(Received for publication, April 1, 1997, and in revised form, May 2, 1997)
From the Department of Pharmacology, University of Washington, Seattle, Washington 98195-7750
The m2 muscarinic acetylcholine receptor (m2
mAChR) belongs to the superfamily of G protein-coupled receptors and is
regulated by many processes that attenuate signaling following
prolonged stimulation by agonist. We used a heterologous expression
system to examine the ability of G protein-coupled receptor kinase-2 (GRK2) and -arrestin-1 to regulate the phosphorylation state and to
promote desensitization and sequestration of the m2 mAChR. Treatment of
JEG-3 cells transiently expressing the m2 mAChR with a muscarinic
agonist induced an ~4- or 8-fold increase in receptor phosphorylation
in the absence or presence of cotransfected GRK2, respectively,
compared with untreated cells transfected with receptor alone. Using
the expression of a cAMP-regulated reporter gene to measure receptor
function, we found that transiently transfected m2 mAChRs underwent
functional desensitization following exposure to agonist. Transfected
GRK2 enhanced agonist-induced functional desensitization in a manner
that was synergistically enhanced by cotransfection of
-arrestin-1,
which had no effect on m2 mAChR function when coexpressed in the
absence of GRK2. Finally, GRK2 and
-arrestin-1 synergistically
enhanced both the rate and extent of agonist-induced m2 mAChR
sequestration. These results are the first to demonstrate that
agonist-induced desensitization and sequestration of the m2 mAChR in
the intact cell can be enhanced by the presence of GRK2 and
-arrestin-1 and show that these molecules have multiple actions on
the m2 mAChR.
The family of muscarinic acetylcholine receptors (mAChRs)1 belongs to the superfamily of G protein-coupled receptors that couple extracellular stimuli to intracellular effector molecules through the actions of heterotrimeric G proteins (1, 2). Five subtypes of muscarinic receptors have been cloned (3-8) and classified according to their ability to couple to different signaling pathways: the m1, m3, and m5 subtypes preferentially couple to activation of phospholipase C via the Gq family of G proteins, whereas the m2 and m4 subtypes preferentially couple to inhibition of adenylate cyclase via the Gi family of G proteins (1, 2). As with other members of the G protein-coupled receptor superfamily, the family of mAChRs is exquisitely regulated by processes that function to attenuate signaling in the presence of prolonged exposure to agonist. These processes, which differ in their time course and mechanism, have been termed desensitization, internalization (or sequestration), and down-regulation (9, 10).
Desensitization, the most rapid of these processes, is dependent upon
receptor phosphorylation mediated either by second messenger kinases or
by members of the family of G protein-coupled receptor kinases (GRKs)
(11). GRK phosphorylation of G protein-coupled receptors, which occurs
only in the presence of agonist, is thought to promote the binding of
one of the members of the arrestin family of molecules to the activated
receptors (12, 13). To date, four members of the arrestin family have
been cloned and characterized: arrestin, cone arrestin, -arrestin-1,
and
-arrestin-2 (or arrestin-3). Arrestin,
-arrestin-1, and
-arrestin-2 each appear to be alternatively spliced, generating at
least two polypeptides for each isoform (11). Together, the actions of
GRK-mediated phosphorylation coupled with arrestin binding lead to
receptor-G protein uncoupling and desensitization. The family of GRKs,
which has at least six members termed GRK1 through GRK6, includes
rhodopsin kinase (GRK1) and
-adrenergic receptor kinase-1 and -2 (GRK2 and GRK3, respectively) (11). The activated m2 mAChR serves as an
excellent substrate in vitro for both GRK2 (14-17) and GRK3
(14) and, to a lesser extent, GRK5 (18) and GRK6 (19). Phosphorylation
of the m2 mAChR by GRK2 and GRK3 promotes desensitization in
vitro (14), whereas blockade of agonist-induced m2 mAChR
phosphorylation in intact cells, either by transfection with
kinase-inactive GRK2 or by removal of the putative phosphorylation
sites by deletion mutagenesis, results in attenuation of
desensitization (20).
Recent evidence suggests that phosphorylation of G protein-coupled
receptors by GRKs (21, 22) and the subsequent binding of -arrestin
(23) play an additional role in receptor internalization, coupling
receptors to a dynamin-dependent pathway in which receptors are targeted for endocytosis via clathrin-coated vesicles (24, 25). The
role of internalization in signal attenuation is not well understood,
but it has been suggested that the primary function of internalization
of the
2-adrenergic receptor is to allow the dephosphorylation of receptors in preparation for their return to the
plasma membrane (26, 27). Tsuga et al. (28) reported that
agonist-induced sequestration of the m2 mAChR, when transiently expressed in COS-7 and BHK-21 cells, could be enhanced by coexpression of GRK2 and attenuated by coexpression of a kinase-inactive GRK2 mutant. In contrast, Pals-Rylaarsdam et al. (20) reported
that coexpression of wild-type and kinase-inactive GRK2 had no effect on agonist-induced m2 mAChR sequestration in a human embryonic kidney
cell line.
