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(Received for publication, November 14,
1995; and in revised form, December 7, 1995) From the
The role of G protein-coupled receptor kinases (GRKs) in the
regulation of dopamine D1A receptor responsiveness is poorly
understood. To explore the potential role played by the GRKs in the
regulation of the rat dopamine D1A receptor, we performed whole cell
phosphorylation experiments and cAMP assays in 293 cells cotransfected
with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5).
The agonist-dependent phosphorylation of the rat D1A receptor was
substantially increased in cells overexpressing GRK2, GRK3, or GRK5.
Moreover, we report that cAMP formation upon receptor activation was
differentially regulated in cells overexpressing either GRK2, GRK3, and
GRK5 under conditions that elicited similar levels of GRK-mediated
receptor phosphorylation. Cells expressing the rat D1A receptor with
GRK2 and GRK3 displayed a rightward shift of the dopamine dose-response
curve with little effect on the maximal activation when compared with
cells expressing the receptor alone. In contrast, cells expressing GRK5
displayed a rightward shift in the EC Phosphorylation is an important mechanism involved in the
regulation of numerous cellular responses, notably the responsiveness
of G protein-coupled receptors(1) . This phosphorylation
process is believed to be the triggering mechanism that leads to
receptor desensitization. The cellular responses elicited upon
activation of G protein-coupled receptors are regulated in a dynamic
fashion by the action of two classes of serine/threonine kinases. The
first class consists of the second messenger-dependent kinases such as
protein kinase A and protein kinase C(1) . The second class
consists of receptor-specific kinases that phosphorylate the
agonist-occupied or activated form of the G protein-coupled
receptors(1, 2, 3) . These receptor kinases
were originally described for rhodopsin (rhodopsin kinase) and the
This large
family of kinases includes six members (GRK1 to GRK6) whose activities
are regulated by phospholipids, post-translational modifications, or G
protein The recent advent of
molecular biology techniques has allowed a better understanding of the
underlying mechanisms of the dopaminergic neurotransmission. So far,
five distinct genes encoding at least six dopamine receptors have been
isolated and characterized(8, 9) . Dopamine receptors
belong to the G protein-coupled receptor superfamily. These dopamine
receptors are grouped into D1- and D2-like receptors based upon their
similarity at the amino acid level and their ability to couple to the
activation (D1A/D1 and D1B/D5) or inhibition (D2short, D2long, D3, and
D4) of adenylyl cyclase(8, 9) . Many of the
neurophysiological effects of dopamine in retina and brain are thought
to be mediated through the activation of dopamine D1A receptor subtype (10, 11, 12, 13, 14, 15) .
However, the mechanisms involved in the regulation of the D1A receptor
responsiveness are poorly understood. Upon exposure to dopamine, D1A
receptors have been shown to undergo a desensitization process as
evidenced in cellular systems expressing endogenous D1A receptors or
heterologous expression
systems(16, 17, 18, 19, 20) .
Furthermore, Zhou et al.(21) demonstrated using a
protein kinase A inhibitor and a GRK inhibitor (heparin) that D1A
receptors, expressed endogenously in SK-N-MC cells, could undergo both
protein kinase A- and GRK-mediated desensitization. Although a recent
study has shown that D1A receptor overexpressed in Sf9 cells can
undergo agonist-dependent desensitization, which was associated with
weak receptor phosphorylation(22) , a convincing demonstration
of a role for direct phosphorylation of the receptor in this process
remains to be clearly established. Most of the regulation studies
performed previously in cellular systems have been limited by the low
levels of receptor
density(16, 17, 18, 19, 20) .
To avoid this difficulty, we have overexpressed the receptor alone or
with various GRKs using a heterologous expression system to investigate
the potential role of the GRK pathway in the phosphorylation and
desensitization of the dopamine D1A receptor as well as to examine the
biochemical and biological specificity of various GRKs. Our results
indicate that the agonist-occupied form of the D1A receptor can serve
as a substrate for a variety of GRKs. Moreover, we show that receptor
phosphorylation by specific members of the GRK family leads to distinct
desensitization patterns of dopamine responsiveness.
