et al. (7) on
the desensitization, internalization, and phosphorylation of the
respective mutant ARs. The serine or threonine residues tentatively
identified as the GRK2 and GRK5 phosphorylation sites were replaced
with alanine. To aid our analysis of the effects of these mutations, we
also replaced the serine residues of the two consensus PKA phosphorylation sites with alanine to eliminate PKA-mediated
desensitization and phosphorylation. The GRK/PKA mutants (designated as
GRK2
or GRK5
), as well as a mutant
AR
containing only the PKA substitutions (PKA
), were
constructed in the WT
AR that had been modified by placement of the
hemagglutinin (HA) antigen at the amino terminus and six histidine
residues at the carboxyl terminus. We recently established that the
desensitization, internalization, and phosphorylation of this double
epitope-modified
AR, stably transfected into HEK 293 cells, was
indistinguishable from the wild type receptor (8). Furthermore, the HEK
293 cell line offers a system in which the effects of overexpressed
GRK2 on
AR phosphorylation and internalization have been studied (9)
and in which endogenous GRK2 expression has been shown (10). Our
results demonstrate that the GRK2 substitutions did not significantly
alter epinephrine-induced desensitization of the
AR, although a
slight reduction of the rate and extent of desensitization was observed
with the GRK5 substitutions. Consistent with these observations, we
found that the mutant
ARs were rapidly phosphorylated and that the
rates of internalization were unimpaired. The lack of any major effects
on these parameters suggests that the GRK site(s) that mediate the
desensitization and subsequent internalization of the
AR do not
involve the sites identified by in vitro
phosphorylation.
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EXPERIMENTAL PROCEDURES |
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Construction of the Mutant ARs--
The construction of the
plasmid containing the HA and six histidine-tagged
AR has been
described previously (8). This plasmid is designated here as WT
AR.
For construction of the mutant
ARs, the HA-His6-tagged
AR was excised from the pBC12B1 plasmid as an
NcoI/SalI fragment, made blunt-ended, and ligated
into the expression vector pKNH that had been
HindIII-digested and blunt-ended. All mutants were
constructed using polymerase chain reaction (PCR) methods. To change
the serines at 261 and 262 to alanines (third intracellular loop PKA
consensus site), a two-step PCR mutagenesis method was used with the
HA-His6
AR in pBC12B1 as template. In the first step,
two independent reactions were carried out, one using a sense
mutagenizing oligonucleotide paired with a downstream oligonucleotide,
and the other using an antisense mutagenizing oligonucleotide paired
with an upstream oligonucleotide. In the second step, the products of
the first PCR reactions were amplified using a pair of oligonucleotides
nested within the upstream and downstream oligonucleotides. The
resulting product was digested with AccI and subcloned into
the plasmid pGEM3Z (Promega). The mutagenized receptor was excised as a
BamHI/HindIII fragment, blunt-ended, and
subcloned into HindIII-digested, blunt-ended pKNH. The S261A
and S262A mutant HA-His6-tagged
AR in pKNH served as a
template for subsequent mutagenesis. All other mutageneses were
performed with single PCR reactions using mutagenizing sense and
antisense oligonucleotides and Pfu I polymerase
(Stratagene). After PCR, digestion with DpnI (which requires
methylated DNA) was performed to remove the non-mutagenized template
DNA, followed by transformation into XL1 Blue competent cells. The
entire length of each mutant
AR was sequenced to verify the changes
and to ensure that no other alterations were introduced by PCR.
Transfection into HEK 293 Cells-- The HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37 °C in 5% CO2. Each mutant plasmid was linearized by PvuI digestion and transfected into the HEK 293 cells using the CaPO4 method. The day after transfection the cells were shocked with 25% glycerol in DMEM and placed in media containing 0.4 mg/ml G418 the day after shocking. Stable transfectants were identified using an intact cell [125I]iodocyanopindolol (125ICYP) binding assay described below.
Measurement of Receptor Levels--
To measure intact cell
receptor number by 125ICYP binding, cells were grown in
12-well dishes. After rinsing with serum-free DMEM, the cells were
removed by pipetting up and down with 200-500 µl of serum-free DMEM.
Triplicate reactions were performed in DMEM containing 200
pM 125ICYP, in a total assay volume of 200 µl. Nonspecific binding was measured with the addition of 1 µM alprenolol. The reactions were incubated on ice for 50 min and terminated by dilution with 2.5 ml of ice-cold 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2.
