(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 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.
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
-adrenergic receptor (
-adrenergic receptor
kinase) and are referred to as the G protein-coupled receptor kinases
or GRKs(
)(1, 2, 3) .
This large
family of kinases includes six members (GRK1 to GRK6) whose activities
are regulated by phospholipids, post-translational modifications, or G
protein
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.
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) 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).
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 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.
To determine the apparent molecular weight of
the HA-rD1A receptor protein expressed in 293 cells, we performed
photoaffinity cross-linking experiments using 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).
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
-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.
To test the ability of the monoclonal antibody
12CA5 to immunoprecipitate the HA-rD1A receptor, 293 cells expressing
the tagged receptor or pCMVGAL (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.
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 (-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.
Figure 4:
Agonist-dependent phosphorylation and
phosphoamino acid analysis of the HA-rD1A receptor expressed in 293
cells. A, cells transfected with -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.
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: -galactosidase (
GAL), 6.7; GRK2, 5.6; GRK3, 5.2; and GRK5, 5.1 pmol/mg
of membrane protein.
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 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.
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 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.
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 ( 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.
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 -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.
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 -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).
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) ; -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.
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
-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.