The rD1A receptor has been shown to be coupled to the
activation of adenylyl cyclase when expressed in 293
cells(26) . To investigate the coupling properties of the
HA-rD1A receptor, dose-response curves to dopamine were performed at
different time points (2, 5, and 10 min) using a whole cell cAMP assay
in the absence of phosphodiesterase inhibitors. The wild-type or
HA-rD1A receptor display similar dose-response curves to dopamine at
all time points investigated (Fig. 1). Over the time course
studied, at similar receptor expression levels for both forms of the
receptor, the maximal activation of adenylyl cyclase (V
Figure 1:
Time course of
dopamine-mediated activation of adenylyl cyclase in 293 cells
transiently transfected with rat dopamine D1A receptor. A,
Wild-type receptor; B, HA-tagged receptor. Whole cell cAMP
assays were performed as described under ``Experimental
Procedures.'' Dose-response curves were performed with increasing
concentrations of dopamine and exposed for 2, 5, and 10 min. Each point
represents the mean of two independent experiments. Curves were fitted
using the ALLFIT program(32) . Determinations of EC
To determine the apparent molecular weight of
the HA-rD1A receptor protein expressed in 293 cells, we performed
photoaffinity cross-linking experiments using
Figure 2:
Photoaffinity cross-linking and
biotinylation of the HA-rD1A receptor expressed transiently in 293
cells. A, photoaffinity cross-linking experiments were
performed in membranes prepared from 293 cells transfected with
To test the ability of the monoclonal antibody
12CA5 to immunoprecipitate the HA-rD1A receptor, 293 cells expressing
the tagged receptor or pCMV
Figure 3:
Time course of agonist-dependent
phosphorylation of HA-rD1A receptor overexpressed alone or with various
GRKs in transiently transfected 293 cells. Cells transfected with the
HA-rD1A receptor alone (
Figure 4:
Agonist-dependent phosphorylation and
phosphoamino acid analysis of the HA-rD1A receptor expressed in 293
cells. A, cells transfected with
Figure 5:
Time course of dopamine-mediated adenylyl
cyclase activation in 293 cells overexpressing the HA-rD1A receptor and
various GRKs. A, basal; B, 10 nM dopamine; C, 10 µM dopamine. Whole cell cAMP accumulation
was measured following exposure to 0.1 mM ascorbate (basal) or dopamine (DA) for 2, 5, and 10 min. Data
are presented as the mean of three independent experiments done in
triplicate determinations. Receptor expression for each of the
experimental procedures were as follows:
To further elucidate the differences in the regulation of
the dopamine D1A receptor responsiveness by GRK2, GRK3, and GRK5, cells
expressing similar levels of the HA-rD1A receptor either alone or with
these kinases were stimulated with increasing concentrations of
dopamine for 5 min. Under these experimental conditions, dopamine
elicited a dose-response curve in cells expressing the receptor alone
with an EC
Figure 6:
Dose-response curves for dopamine-mediated
adenylyl cyclase activation in 293 cells overexpressing the HA-rD1A
receptor alone or with various GRKs. A, Whole cell cAMP assays
were done as described under ``Experimental Procedures.''
Dose-response curves were generated by stimulating cells with
increasing concentrations of dopamine for 5 min. Each point represents
the mean of four to five independent experiments done in duplicate
determinations. Curves were fitted using the ALLFIT program. EC
In this report we demonstrate that the agonist-occupied form
of the D1A receptor can serve as a substrate for various GRKs.
Phosphorylation of the D1A receptor by these GRKs leads to a diminished
ability of the receptor to increase intracellular cAMP levels in
response to dopamine. For an equivalent extent of phosphorylation of
the D1A receptor by various kinases, the attenuation of responsiveness
appears to be more pronounced following phosphorylation by GRK5 than
GRK2 or GRK3. These results provide evidence that specificity of action
can be demonstrated between G protein-coupled receptors and GRKs.
In 293 cells, the HA-D1A receptor behaves identically to the
wild-type receptor. In agreement with the observation that 293 cells
contain low levels of endogenous GRKs(30) , only a very weak
agonist-mediated desensitization of the signal is observed in cells
overexpressing the D1A receptors (data not shown). These findings
correlate with the low agonist-mediated increase (
First, these results
might be explained by the existence of distinct GRK phosphorylation
sites located within the cytoplasmic domains of the D1A receptor.
Indeed, it is possible that phosphorylation of distinct GRK sites leads
to different conformational changes of the phosphorylated D1A receptor,
which may potentially display differences in their ability to activate
adenylyl cyclase. Despite the large amount of evidence for the
phosphorylation of G protein-coupled receptors by GRKs, very little is
known about the exact nature of the phosphorylation sites for the
various characterized GRKs. Distinct specificities have been
demonstrated for Second,
the distinct modulation of D1A receptor responsiveness by the different
GRKs might be explained by the potential role played by arrestin-like
proteins, which have been demonstrated to be essential for the full
extent of receptor desensitization for Previously, selectivity, or the lack thereof, in the ability of the
different GRKs for phosphorylating different receptors has been
documented. Thus, rhodopsin is a better substrate for GRK1 than GRK2 (43, 44) ;
Studies using cellular
systems have helped to delineate the molecular events involved in G
protein-coupled receptor regulation(39) . Several transgenic
studies have now established the relevance of these mechanisms in
various physiological situations(47, 48) . Mice
overexpressing carboxyl-terminal truncated rhodopsin, which lack the
GRK1 phosphorylation sites, display abnormal prolonged flash responses,
suggesting that phosphorylation of rhodopsin is essential for turnoff
of the light signal in vivo(47) . In addition,
transgenic mice overexpressing GRK2 specifically in the heart display a
reduced cardiac function as measured by a diminution of
isoproterenol-stimulated left ventricular contractility, myocardial
adenylyl cyclase activity, and decreased functional coupling of
Volume 271,
Number 7,
Issue of February 16, 1996 pp. 3771-3778
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
value with an
additional 40% reduction in the maximal activation when compared with
cells expressing the receptor alone. Thus, we show that the dopamine
D1A receptor can serve as a substrate for various GRKs and that
GRK-phosphorylated D1A receptors display a differential reduction of
functional coupling to adenylyl cyclase. These results suggest that the
cellular complement of G protein-coupled receptor kinases may determine
the properties and extent of agonist-mediated responsiveness and
desensitization.
-adrenergic receptor (-adrenergic receptor
kinase) and are referred to as the G protein-coupled receptor kinases
or GRKs(
)(1, 2, 3) .
subunits(2, 3, 4, 5, 6) .
The GRKs are widely distributed in brain and periphery, suggesting an
important role in the regulation of responsiveness of various G
protein-coupled receptors(2, 7) . Moreover, Arriza et al.(7) have shown that
-adrenergic receptor
kinase 1 (GRK2) and -adrenergic receptor kinase 2 (GRK3) are found
in presynaptic and postsynaptic localizations in various brain regions
consistent with a general role for these kinases in the desensitization
of neuronal G protein-coupled receptors and their putative role in the
regulation of neuronal activity. However, little information exists as
to the specificity of the various kinases and as to whether
phosphorylation of a given receptor by different kinases results in the
same attenuation of the biological signals.