The 125ICYP-bound receptor protein was isolated by
filtration through Whatman GF/C filters. The filters were rinsed three
times with 2.5 ml of the Tris/MgCl2 buffer and counted in a
Beckman 4000 Gamma counter. Protein was measured in duplicate or
triplicate with 100 µl of cells. To measure
AR levels in
membranes, 5 µg of membrane protein was used per reaction containing
0.1 mM phentolamine, 40 mM Hepes, pH 7.2, 2 mM EDTA, 0.2 mM ascorbate, and 2 mM
thiourea, and
200 pM 125ICYP in the presence
or absence of 1 µM alprenolol. The reactions were
incubated at 30 °C for 50 min and terminated as described for the
intact cell binding.
Measurement of Equilibrium Binding Constants for
Epinephrine--
The Kd values for epinephrine were
determined by displacement of 125ICYP using methods
previously described (8). The 125ICYP was prepared
according to the method of Barovsky and Brooker (11) and Hoyer et
al. (12). Alprenolol (1 µM) was included to measure
nonspecific binding. The reactions were incubated for 50 min at
30 °C and stopped as described above. The reactions included 40-50
pM 125ICYP, 10 µM GTPS, and
concentrations of epinephrine ranging from 0.1 to 100 µM.
The data were fit to a one-component competition sigmoidal curve with a
Hill coefficient of
1 using GraphPad software and
Kd values determined using the Cheng-Prusoff
formulation.
Measurement of Receptor Internalization by
[3H]CGP-12177 Binding in Intact Cells--
Cells were
plated onto 60-mm dishes coated with poly-L-lysine (Sigma)
to improve cell adhesion. The cells were pretreated with 10 µM epinephrine or carrier by additions made directly to growth medium from 100× stock solutions. The 100× epinephrine stock
(1 mM) was prepared in 100× AT carrier, such that the
final concentration of AT was 0.1 mM ascorbate and 1 mM thiourea, pH 7. Controls were treated with the AT
carrier at the same final concentration. Pretreatment was performed at
37 °C for various times and was stopped by removal of media and 6 washes with 2 ml of ice-cold serum-free DMEM, pH 7. Surface receptor
number was then measured with the addition of 2 ml of serum-free DMEM containing 5 nM [3H]CGP-12177, designated CGP
hereafter. Incubations were on ice for 1 h. To measure nonspecific
binding, cells were incubated with 1 µM alprenolol added
to the CGP mix. To measure total receptor number, including
internalized AR, digitonin was added to the binding mix (including
alprenolol controls) to a final concentration of 0.2% as described
previously (8, 13). The reactions were stopped by removal of the
binding mix followed by 3 washes with ice-cold DMEM, 2 ml each. The
cells were scraped into 0.75 ml of trypsin and counted in 5 ml of
scintillation fluid. Measurements were performed in triplicate for each
time point. Additional plates that were washed identically to the
experimental plates were used to measure protein. The surface receptor
number is expressed relative to the AT-treated control in each
experiment. GraphPad software was used to fit the data to an equation
for monoexponential decay and determine the apparent rate of
internalization.
Membrane Preparation--
Cells were plated into 150-mm dishes
coated with poly-L-lysine and were pretreated at 37 °C
with 10 µM epinephrine or AT carrier for the indicated
times. The pretreatment was stopped with 6 washes of 10 ml of 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 bovine serum albumin). The
washed cells were scraped into HME plus 10 µg/ml leupeptin, 20 mM tetrasodium pyrophosphate, and 0.1 µM
okadaic acid and homogenized with 7 strokes in a type B Dounce
homogenizer. The homogenates were layered onto step gradients of 23 and
43% sucrose 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% interface was removed, flash-frozen in liquid nitrogen, and stored at 80 °C.
Adenylyl Cyclase Assay--
Adenylyl cyclase activity was
assayed by a modification of the method described by Salomon et
al. (14). Membranes were diluted to a final protein concentration
of 0.2-0.4 mg/ml and were incubated for 10 min at 30 °C with 40 mM Hepes, pH 7.7, 1 mM EDTA, 1.34 mM MgCl2, 8 mM creatine phosphate,
16 units/ml creatine kinase, 100 µM ATP, 1 µM GTP, 0.1 mM 1-methyl-3-isobutylxanthine, 2 µCi of [-32P]ATP (NEN Life Science Products, 30 Ci/mmol) in a total volume of 100 µl. The final free Mg2+
concentration was calculated to be 0.3 mM to optimize
desensitization measurements (3, 15, 16). Each point was assayed in
triplicate, with 6-8 concentrations of epinephrine bracketing the
EC50. The [32P]cAMP produced in the reaction
was purified over Dowex and alumina columns (17). The
Vmax and EC50 values were determined
with GraphPad software.