Currently, no information exists regarding the ability of specific GRKs
to phosphorylate and promote desensitization of a muscarinic receptor
in an intact cell, and as noted above, the role of GRK phosphorylation
in sequestration of the m2 mAChR remains controversial. Moreover,
blockade of desensitization and/or sequestration by kinase-inactive
GRK2 indicates that a molecule that can interact with GRK is required,
but does not prove that GRK2 itself is required. Whereas both
-arrestin-1 and
-arrestin-2 are known to bind the GRK-phosphorylated m2 mAChR in vitro (29, 30), there have been no reported studies describing the effects of a
-arrestin on
either desensitization or sequestration of muscarinic receptors. We
have used a heterologous expression system in which activation of the
m2 receptor is coupled to the expression of a sensitive, cAMP-responsive reporter gene to examine the combined role of GRK2 and
-arrestin-1 in agonist-induced desensitization of the m2 mAChR in
the intact cell. We report here that GRK2 and
-arrestin-1 can
synergistically promote m2 mAChR desensitization and sequestration in
JEG-3 cells, providing new insight into the regulation of m2 receptor
function in the intact cell.
[3H]quinuclidinyl benzilate (47 Ci/mmol) and N-[3H]methylscopolamine
([3H]NMS; 81-84 Ci/mmol) were purchased from Amersham
Corp., and [32P]orthophosphate
(32Pi) was from NEN Life Science Products.
Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin,
and fetal bovine serum were purchased from Life Technologies, Inc.
Restriction enzymes were from New England Biolabs Inc. Forskolin was
obtained from Calbiochem, and D-luciferin (potassium salt)
was from Analytical Luminescence Laboratory (Ann Arbor, MI). The
anti-FLAG M2 monoclonal antibody was purchased from Eastman Kodak Co.
Pfu polymerase was from Stratagene, whereas other polymerase
chain reaction reagents were from Perkin-Elmer Corp. Electrophoresis
reagents were purchased from Bio-Rad, and Immobilon-P was from
Millipore Corp. Carbamylcholine chloride (carbachol), atropine,
anti-mouse IgG1-agarose, and all other reagents were purchased from
Sigma. Dr. D. Capon (Genentech Inc.) kindly provided the porcine m2
mAChR (Mc7 clone) (6), and Dr. G. S. McKnight (University of
Washington) provided the 168 CRE-luciferase construct (31) and the
RSV-
-galactosidase construct (32). Rat Gi
-2 (33) was
a gift from Dr. R. R. Reed (Johns Hopkins University, Baltimore, MD).
Dr. R. J. Lefkowitz (Duke University) kindly provided the GRK2 and
-arrestin-1 clones. The m2 mAChR, Gi
-2, and GRK2
cDNAs were subloned into the expression vector pCDPS (8), a gift
from Dr. T. Bonner (National Institutes of Health, Bethesda, MD).
JEG-3 cells, a human
choriocarcinoma cell line (American Type Culture Collection, Rockville,
MD), were cultured in DMEM supplemented with 10% fetal bovine serum
and 1% penicillin/streptomycin at 37 °C in a humidified 10%
CO2 environment. Transfection on 24-well plates and
subsequent assays of luciferase and -galactosidase activities were
performed essentially as described (34). Briefly, cells seeded at
18,000/well were transfected 72 h later using the calcium
phosphate precipitation method (35) with 200-315 ng of total DNA/well.
Transfection mixtures included the following expression vectors in the
combinations indicated in the figure legends: 30 ng of receptor
cDNA/well, 0-60 ng of pCDPS-GRK2/well, 0-60 ng of
pCMV5-
-arrestin-1/well, 25 ng of
168 CRE-luciferase/well, 40 ng
of RSV-
-galactosidase/well to correct for transfection efficiency,
and 100 ng of pCDPS-Gi
-2/well. The total DNA for each
transfection mixture in a given experiment was kept the same by
addition of carrier DNA (pCDPS). The medium was changed 20-24 h
after transfection, and cells were treated with various drugs (as
described in the figure legends) an additional 20-24 h later.