Expression Constructs
The dopamine rat D1A
(rD1A) receptor was modified using oligo-directed mutagenesis to
include the sequence for the hemagglutinin (HA) epitope recognized by a
commercially available monoclonal antibody 12CA5(23) . The
nine-residue sequence of the epitope (YPYDVPDYA) was inserted after
Ala, and this residue was repeated after the epitope to
increase the distance between the epitope and the putative N-linked glycosylation site to avoid potential steric
interference. A 67-mer oligonucleotide containing the epitope sequence
as well as the 5` regions of the rat D1A receptor was used to amplify a
modified 5` end of the gene, which was spliced to the wild-type
receptor gene 3` sequence using the internal BglII site. The
sequence of the modified gene was verified by using dideoxy sequencing
method (U. S. Biochemical Corp.) and subcloned into the expression
vector pCMV5. The HA-epitope tagged rat D1A receptor will be referred
to as HA-rD1A receptor. The expression constructs for GRK2, GRK3, and
GRK5 were described previously(7, 24) . The
-galactosidase pCMV expression construct was purchased form
Clonentech.Cell Culture and Plasmid Transfection
Human
embryonic kidney cells (293 cells) were grown in minimal essential
medium with Earle's salts supplemented with heat-inactivated
fetal bovine serum (10% (v/v)) and gentamicin (100 µg/ml) at
37° C in 5% CO atmosphere. Cells seeded in 100-mm
dishes (2.5 
10
cells/dish) were transiently
transfected by a modified calcium-phosphate
method(25, 26) . All experiments were performed with
cells passaged 34-48 times.Radioligand Binding
293 cells seeded in 100-mm
dishes were transfected with 5-10 µg of wild-type rD1A
receptor or the HA-tagged receptor. Following transfection (18-24
h), cells were reseeded in 150-mm dishes and allowed to grow for an
additional 32-48 h. Radioligand binding studies were performed on
membranes expressing either the wild-type or HA-rD1A receptor as
described previously(26) . Protein concentrations were
determined using the Bio-Rad assay kit with bovine serum albumin as
standard.Photoaffinity Cross-linking
Membranes from 293
cells expressing either the -galactosidase alone, wild-type, or
HA-rD1A receptor were resuspended in binding buffer (50 mM Tris-HCl, 120 mM NaCl, 5.0 mM KCl, 4.0 mM MgCl, 1.5 mM CaCl, and 1.0 mM EDTA, pH 7.4) containing protease inhibitors (20 µg/ml
phenylmethylsulfonyl fluoride; 10 µg/ml benzamidine, leupeptine,
and soybean trypsin inhibitor; 5 µg/ml aprotinin; 1 µg/ml
pepstatin A). Photoaffinity cross-linking was performed using
SCH39111(27) , which was radioiodinated by procedures described
by Amlaiky et al.(28) . Membranes (20 µg of
proteins) were incubated with I-SCH39111 (3.0
nM) in the presence or absence of 10 µM flupentixol in a final volume of 200 µl at 25° C for 90
min. At the end of the incubation, membranes were washed by
centrifugation (10,000
g for 15 min) 3 times with
ice-cold 10 mM NaHPO
, 100 mM NaCl (pH 7.4), and resuspended in 1 ml of the same buffer. Under
dark conditions, 10 µl of 2 mM SANPAH (freshly dissolved
in dimethyl sulfoxide) were added to each sample, reacted for 15 min at
25°C, and stopped by the addition of 30 µl of 1 M glycine. The reaction mixture was photolysed as described
previously(29) . Membranes were then pelleted by centrifugation
and solubilized in 60 µl of sample buffer (25 mM Tris-HCl
(pH 6.5), 8% (v/v) SDS, 5% (v/v) 2-mercaptoethanol, 10% (v/v)
glycerol). Photoaffinity cross-linking of proteins were resolved by
SDS-polyacrylamide gel electrophoresis using 10% gels. Bands were
visualized by autoradiography using Biomax films (Eastman Kodak Co.).Biotinylation
293 cells transfected with 10 µg
of -galactosidase, or HA-rD1A receptor were reseeded in 6-well
dishes. Confluent cells were incubated in phosphate-buffered saline (pH
7.65) with 1 mg/ml of Biotin-XX, succinimidyl ester probe (dissolved in N,N-dimethylformamide) for 15 min at 37° C. At
the end of the incubation, dishes were put on ice, and cells were
washed three times with ice-cold phosphate-buffered saline and
processed as described by Freedman et al.(30) . Cells
were solubilized by adding 0.5 ml of RIPA+ buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1%
(v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 10
mM sodium fluoride, and 10 mM disodium pyrophosphate)
containing protease inhibitors (as described above). Solubilized cell
extracts were transferred to 1.5-ml conical tubes in a total volume of
0.8 ml, and solubilized for an additional hour at 4° C using an
inversion wheel. Supernatant fractions were collected by centrifugation
at 200,000 g for 15 min at 4° C in TLA centrifuge
100.2 rotor (Beckman), and 730 µl of solubilized preparations were
transferred to new tubes. Two aliquots (15 µl) were taken for
protein assay using bovine serum albumin as standard (Bio-Rad DC
protein assay kit). Supernatant fractions (700 µl) were
precleared by adding 100 µl of 10% (v/v) protein A-Sepharose beads
(Pharmacia Biotech Inc.) in 2% (w/v) bovine serum albumin and rotated
for 1 h at 4° C, and transferred to new tubes containing 100 µl
of protein A-Sepharose beads and 20 µg of purified monoclonal
antibody 12CA5 (Babco). After 2 h of incubation at 4° C, beads were
pelleted, and the supernatant discarded. The beads were then washed 3
times with 1 ml of RIPA+ buffer and dried by aspiration using a
28-gauge needle (30) . Finally, 60 µl of sample buffer was
added to each tube, and immunocomplexes were dissociated at 25° C
overnight. Samples were resolved by SDS-polyacrylamide gel
electrophoresis using 10% gels. Proteins were transferred to
nitrocellulose membrane (0.45 µm pore size, Bio-Rad) and incubated
with a 1:2000 dilution of streptavidin-horseradish peroxidase conjugate
(Amersham Corp.), and chemiluminescence of immunocomplexes was detected
using an ECL kit (Amersham Corp.).