Quantitation of Desensitization-- As we have previously shown, the expression for coupling efficiency can be combined with that for Vmax to give Equation 1 (8).
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(Eq. 1) |
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(Eq. 2) |
Determination of AR Phosphorylation--
To measure
phosphorylation of the
AR, confluent cells were washed three times
in phosphate-free DMEM, incubated for 3 h with [32P]H3PO4 (0.5-1.0 mCi/100-mm
dish), and pretreated for the indicated times with either 10 µM epinephrine or AT carrier. The cells were solubilized,
and the extracts were subjected to a two-step purification using nickel
nitrilotriacetic acid-agarose and wheat germ agglutinin-agarose (WGA)
as described previously (8) with the following modifications. The
nickel nitrilotriacetic acid eluent fractions containing the
AR were
mixed with 100 µl of WGA (packed volume) and incubated for 90 min at
4 °C with rocking. The WGA/
AR was collected and washed with 5 ml
of nickel column buffer (0.05%
n-dodecyl-
-D-maltoside, 20 mM
Hepes, pH 7.4, and 150 mM NaCl) at 4 °C. The WGA was
further washed twice with 400 µl of 0.5% sodium dodecyl sulfate
(SDS) at 37 °C for a total incubation time of 10 min. The WGA pellet was collected and the
AR eluted with SDS sample buffer (50 mM Tris, pH 6.8, 2% SDS, 0.025% bromphenol blue, 6 M urea, 14.3 mM
-mercaptoethanol). The
receptor was resolved on 7.5% SDS-polyacrylamide gels with the
addition of pre-stained low molecular weight standards (Bio-Rad). The
gels were dried and exposed to a phosphorscreen for 2-24 h, and the
data were analyzed using a Molecular Dynamics Storm PhosphorImager
model 860 and Imagequant software. Autoradiograms of the dried gels
were also obtained (24-48 h). In some experiments the gels following
SDS-PAGE were transferred to 0.22-micron PVDF membranes, and the
identity of the radiolabeled band as the
AR was confirmed by Western
analysis using a primary anti-HA polyclonal antibody (Babco) and a
horseradish peroxidase-conjugated goat anti-rabbit (Bio-Rad) as the
secondary antibody as described previously (8).
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RESULTS |
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Determination of the Coupling Efficiency for Epinephrine Activation
of Adenylyl Cyclase for the Mutant and WTARs--
As we have
previously shown, determination of the coupling efficiency for agonist
activation of adenylyl cyclase requires the measurement of receptor
levels, the low affinity Kd for agonist binding, and
the EC50 for activation of adenylyl cyclase (8). A summary
of these determinations using membranes prepared from each cell line is
shown in Table I. At least two clones expressing each receptor were examined, and those shown in Table I were
selected for all subsequent experiments since they expressed reasonably
similar levels. A representative experiment for the determination of
the low affinity Kd for epinephrine binding is shown
in Fig. 1. We found no significant
differences in the Kd values for the mutant
receptors versus the WT. The mutant and WT cell lines
displayed similar values for basal activity and the
Vmax for epinephrine stimulation. The
EC50 values for epinephrine stimulation of the mutant
receptors were found to be consistently 4-6.5 times higher than that
of the WT
AR (see Fig. 2 for data
summary). Using the formulations we described previously (8, 19), we
calculated from the data in Table I that the coupling efficiencies of
the mutants were reduced by a factor of 2-4-fold relative to
WT
AR.
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Desensitization of the Mutant and WTARs--
To assess
desensitization, HEK 293 cells stably expressing the WT or mutant
ARs were pretreated with 10 µM epinephrine for various
times from 0.5-30 min. Following pretreatment, membranes were prepared
and assayed for epinephrine-stimulated adenylyl cyclase activity using
a range of epinephrine concentrations. The data summary for
desensitization in response to 2 and 30 min pretreatment with 10 µM epinephrine is shown in Fig. 2.
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Internalization of the WT and Mutant
ARs--
Internalization of the mutant and WT
ARs in response to
10 µM epinephrine was measured by CGP binding and is
plotted as the loss of surface receptors (Fig.
