(Received for publication, June 5, 1995; and in revised form, December 15, 1995)
From the
The -adrenergic receptor
(
AR), its truncated mutant T368, different G
protein-coupled receptor kinases (GRK) and arrestin proteins were
transiently expressed in COS-7 or HEK293 cells alone and/or in various
combinations. Coexpression of
-adrenergic receptor kinase
(
ARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced
phosphorylation of the wild type
AR above basal as
compared to that of the receptor expressed alone. On the other hand,
overexpression of the dominant negative
ARK (K220R) mutant
impaired agonist-induced phosphorylation of the receptor.
Overexpression of GRK6 could also increase epinephrine-induced
phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the
AR. Increasing coexpression of
ARK1 or
ARK2
resulted in the progressive attenuation of the
AR-mediated response on polyphosphoinositide (PI)
hydrolysis. However, coexpression of
ARK1 or 2 at low levels did
not significantly impair the PI response mediated by the truncated
AR mutant T368, lacking the C terminus, which is
involved in agonist-induced desensitization and phosphorylation of the
receptor. Similar attenuation of the receptor-mediated PI response was
also observed for the wild type
AR, but not for its truncated mutant, when the receptor was coexpressed with
-arrestin 1 or
-arrestin 2. Despite their pronounced effect
on phosphorylation of the
AR, overexpression of GRK5
or GRK6 did not affect the receptor-mediated response. In conclusion,
our results provide the first evidence that
ARK1 and 2 as well as
arrestin proteins might be involved in agonist-induced regulation of
the
AR. They also identify the
AR
as a potential phosphorylation substrate of GRK5 and GRK6. However, the
physiological implications of GRK5- and GRK6-mediated phosphorylation
of the
AR remain to be elucidated.
Homologous desensitization to the effects of hormones and
neurotransmitters is a ubiquitous regulatory mechanism of receptor
function defined by a rapid and specific loss of responsiveness for
receptors which have been repeatedly stimulated by an
agonist(1) . In the G protein-coupled receptor
family(2) , receptor desensitization has been extensively
characterized for rhodopsin mediating phototransduction in retinal rod
cells and for the -adrenergic receptor
(
AR), (
)which mediates
catecholamine-induced stimulation of adenylyl cyclase. The second
messenger-dependent cAMP-dependent protein kinase can phosphorylate and
desensitize the
AR both in response to its agonist as
well as to other agents increasing the cellular content of cAMP. On the
other hand, a prominent role in homologous desensitization of rhodopsin
and
AR is played by the two second
messenger-independent kinases rhodopsin kinase (3) and the
-adrenergic receptor kinase (
ARK)(4) , respectively.
Once the receptor is occupied by the agonist, it is recognized by the
kinase and becomes phosphorylated. The subsequent uncoupling of the
receptor and G protein is then mediated by arrestin proteins which
specifically bind to the phosphorylated
receptor(5, 6) . Rhodopsin kinase and
ARK are
members of the newly discovered family of G protein-coupled receptor
kinases (GRKs)(7) . These protein kinases have the unique
ability to recognize and phosphorylate their G protein-coupled receptor
substrates only in their active (i.e. agonist-occupied)
conformations. Molecular cloning techniques have revealed that the
current members of the GRK family include rhodopsin kinase
(GRK1)(8) ,
ARK 1 and 2 (GRK2 and 3,
respectively)(9, 10) , the human gene IT11 (GRK4)(11) , GRK5 (12) , GRK6 (13) and
several homologs from Drosophila(14) .
ARK
isozymes can phosphorylate a variety of receptors in vitro including the
AR(9, 10) , the
AR(15) , the M2 muscarinic cholinergic
receptor (M2-AchR) (16) and, at lower stoichiometry,
rhodopsin(9, 10) . The lack of proportion between the
small number of GRKs and the large number of G protein-coupled
receptors suggests that different GRKs can recognize several receptors in vivo.