Whole Cell Phosphorylation
293 cells were
transfected with a total of 15 µg of DNA. For control, GRK2, or
GRK3 experimental conditions, 293 cells were cotransfected with 2.5
µg of HA-rD1A receptor expression construct and 12.5 µg of
-galactosidase (control), GRK2, or GRK3 expression construct. In
the case of GRK5 experimental conditions, 293 cells were cotransfected
with 5 µg of HA-rD1A receptor expression construct and 10 µg of
GRK5 expression construct. Under these different experimental
conditions, the expression levels for the HA-rD1A receptor were
similar. A day prior to the assay, transfected cells were reseeded in
6-well dishes with 1-1.5 10 cells/well and
grown for an additional 18 h. Cells were labeled for 90 min at 37°
C, in 20 mM HEPES-buffered phosphate-free Dulbecco's
modified Eagle's medium (pH 7.4), and gentamicin containing 0.2
mCi/ml of P
. At the end of the labeling
period, cells were incubated in the presence or absence of 10
µM dopamine. Solubilization and immunoprecipitation were
carried out as described above. Phosphorylated receptors were resolved
by SDS-polyacrylamide gel electrophoresis using 10% gels. Gel lanes
were loaded with volumes giving equivalent amounts of HA-rD1A receptor
as assessed by ligand binding. Gels were dried and exposed to Biomax
film at room temperature for 4-24 h. The extent of receptor
phosphorylation was quantitated with a PhosphorImager (Molecular
Dynamics).Immunoblotting
Transfected 293 cells reseeded in
6-well dishes (see above) were washed with phosphate-buffered saline,
harvested in ice-cold lysis buffer (10 mM Tris-HCl, 5 mM EDTA, pH 7.4), containing protease inhibitors, and homogeneized
with a plastic pestle. 20 µg of proteins from total cell extracts
were resolved by SDS-polyacrylamide gel electrophoresis using 10% gels.
Moreover, 5 µg of proteins from total Sf9 cell extract
overexpressing GRK2, GRK3, or GRK5 were used as positive controls.
Proteins were transferred to nitrocellulose membrane and incubated with
a 1:2000 dilution of either GRK2/GRK3 or GRK5 polyclonal
antibodies(7, 24) , and the chemiluminescence of
immunocomplexes was detected using an ECL kit (Amersham Corp.).Phosphoamino Acid Analysis
Phosphoamino acid
analysis was performed with gel used for whole cell phosphorylation
experiments. Phosphorylated protein bands were identified by lining up
the autoradiogram with the gel. The bands were excised from the gel,
eluted in 50 mM NHHCO
, 0.1% (v/v) SDS,
0.5% (v/v) 2-mercaptoethanol, precipitated 1 h on ice, and hydrolyzed
in 50 µl of 6 N HCl for 1 h at 110° C as described
previously(31) . Phosphoamino acids were resolved by
two-dimensional electrophoresis on thin layer cellulose plates (Eastman
Kodak Co.). The first dimension was carried out in pH 1.9 buffer (2.2%
(v/v) formic acid, 1.38 M glacial acetic acid), and plates
were run for 1.5 h at 900 V. The second dimension was carried out in pH
3.5 buffer (0.5% (v/v) pyridine, 0.87 M glacial acetic acid),
and plates were run for 45 min at 900 V. Finally, thin layer cellulose
plates were air-dried, stained with 1% (v/v) ninhydrin to visualize
phosphoamino acid standards, and exposed on Biomax film for 48 h at
-80° C.cAMP Accumulation Assay
Following transfection
(18-24 h), 293 cells were reseeded in 6-well dishes (wild-type versus HA time course experiments) or 12-well dishes (GRK
experiments). The next day, culture medium was replaced by fresh
minimum essential medium containing 5% (v/v) fetal bovine serum,
gentamicin, and labeled with [H]adenine (1.5
µCi/ml) for 18-22 h. Determinations of intracellular cAMP
were assessed by incubating cells in 10 mM HEPES-buffered
minimum essential medium (with no phosphodiesterase inhibitors) in the
absence or presence of dopamine at 37° C for various periods of
time(26) . Assays were terminated by transferring dishes on
ice, aspirating the medium, and adding 1 ml of stop solution (2.5%
(v/v) perchloric acid, 100 µM cAMP, and 10,000 cpm of
[
C]cAMP). After 20-30 min in the cold, the
acid-cell lysates were transferred to tubes containing 0.1 ml of
neutralizing solution (4.2 M KOH). The salt precipitates were
pelleted by centrifugation, and separation of
[H]cAMP in the cell lysate supernatants was done
using a sequential chromatography on Dowex and alumina columns. Data
are presented as 1000 times the ratio [H]cAMP
formed over the total uptake measured in the well.Data Analysis
Radioligand binding and
dose-response curves were analyzed using the curve-fitting programs
ALLFIT and LIGAND(32, 33) . Statistical analysis of
data were performed using analysis of variance. Pairwise comparisons
were assessed either by the t distribution test or
GT2-method(34) . Homogeneity of variances was assessed by the F
test. The level of significance was
established at 5% using one-tailed test. Results are expressed as the
mean ± S.E.Materials
Human embryonic kidney cells (293) were
obtained from American Tissue Culture Collection (CRL 1573). Tissue
culture reagents were from Life Technologies, Inc.