4). The apparent rate of internalization was determined by fitting the data shown in Fig. 4 to an equation for
monoexponential decay. The fit to monoexponential decay did not take
into account the initial lag observed for internalization of all the
receptor types. This method, however, allowed calculation of
approximate apparent rates of internalization for comparison of the WT
and mutant
ARs. The apparent rate of internalization of the WT
AR
(2.96 min ± 0.17, n = 3) was found to be similar to those measured for the GRK2
(2.96 min ± 0.30, n = 3), the GRK5
(3.76 min ± 0.25, n = 3), and the PKA
(3.69 min ± 0.28, n = 3)
AR mutants. The extent of
internalization was also similar, with 80% ± 1.9 (n = 9) of the WT
AR internalized after 30 min of 10 µM
epinephrine pretreatment compared with 84% ± 0.5 (n = 6), 82% ± 1.1 (n = 5), and 77% ± 0.5 (n = 4) for the GRK2
, GRK5
,
and PKA
mutants, respectively. The internalization was
not the result of receptor loss or down-regulation, as determined by
CGP binding in the presence and absence of digitonin as described under
"Experimental Procedures" (data not shown).
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Phosphorylation of the Mutant and Wild Type ARs in Response to
10 µM Epinephrine--
Cells expressing the WT,
GRK5
, and PKA
ARs were labeled with
32P for 3 h and subsequently treated with either
carrier or 10 µM epinephrine for 1 min. Phosphorylation
of the
AR was assessed by solubilization and purification of the
receptors using the two-step affinity chromatography procedure
described under "Experimental Procedures." The purified receptor
was subjected to SDS-PAGE, and the proteins were transferred to PVDF
membranes as described under "Experimental Procedures." A
representative experiment performed in duplicate is shown in Fig.
5. The PhosphorImage scan of the gel
after transfer to the PVDF membrane is shown in Fig. 5A, and the Western blot of the same membrane is seen in Fig. 5B.
The time course of phosphorylation for the mutant
ARs was similar to
what we have previously reported for the WT
AR (8), with the peak at
about 1 min, declining after 5 min (data not shown).
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DISCUSSION |
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Our experiments demonstrate that mutant ARs containing alanine
substitutions for the serine/threonine residues, tentatively identified
by in vitro phosphorylation as the sites of GRK2 or GRK5
phosphorylation (7), undergo extensive and rapid agonist-induced desensitization and internalization. We had expected that these substitutions of the putative GRK and PKA sites would eliminate the
desensitization of the
AR. Consistent with the desensitization data,
we found that the GRK5
mutant was rapidly phosphorylated.
We propose that sites other than or in addition to those identified
in vitro by Fredericks et al. (7) are required
for in vivo GRK2 or GRK5 phosphorylation and desensitization
of the
AR.
It is possible that one or more of the crucial GRK sites involved in
the functional desensitization of the receptor were missed in the
in vitro study of GRK2 and GRK5 phosphorylation (7). The
sequencing of peptides in this study was focused on a fragment located
in the distal portion of the receptor carboxyl terminus (residues
374-413). Thus, it remains possible that serines 355, 356, and 364 and
threonine 360 residues located in the proximal portion of the receptor
carboxyl terminus are involved in GRK phosphorylations in
vivo. All 11 of the serine/threonine residues found in the
receptor carboxyl terminus, from amino acid 355 to 413, have been
implicated as possible sites of GRK phosphorylation. Decreased
desensitization and phosphorylation was reported for a mutant AR
containing substitutions at all 11 carboxyl-terminal serine/threonine
sites (5, 6). A mutagenesis study in which only serines 355, 356, and
364 and threonine 360 were substituted for either glycine or alanine
suggested that the in vivo site(s) of GRK phosphorylation
may be among these residues (20). This study demonstrated that
substitution of the four residues resulted in a mutant
AR almost
completely defective in the rapid agonist-induced desensitization,
internalization, and phosphorylation. The authors speculated that the
presence of desensitization, partial phosphorylation, and
internalization observed in the
AR containing substitutions at all
11 residues resulted from relief of a conformational inhibition present
in the mutant with only four substitutions (20). Their data, however,
are consistent with the possibility that serines 355, 356, and
364 and threonine 360 may include the in vivo sites of GRK
phosphorylation.
Another possible explanation for the discrepancy we have found between
the in vitro phosphorylation of the AR and our studies of
the functional effects of these mutations when expressed and analyzed
following intact cell treatment is that additional sites may be
nonspecifically phosphorylated by GRK2 or GRK5 in vitro. Precedent for this possibility is found in recent studies of the rhodopsin receptor that demonstrated important differences between in vivo and in vitro identification of GRK
phosphorylation sites. Chemical identification of the sites
phosphorylated in vivo by a member of the GRK family has
been described for the rhodopsin receptor by Ohguro et al.