Despite the large amount of information about the
adenylyl cyclase-linked AR, much less is known about
the molecular mechanisms involved in desensitization of G protein
coupled receptors which activate polyphosphoinositide (PI) hydrolysis
via phospholipase C (PLC). Protein kinase C inhibitors or phorbol
ester-induced depletion of cellular protein kinase C do not alter
homologous desensitization of various receptors including the receptors
for thrombin(17) , bombesin(18) , histamine and
ATP(19) . We have recently shown that agonist-induced
phosphorylation and desensitization of the cloned
AR
expressed in Rat-1 fibroblasts as well as in COS-7 cells does not
primarily involve phorbol ester-sensitive protein kinase
C(20) . However, the protein kinases and, in more general
terms, the biochemical mechanisms involved in agonist-induced
regulation of G protein-coupled receptors linked to the PLC signaling
pathway remain to be assessed. Potential protein kinase candidates
might belong to the GRK family. A role of
ARK in the regulation of
PLC-linked receptors has been suggested by few studies. Both the
receptor for substance P (21) and the M3 muscarinic cholinergic
(M3-AchR) (22) have been shown to be substrates for
ARK-mediated phosphorylation in vitro. A more recent
study has shown that the thrombin receptor-mediated response on
intracellular calcium was impaired after coexpression of the receptor
with
ARK2 in Xenopus oocytes(23) .
In the
present study, we wished to investigate whether protein kinases
belonging to the GRK family play a role in the regulation of the the
AR. For this purpose, we transiently coexpressed
different GRKs with the
AR and its truncated mutant
T368 (20) in COS-7 or HEK293 cells to assess their effects on
agonist-induced phosphorylation and desensitization of the receptors.
We also investigated the effect of two arrestin proteins on
receptor-mediated response on PI hydrolysis. Our findings indicate that
distinct GRKs might be differently involved in agonist-induced
phosphorylation and regulation of the
AR.
HEK293
cells were transfected with different DNAs by calcium phosphate
precipitation. HEK293 cells (5 10
) grown in 100-mm
dishes were transfected with 1 and 3 µg of DNA/1,000,000 cells for
the
ARs and different GRKs, respectively. The total
amount of DNA transfected was kept constant (4 µg/1,000,000 cells)
under different conditions adding pRK5 or pCMV. 24 h after the
transfection, cells were trypsinized and seeded in 55- or 35-mm dishes
for phosphorylation experiments and inositol phosphate determination,
respectively. HEK293 cells were harvested 72 h after their
transfection.
The expression of rat ARK1 and 2
in COS-7 and HEK293 cells was assessed by Western blot analysis of the
cytosolic proteins with an antiserum which could equally recognize
ARK1 and 2(26) . These experiments indicated that equal
amounts of transfected DNA resulted in similar expression of rat
ARK1 and 2 both in COS-7 (Fig. 1, panel A) and
HEK293 cells (results not shown). The expression of both kinases was
similar in COS-7 cells coexpressing the wild type
AR
or its truncated mutant (results not shown). Similarly to what observed
with the rat
ARKs, equal amounts of DNA resulted in similar
expression of bovine
ARKs as well as of their dominant negative
mutants K220R (Fig. 1, panel B). Some
ARK
immunoreactivity was also detected in the crude membrane fractions of
transfected cells, and it was similar for both kinases (results not
shown). The comparison of the signals corresponding to the cytosolic
rat
ARKs expressed in COS-7 cells with those of known amounts of
ARKs purified from Sf9 cells (Fig. 1, panel A)
suggests that the expression of cytosolic rat
ARK1 or 2 is about
3.5 pmol/1,000,000 cells. This level of expression is about
10-15-fold higher than that of the
AR as
assessed by ligand binding on cell membranes (200-300
fmol/1,000,000 cells) when both values are normalized/1,000,000 cells.