[H]Adenine, [C]cAMP, and P
were purchased from DuPont NEN. Dopamine-HCl
and flupentixol-HCl were from Research Biochemical International.
Protease inhibitors were obtained from Sigma. Nonidet P-40 was
purchased from Calbiochem. SANPAH was purchased from Pierce. Biotin-XX
succinimidyl ester probe was procured from Molecular Probes. SCH39111
was obtained from Schering-Plough Corp.
Molecular and Biochemical Characterization of the
Hemagglutinin Epitope-tagged Dopamine D1A Receptor
Using
polymerase chain reaction-based methodology, we have engineered the
sequence coding for the hemagglutinin epitope (HA) in the amino
terminus of the rat dopamine D1A (rD1A) receptor. To verify that the
receptor function was not impaired by the insertion of the epitope, we
characterize the binding and coupling properties of the HA-rD1A
receptor. Saturation studies revealed that equilibrium dissociation
constant (K
) of the antagonist I-SCH23982 for 293 cells expressing either the wild-type
or the HA-rD1A receptor was similar (0.51 ± 0.03 nMversus 0.46 ± 0.05 nM, respectively). We
subsequently investigated the binding properties of agonists and
antagonists. The affinity constants (K
) of the
agonist dopamine and the antagonist were identical at both the
wild-type or HA-tagged receptor (data not shown). These results suggest
that insertion of HA in the amino terminus does not affect the binding
properties of agonists and antagonists displayed at the wild-type rD1A
receptor.) increased to similar extent, while the basal
activity was not statistically different. As depicted in Fig. 1,
the effective concentration (EC) measured for the HA-rD1A
receptor was not statistically different from the wild-type receptor
(
15 nM).
values at each stimulation times for either the wild type or
HA-tagged rD1A receptor were not statistically different (Wild type,
EC
= 15.5 ± 0.9 nM; HA, 15.6
± 1.5 nM). Maximal activation values for the wild type
(
, 7.2 ± 0.2; , 9.9 ± 0.2;
10.4
± 0.2) and HA-tagged (, 7.6 ± 0.2; s, 9.9
± 0.2;
, 11.6 ± 0.2) receptors were increased
significantly over the time course studied. Expression levels for the
wild type and HA-tagged were 10.6 and 11.1 pmol/mg of membrane protein,
respectively.
I-SCH39111(27) . As shown in Fig. 2A, the HA-rD1A receptor is expressed at the
plasma membrane of 293 cells as a broad protein band of about 80 kDa,
an electrophoretic mobility similar to that of the wild-type rD1A
receptor. These results suggest that insertion of the epitope in the
amino terminus does not interfere with the glycosylation of the D1A
receptor. This photoaffinity labeling was specific to the expression of
the wild-type or tagged receptor, since no detectable labeling was
observed in mock-transfected cells (Fig. 2A). The
post-translational modification of the human homologue of the D1A
receptor expressed in 293 cells was identical to its rat counterpart
(data not shown).
-galactosidase alone (MOCK), HA-tagged rD1A (HA-rD1A) or wild-type receptor (WT-rD1A). Membranes were incubated with I-SCH39111 in the absence or presence of 10 µM flupentixol (FLU) as described under ``Experimental
Procedures.'' No detectable binding was measured in mock
transfected cells, whereas cells harboring HA-rD1A or wild type
receptor expressed 5.6 and 6.5 pmol/mg of membrane protein,
respectively. Shown is a representative example of an experiment
repeated 3 times. B, cells were transfected with
-galactosidase alone (M) or HA-tagged rD1A receptor (HA), biotinylated, solubilized, and immunoprecipitated using
the purified monoclonal antibody 12CA5 as described under
``Experimental Procedures.'' Immunocomplexes were resolved by
SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and
blotted with streptavidin-horseradish peroxidase conjugate. The
expression level for cells harboring the HA-rD1A receptor was 9.4
pmol/mg of membrane protein.
GAL (mock) were conjugated with a
succinimidyl ester probe (biotin-XX), solubilized, and subjected to
immunoprecipitation as described under ``Experimental
Procedures.'' Fig. 2B shows that the 12CA5
monoclonal antibody specifically immunoprecipitated protein, which can
be visualized as a broad band of about 80 kDa. No such broad band could
be detected by immunoprecipitation from mock transfected cells (Fig. 2B). Similar findings were obtained by photoaffinity
cross-linking experiments using the I-SCH39111 (data not
shown). Thus, we have shown that the molecular and biochemical
characteristics of the HA-rD1A receptor are similar to that of the
wild-type receptor.