(21). They found that two sites in the receptor carboxyl terminus were
phosphorylated by rhodopsin kinase (GRK1). The two serines they
identified were differentially regulated; serine 338 was phosphorylated
in response to flashes of light, whereas serine 334 was phosphorylated
more slowly, and only after continuous light exposure. In rather
striking contrast to these studies, in vitro phosphorylation
consistently identified 7-8 mol of phosphate/mol of rhodopsin receptor
(22, 23). Based on these studies of rhodopsin at least, it is
reasonable to expect that there may be substantial differences between
in vitro and in vivo phosphorylations of the
AR.
Still another possibility to consider is that the numerous
substitutions we have made in the GRK2 and GRK5 mutants have in some
way altered the specificity of GRK phosphorylation through de-localized
effects. Although we may have eliminated one or more sites of GRK
phosphorylation, the amino acid changes in the mutants here may allow
inappropriate GRK phosphorylation at other sites. However, de-localized
effects are unlikely because of the similar functional properties of
the WTAR and mutant receptors. The desensitization, internalization,
and agonist binding affinity of the mutant receptors were similar to
those of the WT
AR, and only modest reductions in coupling efficiency
were observed.
The similar desensitization we observed for the PKA and
the WT
AR (Fig. 2B and Fig. 3) was also unexpected, since
it has been suggested that receptor phosphorylation by PKA is necessary
to achieve maximum desensitization in response to high agonist
concentrations. Hausdorff et al. (5) reported that a mutant
AR containing alanine substitutions for the serines of the two
consensus PKA sites showed decreased desensitization upon exposure to
high concentrations of isoproterenol relative to the wild type receptor
in Chinese hamster fibroblast cells. Similarly, Moffett et
al. (24) found that a mutant
AR with alanine substitutions for
the serines of the carboxyl-terminal PKA site was subject to less
desensitization than the wild type receptor in mouse Ltk
cells. In contrast, our results indicate that PKA-mediated receptor phosphorylation is not required for maximum desensitization in response
to high agonist exposure. These results agree with studies we performed
with cyc
and kin
mutants of the S49 wild
type lymphoma cell line in which we found no alteration in the extent
of agonist-induced homologous desensitization relative to the wild type
(25). Internalization of the PKA
AR was also similar to
that of the WT
AR. We speculate that our results may be caused by
redundancy of PKA-mediated phosphorylation/desensitization with the
GRK/
-arrestin/internalization pathway as has been previously suggested (26). Alternatively there may be cell-specific factors that explain these discrepancies.
To conclude that the sites phosphorylated by GRK2 and GRK5 in vivo do not correspond with the sites identified in vitro by Fredericks et al. (7) requires that GRK2 and GRK5 are expressed in HEK 293 cells. That GRK2 is present in HEK 293 cells has been shown by Menard et al. (10) using Western blot analysis and by other investigators using reverse transcription-coupled PCR as well as Western blots.2 GRK5 is either absent or expressed at low levels in HEK 293 cells using reverse transcription-PCR.2 The levels of GRK expression needed in vivo to mediate receptor phosphorylation are unknown. The absence or low expression of GRK5 in HEK 293 cells makes it difficult to determine the functional significance in vivo of the sites phosphorylated by GRK5 in vitro. However, since GRK2 expression in HEK 293 cells has been shown, the work presented here conclusively demonstrates the lack of in vivo functional significance for the six serine/threonine residues identified in vitro as sites of GRK2 and/or GRK5 phosphorylation.
Identification of the AR sites phosphorylated in vivo is
important for a more complete understanding of the complex processes of
desensitization. With the exception of rhodopsin, G protein-coupled receptors have been notoriously refractory to chemical analysis of
phosphorylation sites in vivo due to their extremely low
concentrations in the cell. This approach, however, may ultimately be
required for resolution of the molecular actions of the GRKs, PKA, and other protein kinases that have been implicated in the regulation of
the
AR.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 31208.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 Texas at
Houston Medical School, Dept. of Integrative Biology, Pharmacology, and
Physiology, P. O. Box 20708, Houston, TX 77225. Tel.:
713-500-7490; Fax: 713-500-7455.
1
The abbreviations used are: AR, human
2-adrenergic receptor; HA, hemagglutinin; GRK, G
protein-coupled receptor kinase; WT, wild type; PKA,
cAMP-dependent protein kinase; 125ICYP,
[125]iodocyanopindolol; PAGE, polyacrylamide gel
electrophoresis; CGP, [3H]CGP-12177; DMEM, Dulbecco's
modified Eagle's medium; AT, ascorbic acid/thiourea; WGA, wheat germ
agglutinin; PVDF, polyvinylidene difluoride; PCR, polymerase chain
reaction; GTP
S, guanosine 5'- 3-O-(thio)triphosphate.
2 M. Ascoli and R. Premont, personal communication.
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REFERENCES |
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