Figure 1:
Expression of GRKs and -arrestin
in Cos-7 cells. A, Western blot analysis of bovine
ARK1
and 2 (lanes 1 and 2) purified from Sf9 cells and of
cytosolic proteins from COS-7 cells transfected with the DNAs encoding
the
AR alone (0.2 µg/1,000,000 cells) (lane
3) or in combination with increasing amounts of DNA
(µg/1,000,000 cells) encoding rat
ARK1 (lanes
4-6) or rat
ARK2 (lanes 7-9). Each
purified kinase was 150 ng. B, Western blot analysis of
cytosolic proteins from COS-7 cells transfected with the DNAs encoding
the
AR alone (0.2 µg/1,000,000 cells) (lane
1) or in combination with increasing amounts of DNA
(µg/1,000,000 cells) encoding bovine
ARK1 (lanes 2 and 3), its K220R mutant (lanes 4 and 5), bovine
ARK2 (lanes 6 and 7), or its
K220R mutant (lanes 8 and 9). C, Western
blot analysis of GRK5 (lane 1) and GRK6 (lane 2)
purified from from Sf9 cells and of cytosolic proteins from COS-7 cells
transfected with the DNAs encoding the
AR alone (0.2
µg/1,000,000 cells) (lane 3) or in combination with the
DNA (2 µg/1,000,000 cells) encoding GRK5 (lane 4) or GRK6 (lane 5). Each purified kinase was 20 ng. D, Western
blot analysis of in vitro translated
-arrestin 1 (lane 1) and cytosolic proteins from COS-7 cells transfected
with the DNAs encoding the
AR alone (0.2
µg/1,000,000 cells) (lane 2) or in combination with the
DNA (2 µg/1,000,000 cells) encoding
-arrestin 1 (lane
3). The in vitro translated
-arrestin 1 was 100 ng.
In all the experiments the cytosolic proteins were 25 µg (derived
from 0.3-0.5 1,000,000 cells). The results are representative of
several experiments.
We also tested the cytosolic kinase activity of COS-7 cells
expressing different ARKs on urea-treated ROS. Despite the
similarity of their protein expression detected by Western blot
analysis, the cytosolic kinase activity on ROS of rat
ARK2 was
5-fold greater as compared to that of rat
ARK1 (results nor
shown). Our results are in agreement with those of a previous study in
which the cytosolic activity on ROS of rat
ARK2 expressed in Xenopus oocytes was greater than that of rat
ARK1(23) . On the other hand, the bovine
ARK1 and 2
did not differ in their cytosolic kinase activity on ROS (results not
shown).
The expression of GRK 5 and 6 was assessed by Western blot analysis on cytosolic proteins from COS-7 cells with an antiserum raised against the C-terminal portion of GRK5. This antiserum seems to recognize about 2-3 times better purified GRK5 than GRK6 (Fig. 1). These experiments indicated that the expression of GRK6 in COS-7 cells was higher than that of GRK5. Some immunoreactivity was also detected in the crude membrane fractions, and it was also higher for GRK6 than GRK5 (results not shown).
Figure 2:
Phosphorylation of the
AR in COS-7 cells. A, COS-7 cells expressed
the
AR alone (lanes 1 and 2) or in
the presence of rat
ARK1 (lanes 3 and 4), GRK5 (lanes 5 and 6), and GRK6 (lanes 7 and 8). The transfected DNA was 1 and 5 µg/1,000,000 cells for
the
AR and the GRKs, respectively. After labeling
with
P
, cells were treated for 5 min in the
absence(-) or presence (+) of 10
M epinephrine. The phosphorylated receptors were immunoprecipitated
with the antiserum against the C terminus of the
AR,
as described under ``Experimental Procedures.'' 0.25 pmol of
receptors were loaded in each lane. Receptors were resolved by 10%
SDS-PAGE. Positions of prestained molecular mass markers are indicated
in kDa. Results are from a representative experiment in which the
effects of different GRKs were tested in parallel. B, COS-7
cells expressing the
AR in the absence (CON)
or presence of different GRKs were labeled with
P
and treated with 10
M epinephrine (EPI) for different times. The
P content of the
phosphorylated receptors was quantified as described under
``Experimental Procedures.'' Basal indicates the
receptor phosphorylation of untreated cells. Results are the mean
± S.E. of five or three independent experiments for the control
(CON) and for the other conditions, respectively. *, p <
0.05 as compared to epinephrine-induced phosphorylation of control (CON); $, p < 0.05 as compared to the basal
phosphorylation of control (CON).