Agonist-occupied D1A Receptor Is Phosphorylated by
GRKs
To investigate the potential role of GRK-mediated
phosphorylation in the regulation of the dopamine D1A receptor
responsiveness, 293 cells transfected with the HA-rD1A receptor alone
or with either GRK2, GRK3 or GRK5 were exposed to 10 µM dopamine for various times, and the agonist-dependent
phosphorylation of the HA-rD1A receptor was monitored (Fig. 3).
The time course of the GRK-mediated phosphorylation of the HA-rD1A
receptor revealed that overexpression of the GRKs increase both the
rate and extent of receptor phosphorylation when compared with cells
overexpressing the receptor alone (Fig. 3). Maximal receptor
phosphorylation was observed following 5 min of agonist exposure.
Phosphorylation of HA-rD1A receptor by various GRKs upon exposure to
dopamine for 5 min is illustrated in Fig. 4A. In cells
expressing the receptor alone, the extent of agonist-dependent
phosphorylation was approximately 150% above control basal conditions.
In cells overexpressing either the GRK2, GRK3, and GRK5, the HA-rD1A
receptor displayed an increase in agonist-dependent phosphorylation of
approximately 350-450% (Fig. 4B). Increased
agonist-dependent receptor phosphorylation could also be visualized by
an upshift in the electrophoretic mobility of the broad 80 kDa band (Fig. 4A). Augmentation of agonist-dependent receptor
phosphorylation was correlated with increased GRK expression as
detected by Western blot analysis (Fig. 4C). Moreover,
phosphoamino acid analysis showed that GRK-mediated receptor
phosphorylation occurs exclusively on serine residues (Fig. 4D). These data represent the first evidence for
a direct role of GRK2, GRK3, and GRK5 in mediating dopamine D1A
receptor phosphorylation.
-galactosidase (GAL) or with
GRK2, GRK3, or GRK5 were labeled with P
(0.2
mCi/ml) for 90 min. The cells were then treated with or without 10
µM dopamine for various periods of time. The
phosphorylated receptors were solubilized and immunoprecipitated as
described under ``Experimental Procedures.'' The amount of
receptor phosphorylation was quantitated by PhosphorImager, and data
were expressed as percentage above control basal (as measured in cells
transfected with HA-rD1A receptor and GAL incubated in the absence
of agonist). Each curve represents the mean of three to four
independent experiments. The receptor expression obtained under the
transfection conditions were similar and as follows:
-galactosidase, 11.2; GRK2, 11.6; GRK3, 10.0; and GRK5, 5.2
pmol/mg of membrane protein.
-galactosidase alone (MOCK), or transfected with the HA-rD1A receptor alone (GAL) or with GRK2, GRK3, or GRK5 were treated with or
without 10 µM dopamine (DA) for 5 min were
subjected to immunoprecipitation as described under ``Experimental
Procedures.'' The immunocomplexes were then resolved by
SDS-polyacrylamide gel electrophoresis using 10% gels. The increase in
receptor phosphorylation was visualized by autoradiography. Shown is a
representative example of an experiment repeated 8-10 times. The
receptor expression obtained under the different experimental
conditions was similar and was as follows: -galactosidase, 12.4;
GRK2, 15.1; GRK3, 14.0; GRK5; 11.4 pmol/mg membrane protein. B, the receptor phosphorylation obtained under the
experimental conditions described in A was quantitated by
PhosphorImager. The results are expressed as the mean of 8-10
independent experiments. Data are presented as percentage above control
basal phosphorylation (as measured in cells transfected with HA-rD1A
and -galactosidase in the absence of agonist). The receptor
expression level obtained under the different experimental conditions
were as follows: -galactosidase, 14.8; GRK2, 15.2; GRK3, 13.3; and
GRK5, 11.5 pmol/mg of membrane protein. C, cell lysates were
prepared from 293 cells overexpressing the -galactosidase only (MOCK), or the HA-rD1A receptor alone (GAL) or
with various GRKs. Cell lysates prepared from Sf9 cells overexpressing
GRK2, GRK3, or GRK5 were used as controls. Immunoblotting was performed
as described under ``Experimental Procedures.'' D,
phosphorylated bands obtained upon exposure to 10 µM dopamine shown in A were excised, and the proteins were
eluted and hydrolyzed with 6 N HCl. Phosphoamino acids were
separated by two-dimensional electrophoresis on thin layer cellulose
plates. The positions of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) standards are circled. The HA-rD1A receptor immunoprecipitated from cells
coexpressing various GRKs were phosphorylated only on serine
residues.
Overexpression of GRKs Diminishes the Dopamine D1A
Receptor Activation of Adenylyl Cyclase
To assess the functional
importance of the GRK-mediated phosphorylation of the D1A receptor, we
assessed the accumulation of cAMP in intact cells expressing the
receptor alone or with different GRKs (Fig. 5). The basal
adenylyl cyclase activity of cells harboring the receptor alone or
co-transfected with different GRKs was not significantly modified under
the same experimental conditions (Fig. 5A). However, a
time course of intracellular cAMP accumulation reveals that activation
of the D1A receptor by 10 nM dopamine (EC
dose) was dampened in cells overexpressing different GRKs at each
time point studied (Fig. 5B). A time course of the
intracellular cAMP formation elicited by stimulation of the HA-rD1A
receptor using a maximal concentration of dopamine (10 µM)
was not modified in cells overexpressing GRK2 or GRK3 (Fig. 5C). Interestingly, however, cells overexpressing
GRK5 display a significant blunting of the adenylyl cyclase response
under these conditions (Fig. 5C). These results suggest that
phosphorylation of the D1A receptor by various GRKs leads to functional
uncoupling from the G
protein as evidenced by a decrease in
the rate and extent of adenylyl cyclase activation. Moreover, at a high
concentration of dopamine, the rate and extent of adenylyl cyclase
activation appears to be affected only in those cells overexpressing
the GRK5.