For whole cell phosphorylation and sequestration assays, JEG-3 cells
(~80% confluent) were transfected according to the calcium phosphate
precipitation method (35) and treated with 100 µM chloroquine for 3-4 h, followed by a 3-min treatment with 15% glycerol in HEPES-buffered saline (20 mM HEPES, 0.7 mM Na2HPO4, 137 mM
NaCl, 5.0 mM KCl, and 5.6 mM dextrose, pH
7.05). For whole cell phosphorylation experiments, 150-mm tissue
culture dishes were transfected with the following amounts of
expression vectors as indicated below: 25 µg of receptor cDNA, 25 µg of pCDPS-GRK2, 25 µg of pCMV5--arrestin-1, 10 µg of
pCDPS-Gi
-2, and pCDPS as needed to maintain a constant
amount of DNA in each of the transfection mixtures in a given
experiment. Cells were transfected in a similar fashion for receptor
sequestration experiments except that the following amounts of
expression vectors were used: 8 µg of receptor cDNA, 16 µg of
pCDPS-GRK2, 16 µg of pCMV5-
-arrestin-1, and 20 µg of
pCDPS-Gi
-2.
Polymerase chain reaction was used to engineer an m2 mAChR
tagged at its N terminus with a modified FLAG epitope:
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Ala. Using the upstream primer
5-GGTACCGGTACCATGGACTACAAGGACGATGATGACGCCGTGAATAACTCCACCAACCTCC-3
, which encodes the FLAG epitope and contains a KpnI
site at its 5
-end, and the downstream primer
5
-GGTTCTTGGTTGGCCACAGGCTCC-3
, which corresponds to bases
666-682 of the wild-type sequence and flanks an endogenous
MscI site, a 730-base pair polymerase chain reaction product
was generated that contains the sequence encoding the FLAG epitope
immediately following the ATG initiation codon. This polymerase chain
reaction product was digested with KpnI and MscI
and then ligated to pCDPS-m2, which had been previously digested with
KpnI and MscI, to generate pCDPS-FLAG-m2. The
resulting construct was sequenced on an Applied Biosystems 373A DNA
sequencing system to verify the presence of the FLAG epitope and to
ensure that mutations were not introduced during amplification. The
ability of the pCDPS-FLAG-m2 construct to inhibit forskolin-stimulated changes in CRE-luciferase expression was identical to that previously described for nontagged receptors (36).
The deletion mutant m2 mAChR was generated by digestion of
pCDPS-FLAG-m2 with MscI and SmaI, which excises
bases 677-1141 of the m2 mAChR coding sequence. The remaining fragment
containing the 5- and 3
-ends of the m2 mAChR in pCDPS was ligated
together to generate pCDPS-FLAG-m2
3. This construct encodes an m2
mAChR that is missing amino acids 227-381 and converts Ala-226 to Gly. Expression of this construct in transiently transfected JEG-3 cells was
comparable to that seen with full-length pCDPS-FLAG-m2.
JEG-3 cells on 24-well culture
plates were transiently cotransfected with 30 ng of receptor
cDNA/well, 25 ng of 168 CRE-luciferase/well, 40 ng of
RSV-
-galactosidase/well, 0-60 ng of pCDPS-GRK2/well, 0-60 ng of
pCMV5-
-arrestin-1/well, and 100 ng of
pCDPS-Gi
-2/well. Approximately 40-48 h after
transfection, cells were pretreated without or with 0.25 ml of complete
DMEM containing carbachol for 15 or 30 min at 37 °C and washed twice
with 1 ml of phosphate-buffered saline (PBS; 4.3 mM
Na2HPO4, 1.4 mM
KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4), and then triplicate wells were treated
with 0.25 ml of complete DMEM without drugs (background) or with 2 µM forskolin and various concentrations of carbachol or
PBS (control) for 60 min at 37 °C. In some experiments, control
cells were pretreated with drug-free DMEM for either 15 or 30 min;
signaling by these cells was identical to non-pretreated cells.
Following pretreatment, cells were washed twice with PBS, treated with
0.25 ml of complete DMEM without drugs, and incubated an additional
4 h at 37 °C. Cells were lysed and assayed for luciferase and
-galactosidase activities as described previously (34). Data were
normalized for transfection efficiency, corrected for background, and
expressed as the percent of signal seen with forskolin alone
(control).