Treatment of
COS-7 cells expressing the AR alone with epinephrine
(10
M) resulted in a time-dependent
increase of receptor phosphorylation which reached its maximal increase
of about 55% above basal after 15 min (Fig. 4). In cells
overexpressing rat
ARK1, agonist-induced phosphorylation of the
AR above basal was about 180% greater than that of
cells expressing the receptor alone (Fig. 2). Also
overexpression of GRK6 could enhance agonist-induced phosphorylation of
the
AR. Following 5 min of stimulation with
epinephrine, agonist-induced phosphorylation of the receptor above
basal was 230% greater than that of the receptor expressed alone (Fig. 2). On the other hand, coexpression of GRK5 resulted in a
60% increase of basal phosphorylation of the
AR, as
compared to the basal level of the receptor expressed alone, without
significantly affecting the maximal level of epinephrine-induced
phosphorylation (Fig. 2).
Figure 4:
Effect of bovine ARKs and their
dominant negative mutants on phosphorylation of the
AR. A, COS-7 cells expressed the
AR alone (lanes 1-3) or in the
presence of bovine
ARK1 (lanes 4-6), bovine
ARK2 (lanes 7-9), the
ARK1 mutant K220R (lanes 10-12), or the
ARK2 mutant K220R (lanes
13-15). The transfected DNA was 1 and 5 µg/1,000,000
cells for the
AR and the
ARKs, respectively.
After labeling with
P
, cells were not treated (0) or stimulated with 10
M epinephrine for 5 and 15 min. The other experimental conditions
are as in Fig. 2. B, COS-7 cells expressing the
AR in the absence (CON) or presence of
different
ARKs were labeled with
P
and
stimulated with 10
M epinephrine (EPI) for different times as indicated. Receptor numbers
measured in membrane preparations were similar under the different
conditions and in the range of 1-2 pmol/mg of protein
(200-300 fmol/1,000,000 cells). Receptor numbers measured in
intact cell monolayers were also similar under the different conditions
and in the range of 110-125 fmol/1,000,000 cells. Results are the
mean ± S.E. of three independent experiments. *, p <
0.05 as compared to epinephrine-induced phosphorylation of control (CON) for each time of
stimulation.
To further characterize the
GRK-mediated phosphorylation of the AR, the kinases
were coexpressed with the truncated receptor mutant T368. Coexpression
of different GRKs in COS-7 cell was not able to increase either basal
or epinephrine-induced phosphorylation of the T368 mutant (results not
shown). This observation is in agreement with our previous findings
indicating that phosphorylation of the
AR requires
the integrity of its C-terminal portion(20) .
The effect of
different GRKs on agonist-induced phosphorylation of the
AR was also investigated in HEK293 cells. As observed
in COS-7 cells, coexpression of rat
ARK1 or GRK6 could also
increase epinephrine-induced phosphorylation of the
AR expressed in HEK293 cells, whereas coexpression of
GRK5 enhanced its basal phosphorylation, but not that induced by the
agonist (results not shown). The increasing effect of rat
ARK2 on
agonist-induced phosphorylation of the
AR could only
be observed using low amounts of transfected DNA (0.1-0.3
µg/1,000,000 cells) (Fig. 3). These amounts of DNA were
lower than those used for rat
ARK1. We then discovered that
overexpression of rat
ARK2 obtained using 2 µg of transfected
DNA/1,000,000 cells induced a 45% decrease of the cell surface
ARs measured by [
H]Prazosin
binding at 4 °C on intact COS-7 cells (Fig. 3), whereas the
receptor number measured on cell membranes was similar to that of cells
expressing the receptor alone. A decrease of cell surface receptors was
also observed for the truncated mutant T368 in cells overexpressing rat
ARK2 (results not shown). Similar results were obtained for both
the wild type and truncated
AR in HEK293 cells
overexpressing rat
ARK2 (results not shown). However, no change in
cell surface receptors was observed in cells coexpressing the
AR with any of the other GRKs tested (results not
shown).