-galactosidase (GAL), 6.7; GRK2, 5.6; GRK3, 5.2; and GRK5, 5.1 pmol/mg
of membrane protein.
of 23 nM with a maximal stimulation of
10-15 fold above basal activity (Fig. 6A). In
cells expressing either GRK2 or GRK3, dose-response curves display a
statistically significant 3- and 7-fold rightward shift in the
EC
with values corresponding to 68 nM and 157
nM for GRK2 and GRK3, respectively (Fig. 6A).
Expression of GRK2 or GRK3, however, had no significant effect on
either the -fold activation or V
(Fig. 6A). In contrast to the GRK2 and GRK3
situation, cells overexpressing the GRK5 displayed the most striking
change in the HA-D1A receptor responsiveness depicted by a marked
decrease in the maximal stimulation of intracellular cAMP (Fig. 6A). A significant 2-fold rightward shift in the
EC for dopamine was also observed (Fig. 6A). Concomitant to these dose-response curves,
phosphorylation experiments were performed in the same transfected
cells used for the whole cell cAMP assay. Interestingly, stimulation of
the HA-rD1A receptor by 10 µM dopamine for 5 min leads to
a similar amount of the agonist-induced receptor phosphorylation by all
the GRK isoforms utilized (Fig. 6B). This suggests that
functional differences observed for the D1A receptor responsiveness can
not be explained by differences in the extent of receptor
phosphorylation.
values ± standard errors from the fits obtained for the
different experimental procedures were as follows:
-galactosidase (GAL), 22.9 ± 3.6 nM; GRK2, 67.9 ±
11.9 nM; GRK3, 157 ± 29.6 nM; and GRK5, 43.2
± 12.8 nM. Dose-response curves obtained in cells
overexpressing GRK2, GRK3, and GRK5 all display a significant rightward
shift. The maximal activation obtained in cells overexpressing GRK5
(6.2 ± 0.2) was significantly reduced in comparison with the
control curve (-galactosidase; 11.0 ± 0.2) or GRK2 (10.5
± 0.3) and GRK3 (10.6 ± 0.3) curves. B, whole
cell phosphorylation were performed in the same transfected pool of
cells used in (A) as described under ``Experimental
Procedures.'' Upon exposure to 10 µM dopamine for 5
min, the extent of agonist-dependent phosphorylation obtained between
the cells overexpressing the various GRKs was not statistically
different but significantly higher than the one measured in control
cells (HA-rD1A receptor and -galactosidase). The receptor
expressions were not statistically different between the four
experimental conditions tested. Receptor levels were as follows:
-galactosidase, 9.6; GRK2, 9.3; GRK3, 8.5; and GRK5, 7.5 pmol/mg
of membrane protein.
Direct Phosphorylation of Dopamine D1A Receptor by GRKs
Regulates Signaling Function
Previous evidence for a regulatory
role of phosphorylation in the function of the dopamine D1A receptor
has come mostly through indirect means. Zhou et al.(21) used kinase inhibitors to imply a role for protein
kinase A- and GRK-mediated phosphorylation of the D1A receptor in the
process of agonist-mediated desensitization. In the latter and other
desensitization studies, direct phosphorylation of the D1A receptor
could not be demonstrated presumably due to low levels of receptor
expression in the various systems
used(16, 17, 18, 19, 20) .
To circumvent these difficulties, we have overexpressed an
epitope-tagged D1A receptor in 293 cells co-transfected with various
GRKs. 50%) in
phosphorylation of the D1A receptor in the absence of exogenous kinases (Fig. 4C). In contrast, overexpression of various GRKs
(>20-fold as assessed by immunoblotting; Fig. 4C)
increases both the rate and extent of the agonist-dependent
phosphorylation of the transfected receptor, suggesting that this
cellular system can be used to study the actions of the various GRKs.
Indeed, for each transfected GRK, the extent of agonist-dependent
phosphorylation is significantly greater than that observed as a result
of the endogenous kinases only. Moreover, the D1A receptor undergoes a
rapid loss of responsiveness, which is detectable as soon as 2 min
after stimulation and remains relatively constant for at least 10 min.
These results are consistent with the time course reported for
GRK-mediated desensitization(35) . This modification of the
transfected D1A receptor by the various GRKs supports a role of
phosphorylation in regulating the functional state of the D1A receptor.
Distinct Modulation of D1A Receptor Responsiveness
Suggests Specificity of GRK Actions
Phosphorylation of the D1A
receptor by different GRKs resulted in attenuation of responsiveness
with distinguishing characteristics. Indeed, phosphorylation of the D1A
receptor by GRK5 afforded a dramatic attenuation of response with both
a shift in the EC for dopamine and a marked 40% decrease
in maximum response. In contrast, GRK2- and GRK3-mediated receptor
phosphorylation led to significant rightward shifts of the
dose-response curves for dopamine with no change in the maximal
response (Fig. 6A). Therefore, GRK5 phosphorylation of
the D1A receptor engenders a more profound desensitization than
phosphorylation by either GRK2 or GRK3. These differences could not be
attributed to differences in the extent of phosphorylation of the D1A
receptor as similar levels of phosphorylation were achieved with all
three kinases. Taken together, these results suggest that the D1A
receptor responsiveness can be regulated differentially according to
the GRK subtype expressed in a particular cell. What possible
underlying mechanisms could explain such differential modulation of the
D1A receptor function by the various GRKs?
-adrenergic receptor kinase 1 (GRK2) and GRK5
using peptide substrates(36, 37) . Thus,
phosphorylation of distinct sites by various GRKs could result in
different extent of attenuation of the response. It is interesting to
note that, although GRKs are serine/threonine kinases, of the 22 serine
and 14 threonine residues present in the cytoplasmic domains of the D1A
receptor, only serine residues appear to be phosphorylated (Fig. 4D). Further studies using purified and
GRK-phosphorylated D1A receptor will be required to determine the exact
nature of the sites phosphorylated by GRK2, GRK3, and GRK5.-adrenergic receptor kinase
1 (GRK2) (3) and rhodopsin kinase (GRK1)(38) .
Meanwhile, no such data exist for GRK5-mediated desensitization. Under
normal conditions, the levels of arrestin proteins are unlikely to be
limiting(39) ; however, under conditions of overexpression of G
protein-coupled receptors in heterologous systems, kinase and arrestin
proteins may become limiting(40) . Thus, the absence of a
diminished V in cells overexpressing GRK2 and
GRK3 in our studies might be explained by a limiting level of arrestin
proteins. However, this raises the intriguing question about the
potential role arrestin proteins play in GRK5-mediated desensitization
of the D1A receptor. GRK5 belongs structurally to a different subfamily
of kinases than GRK1, GRK2, and GRK3(2, 41) , and
therefore receptor desensitization by GRK5 may be potentially elicited
independent of the binding of arrestin proteins. In addition, several
forms of arrestin proteins have been isolated(3) , and it is
interesting to speculate that different phosphorylated sites may
provide different interaction sites for the various arrestin proteins.
Further studies are required to establish the precise role of arrestin
proteins in the modulation of D1A receptor function upon its
phosphorylation by GRKs. Finally, it is worth mentioning that these
effects were observed in whole cell preparations, and therefore we
cannot rule out that GRK5 also regulates a downstream effector
important for D1A receptor signaling. Studies performed using added
GRKs to membranes expressing D1A receptors may help to elucidate
potentially these different effects(42) . Regardless of the
basis for the observed differences, our data document that under
identical conditions, the effect of these various kinases can be
significantly different (i.e. selectivity of action exists). -adrenergic and
m-muscarinic receptors are better substrates for
-adrenergic receptor kinase 1 (GRK2) than
GRK5(37, 42) , whereas the
-adrenergic receptor appears to be as effectively
phosphorylated by either GRK2, GRK3, or GRK5(30) . The dopamine
D1A receptor represents yet a different type of selectivity in that the
receptor appears to be covalently modified by the various kinases to a
similar extent, but the biological consequence of that phosphorylation (i.e. desensitization) differs.Physiological Relevance of D1A Receptor
Phosphorylation
The demonstration of a role for the GRKs in
regulating D1A receptor responsiveness in a heterologous mammalian
expression system raises the issue of the physiological relevance of
the differential modulation of D1A receptor function by GRK2, GRK3, and
GRK5. Cellular co-localization of the D1A receptor (or any other G
protein-coupled receptors) with different GRKs is currently unknown.
However, in situ hybridization and immunohistochemistry
studies have shown that GRK2 and GRK3 are expressed in brain regions
that have been shown to contain D1A receptors(7) . In addition,
Arriza et al.(7) have shown that GRK2 and GRK3 appear
to be associated with presynaptic and more predominantly postsynaptic
localizations in various brain regions, consistent with a putative role
of these two GRKs in the desensitization of synaptic G protein-coupled
receptors. Moreover, in support of a physiological relevance for D1A
receptor regulation by GRK5, the GRK5 mRNA has been found in cortex and
in retina that also express the D1A receptor(24, 45) .
Recently, it has been shown that exposure of D1A receptors to
dopaminergic agonists leads to a greater desensitization of the D1A
receptor in the retina than in the striatum(46) . Thus, it
would appear that regulation of D1A receptor function in these two
tissues is different, and our results may provide the molecular and
biochemical basis for this observation.-adrenergic receptors(48) . Recent studies have also shown
that levels and activities of GRKs can be modulated by physiological or
pharmacological situations that modulate the levels of hormonal or
neuronal input(40, 49) . Since the D1A receptor
mediates several behavioral paradigms and responses to
psychostimulants, the regulation of its function by GRK-dependent
events is a question of interest that will require further
investigation of the underlying mechanisms possibly using genetically
altered animals. The present study illustrates the functional
importance the multiplicity of GRKs may play in regulating receptor
responsiveness in these various physiological situations.
)
We thank Drs. Julie Pitcher and Neil Freedman for
helpful discussions during the course of this study.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. L. Schlador and N. M. Nathanson Synergistic Regulation of m2 Muscarinic Acetylcholine Receptor Desensitization and Sequestration by G Protein-coupled Receptor Kinase-2 and beta -Arrestin-1 J. Biol. Chem., July 25, 1997; 272(30): 18882 - 18890. [Abstract] [Full Text] [PDF]
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N. J. Freedman, A. S. Ament, M. Oppermann, R. H. Stoffel, S. T. Exum, and R. J. Lefkowitz Phosphorylation and Desensitization of Human Endothelin A and B Receptors. EVIDENCE FOR G PROTEIN-COUPLED RECEPTOR KINASE SPECIFICITY J. Biol. Chem., July 11, 1997; 272(28): 17734 - 17743. [Abstract] [Full Text] [PDF]
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