Figure 3:
Effect of rat ARK2 on
phosphorylation of the
AR and cell surface receptors.
COS-7 cells were transfected with the DNA encoding the
AR alone (0.5 µg/1,000,000 cells) or in
combination with increasing amounts of DNA (µg/1,000,000 cells)
encoding rat
ARK2. A, phosphorylation of the
AR was measured as in Fig. 2. Results are the
mean ± S.E. of three independent experiments. *, p <
0.05 as compared to epinephrine-induced phosphorylation of control (CON). B, [
H]Prazosin binding
to intact cell monolayers was measured as described under
``Experimental Procedures.'' Control indicates the B
in cells expressing the
AR
alone (CON), which was 133 ± 30 fmol/1,000,000 cells
(means ± S.E. of three experiments). Receptor numbers measured
in membrane preparations were similar under the different conditions
and were in the range of 1-2 pmol/mg of protein (200-300
fmol/1,000,000 cells). *, p < 0.05 as compared to control (CON).
We are currently unable to understand the biochemical
mechanisms underlying the effect of rat ARK2 on the expression of
cell surface receptors. However, our findings indicate that
ARK2-mediated increase of receptor phosphorylation could be
observed only when the cell surface
ARs were similar
to those of the control cells expressing the receptors alone. Our
hypothesis is that a loss of cell surface receptors resulting from the
overexpression of rat
ARK2 might decrease the availability of the
receptors for agonist binding. This might result in the underestimation
of agonist-induced phosphorylation of the
AR
coexpressed with
ARK2.
To better assess the role of ARK in
agonist-induced phosphorylation of the
AR, we
performed a new series of experiments using the
ARK 1 and 2 from
the bovine species, which have been more extensively characterized in a
variety of G protein-coupled receptor systems. The effect of bovine
ARK 1 and 2 on receptor phosphorylation was also compared with
that of their dominant negative mutants K220R lacking their kinase
activity(27) . The expression of the
AR both
in cell membranes and at the cell surface was similar in cells
overexpressing bovine
ARKs or their mutants as compared to that of
cells expressing the receptor alone (results not shown). Overexpression
of both bovine
ARK 1 and 2 resulted in a pronounced increase of
epinephrine-induced phosphorylation of the
AR (Fig. 4). On the other hand, in cells overexpressing the
dominant negative kinase mutants K220R agonist-induced phosphorylation
of the receptor was greatly impaired (Fig. 4). Our hypothesis is
that the dominant negative kinase mutants can inhibit the effect of the
endogenous kinases involved in agonist-induced phosphorylation of the
AR in COS-7 cells. These findings have two main
implications. First, they indicate that both
ARK1 and 2 can
increase agonist-induced phosphorylation of the
AR
with an apparently similar affinity for the receptor. Second, they
strongly suggest that agonist-induced phosphorylation and regulation of
the
AR occurring in a variety of cells is mediated,
at least in part, by
ARK.
Figure 5:
Overexpression of rat ARK1 and 2
attenuates
AR-mediated PI hydrolysis. COS-7 cells
(500,000-1,000,000) grown in 35-mm dishes were transfected with
the DNAs (0.2 µg/1,000,000 cells) encoding the
AR
or the T368 receptor alone or in combination with increasing amounts of
DNA (µg/1,000,000 cells) encoding rat
ARK1 or 2. Total
inositol phosphates were measured as described under
``Experimental Procedures.'' Receptor numbers measured in
membrane preparations were similar under the different conditions for
both receptors and in the range of 1-2 pmol/mg of protein
(200-300 fmol/1,000,000 cells). Control indicates the increase of
inositol phosphates induced by 20 min of stimulation with epinephrine
(10
M) in cells expressing the receptor
alone (CON), which was 273 ± 48 and 321 ± 47%
over basal (mean ± S.E. of eight independent experiments) for
the
AR and the T368, respectively. The results are
the mean ± S.E. of three independent experiments done in
triplicate.
Fig. 6shows that
coexpression of rat ARK1 with the
AR induced
both a decrease of the maximal effect of epinephrine and 100-fold
increase of its EC
to stimulate receptor-mediated PI
response. Similar results were obtained when the
AR
was coexpressed with rat
ARK2 (results not shown). Decreased
sensitivity to the agonist and reduced ability to mediate the maximal
response are two properties of G protein-coupled receptors following
agonist-induced desensitization. Thus, overexpression of
ARK1 and
2 seems to result in biochemical modifications similar to those
occurring during homologous desensitization of the
AR. To further test this hypothesis, rat
ARK1
and 2 were coexpressed with the truncated
AR mutant
T368. We had previously shown that the T368 receptor was impaired in
its ability to undergo agonist-induced desensitization and
phosphorylation. In agreement with these findings, the PI response
mediated by the T368 mutant was only slightly impaired by
overexpression of rat
ARK1 or 2 ( Fig. 5and Fig. 6).
Figure 6:
Dose response of epinephrine-stimulated PI
hydrolysis. COS-7 cells (500,000-1,000,000) grown in 35-mm dishes
were transfected with the DNAs (0.2 µg/1,000,000 cells) encoding
the AR or the T368 receptor alone or in combination
with the DNA (1 µg/1,000,000 cells) encoding rat
ARK1. The
experimental conditions are as in Fig. 5. % of max indicates the response induced by 10
M epinephrine (EPI) for each dose response. The results are
representative of two experiments.
This strongly suggests that for both rat ARK1 or 2 the most
probable mechanism underlying their facilitating effect on
desensitization of the
AR-mediated response is
receptor phosphorylation. As shown in Fig. 3, overexpression of
rat
ARK2 (but not that of rat
ARK1) achieved using 2
µg of DNA/1,000,000 cells resulted in a 45% decrease of cell
surface receptors. However, two observations seem to rule out that rat
ARK2-induced decrease of cell surface receptors is responsible for
the desensitization of the
AR. First, overexpression
of rat
ARK2 could also decrease the cell surface expression of the
truncated mutant T368 without impairing its response. In addition, in
separate experiments using smaller amounts of transfected DNA encoding
the
AR, we observed that a 40-50% lower
expression of cell surface receptors did not result in a
reduction of the receptor-mediated response because a large portion of
receptor in COS-7 cells are spare (results not shown).
In
conclusion, our results suggest that coexpression of ARK1 and 2
can promote desensitization of the wild type
AR. On
the other hand, GRK5 and GRK6 can increase the phosphorylation of the
receptor without inducing any desensitization.
Because of this
apparently conflicting result concerning ARK1 and 2 versus GRK5 and GRK6, we wished to further assess whether
ARK-induced desensitization of the
AR was truly
mediated by the phosphorylation of the receptor. Thus, the
AR and its truncated mutant T368 were cotransfected
with the dominant negative mutants K220R of
ARKs lacking their
kinase activity. For these experiments both the wild type and mutant
ARK 1 and 2 were from the bovine species. Transfection of COS-7
cells with increasing amounts of DNA encoding bovine
ARK1 or 2 or
their mutants resulted in their progressive expression, which was
comparable for all the kinases (Fig. 1). Low expression of
bovine
ARK1 (obtained using 0.2 µg of DNA/1,000,000 cells)
resulted in about 40% impairment of
AR-mediated PI
response, without any significant effect on the response mediated by
the T368 mutant (Fig. 7). However, higher expression of
ARK1 could also impair the T368-mediated response even if at a
smaller extent as compared to the wild type receptor (35% versus 75% of impairment for the T368 and wild type receptor,
respectively). For both the wild type and truncated
AR, a low expression of the dominant negative mutant
ARK1 did not impair the PI response, whereas its high expression
could impair about 40% of both receptor-mediated response. Similar
results were obtained when both receptors were expressed with the wild
type
ARK2 or its dominant negative mutant (results not shown).
These results indicate that overexpression of
ARK can impair the
AR-mediated response by at least two mechanisms
depending on the expression level of the kinase. The first, occurring
at lower level of expression, might be truly mediated by receptor
phosphorylation because it is not observed with similar expression
levels of the kinase-deficient mutant K220R. The second, occurring at
higher expression of
ARK, seems independent from receptor
phosphorylation. This is supported by the fact that at higher
expression both the wild type
ARK and its dominant negative mutant
exert similar effects on the phosphorylation-deficient T368 mutant.
Figure 7:
Effect of bovine ARK1 and its
dominant negative mutant on
AR-mediated PI
hydrolysis. COS-7 cells were transfected with the DNAs encoding the
AR or the T368 receptor alone or in combination with
increasing amounts of DNA (µg/1,000,000 cells) encoding bovine
ARK1 or its dominant negative mutant K220R. The experimental
conditions are as in Fig. 5. Receptor numbers measured in
membrane preparations were similar under the different conditions for
both receptors and in the range of 1-2 pmol/mg of protein
(200-300 fmol/1,000,000 cells). [
H]Prazosin
binding to intact cell monolayers indicated that the expression levels
of the receptors coexpressed with different
ARKs were similar to
those of the receptors expressed alone which were 92 ± 8 and 120
± 15 fmol/1,000,000 cells (mean ± S.E. of three
independent experiments) for the wild type and T368 receptor,
respectively. The results are the mean ± S.E. of three
independent experiments done in triplicate.
Altogether, these results provide strong evidence that both
ARK1 and 2 can promote desensitization of the
AR
and that this is mediated by their ability to phosphorylate the
receptor.
Figure 8:
Overexpression of -arrestin 1
attenuates
AR-mediated PI hydrolysis. COS-7 cells
were transfected with the DNAs encoding the
AR or the
T368 receptor alone or in combination with increasing amounts of DNA
(µg/1,000,000 cells) encoding
-arrestin 1. The experimental
conditions are as in Fig. 5. The results are the mean ±
S.E. of three independent experiments done in
triplicate.
This study provides the first evidence that members of the
GRK family as well as arrestin proteins might play a role in
agonist-induced regulation of the AR. This is mainly
supported by the finding that cellular overexpression of
ARK1 or 2
can both increase agonist-induced phosphorylation of the
AR and promote desensitization of the
receptor-mediated PI response. Coexpression of
-arrestin 1 or 2
could also desensitize the
AR-mediated activation of
PLC. On the other hand, the truncated
AR mutant T368,
which was unable to undergo homologous desensitization in Rat1 cells,
was largely insensitive to the effect of both
ARKs and arrestins.
The experimental approach undertaken in our
present study consisted in coexpressing different GRKs or arrestin
proteins with the AR or its truncated mutant T368 in
two different cell systems, COS7 and HEK293 cells. Overexpression of
both
ARK1 and 2 with the
AR resulted in two of
the most common biochemical modifications occurring during homologous
desensitization of G protein-coupled receptors, namely a decreased
sensitivity of the receptor to the agonist and its reduced ability to
mediate the maximal response (Fig. 6). In addition,
overexpression of both
ARK1 and 2 could increase the
agonist-induced phosphorylation of the
AR above basal
of almost 2-fold as compared to that of the receptor expressed alone (Fig. 2Fig. 3Fig. 4). Two lines of evidence
support the notion that
ARK-induced desensitization of the
AR is, at least in part, mediated by phosphorylation
of the receptor. First, the phosphorylation-deficient T368 receptor
mutant, which could mediate the activation of PI response as well as
the wild type
AR, was largely insensitive to both
ARK1 and 2 (Fig. 5). Second, when the kinases were
overexpressed at low level, the
AR-mediated response
could be inhibited by wild type
ARK1 or 2, but not by
their dominant negative mutants lacking the kinase activity (Fig. 7).
In cotransfection experiments, to assess the role
of a single biochemical component, such a specific receptor kinase or
arrestin, this latter must be overexpressed to overcome the different
endogenous mechanisms involved in receptor function and regulation.
Thus, in our experiments the AR was expressed at a
constant level of 200-400 fmol/1,000,000 cells, whereas the
cytosolic expression of
ARK was about 10-fold higher of that of
the receptor (see ``Results''). These experimental conditions
might be considered far from being physiological. However, our findings
provide also the evidence that
ARK might play a general role in
homologous desensitization of the
AR. This is
supported by the finding that overexpression of the dominant negative
ARK mutants could inhibit the agonist-induced phosphorylation of
the
AR mediated by the endogenous kinases in COS-7 (Fig. 4). Thus, agonist-induced phosphorylation and regulation
of the
AR occurring in a variety of cells might be
mediated, at least in part, by
ARK.
In a previous
study(15) , we were unsuccessful at phosphorylation of the
AR purified from DDT1 MF-2 smooth muscle cells
reconstituted in vitro with
ARK purified from bovine
brain. An explanation of our previous lack of success might be that the
reconstitution procedure impaired the receptor's ability to bind
the agonist or to be stabilized in its ``active''
conformation, which is the substrate conformation required by
ARK.
This hypothesis can be supported by the fact that the efficiency of
reconstitution in phospholipid vesicles (assessed as the ratio between
ligand binding activity and the amount of reconstituted protein) was
always much lower for the
AR as compared to other
receptors like the
AR suggesting that only a very
small fraction of the reconstituted
AR was
functional. (
)Thus, future in vitro studies should
first attempt to optimize the experimental conditions which can
preserve the functional properties of the purified
AR.
Our results are in agreement
with previous studies showing that overexpression of arrestin could
impair the cAMP response mediated by the AR (34) and, more recently, by the
AR(35) . However, these findings are not
simple to interpret from a mechanistic point of view. For non-visual
arrestin, very little is known about the molecular mechanisms
underlying its interaction with the receptors. An important
contribution has been provided by a recent study(36) , which
has proposed a kinetic model of non-visual arrestin interaction with
receptors based on a detailed binding analysis of the different
purified components. The model of arrestin-receptor interaction
proposed is analogous to the current model of G protein-receptor
interaction (33) in several aspects. Thus, similarly to the G
protein, an excess of arrestin can drive all the agonist-receptor
complexes to bind arrestin. Cotransfection experiments in intact cells
cannot provide precise mechanistic information because it is difficult
to assess the precise stoichiometry of the expressed proteins as well
as to which extent they are functionally active. Our hypothesis is
that, in cotransfection experiments with different
receptors(34, 35) , arrestin can induce receptor
desensitization independently of the phosphorylation state of the
receptor because of its stoichiometric excess over the G protein.
Another important finding of our study is that the integrity of the
C-terminal portion of the AR is required for its
interaction with arrestin. This was demonstrated by the fact that
overexpression of
-arrestin could impair the wild type
receptor-mediated response, but not that of its truncated
mutant T368 (Fig. 8). Virtually nothing is known about the
structural domains of G protein-coupled receptors interacting with
non-visual arrestins. It is well documented that arrestin
preferentially binds to the phosphorylated receptor(36) . This
cannot be demonstrated by our study because the amount of
phosphorylated versus non-phosphorylated
AR
is not known and cannot be unequivocally established in cells
overexpressing the receptors. However, our findings indicate that,
independently of the phosphorylation state of the receptor, a main
structural determinant of the receptor binding site for arrestin is
located in the C terminus of the
AR, which is also an
important phosphorylation domain of the receptor.
However, despite their effect
on phosphorylation of the AR, coexpression of GRK5 or
GRK6 either with or without
-arrestin did not induce any change of
the receptor-mediated response. Thus, the functional correlates of GRK5
and GRK6-mediated phosphorylation of the
AR remain
unknown. One possibility is that COS-7 or HEK293 cells are missing a
yet unidentified component essential for the full regulatory activity
of GRK5 and GRK6. Alternatively, our measurement of the
AR-mediated response is not sufficiently sensitive to
assess more rapid or subtle regulatory events.