Each 150-mm culture dish of transiently cotransfected JEG-3 cells was subcultured onto three 100-mm culture dishes 16-20 h after transfection. After 20-24 h, cells from one 100-mm plate were scraped into 0.8 ml of buffer A (20 mM KH2PO4, pH 7, 20 mM NaF, 5 mM EGTA, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml benzamidine, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 µg/ml pepstatin) and then used to determine receptor expression. Cells on the two remaining 100-mm plates were washed twice with phosphate-free DMEM, incubated for 2-3 h in phosphate-free DMEM, and then labeled an additional 2-3 h with 0.12-0.20 mCi/ml 32Pi in phosphate-free DMEM. The cells were stimulated with 1 mM carbachol for 15 min at room temperature, washed three times with 5 ml of ice-cold PBS, and then scraped into 0.8 ml of buffer A. The cell suspension was centrifuged for 10 min in a microcentrifuge at 4 °C, and the resulting cell pellet was resuspended in 0.25 ml of ice-cold buffer B (buffer A containing 0.5% digitonin and 0.05% cholate) and then incubated for 60 min at 4 °C with constant mixing. Cellular debris was sedimented by centrifugation for 10 min in a microcentrifuge at 4 °C. The resulting lysate was transferred to fresh tubes and precleared by incubation with 25 µl of IgG1-agarose for 30 min at 4 °C. Following removal of the beads by centrifugation, the m2 mAChRs were immunoprecipitated from the cleared lysate by an overnight incubation with 25 µl of IgG1-agarose previously coupled to 2.5 µg of the anti-FLAG M2 monoclonal antibody. The following day, immunoprecipitates were washed six to eight times with 0.5 ml of buffer C (buffer B containing 200 mM NaCl) and twice with 0.5 ml of PBS to remove nonspecifically bound proteins. The specifically adsorbed proteins were eluted from the anti-mouse IgG1-agarose by incubation in SDS-polyacrylamide gel electrophoresis sample buffer containing 8 M urea and then subjected to SDS-polyacrylamide gel electrophoresis on 9% gels containing 4 M urea as described (37), followed by electrophoretic transfer to Immobilon-P. The m2 mAChRs were analyzed by immunoblotting and autoradiography. Specifically labeled bands in the autoradiograms were analyzed by densitometry scanning on a Bio-Rad GS-670 imaging densitometer and then corrected for transfection efficiency using the amount of receptor expression as determined by [3H]quinuclidinyl benzilate binding assays on parallel plates performed as described previously (38).
Receptor Sequestration AssaysDetermination of m2 mAChR sequestration using the binding of [3H]NMS, a membrane-impermeable ligand, to intact cells has been previously described (39). Briefly, 20-24 h after transfection, JEG-3 cells from one 150-mm culture dish were subcultured onto five 6-well plates and allowed to attach for an additional 24 h. The cells were treated with various concentrations of carbachol for 0-60 min (6 wells/condition), washed three times with 3 ml of ice-cold PBS, and labeled with ~1.4 nM [3H]NMS in PBS for 4-5 h at 4 °C. Nonspecific binding was determined in the presence of 1 µM atropine. Labeled cells were washed three times with 3 ml of ice-cold PBS, solubilized in 0.5 ml of 1% Triton X-100, and combined with 3.5 ml of scintillation mixture prior to the determination of radioactivity by scintillation counting. Data are expressed as the percent of [3H]NMS binding seen with untreated cells.
A previous
report by Richardson et al. (14) demonstrated that GRK2
could phosphorylate the m2 receptor in vitro. We wanted to
determine whether GRK2 could phosphorylate and promote desensitization of the m2 receptor in the more physiologically relevant context of an
intact cell. In addition, we were interested in the effects, if any, of
-arrestin-1 coexpression on m2 mAChR phosphorylation and function.
To test these questions directly, JEG-3 cells were transiently
cotransfected with various combinations of expression vectors encoding
GRK2,
-arrestin-1, and an m2 mAChR construct tagged at its N
terminus with a FLAG epitope (termed FLAG-m2) and then metabolically
labeled with [32P]orthophosphate. Following a brief
exposure to 1 mM carbachol, a muscarinic agonist, cells
were lysed, and FLAG-tagged m2 mAChRs were immunoprecipitated. In cells
transfected with FLAG-m2 alone, immunoprecipitated receptors migrated
as a broad band with an apparent molecular mass of ~86-110 kDa (Fig.
1A). In cells transfected with receptor
alone, treatment with carbachol led to an ~4-fold increase in
receptor phosphorylation (Fig. 1, A and B).
Agonist-dependent phosphorylation of these receptors was
enhanced by cotransfection with GRK2, increasing the level of
phosphorylation ~8-fold over that measured in cells transfected with
receptor alone in the absence of carbachol (Fig. 1, A and
B). Basal levels of m2 receptor phosphorylation also
appeared to be modestly increased in cells expressing GRK2.
Coexpression of
-arrestin-1 had negligible effects on receptor
phosphorylation, leading to a slight reduction in agonist-induced
phosphorylation in cells coexpressing GRK2 (Fig. 1B).
Together, these data suggest that exposure to agonist promotes phosphorylation of the m2 mAChR by GRK2 in transiently transfected JEG-3 cells.
Functional Desensitization of the m2 mAChR in Transiently Transfected JEG-3 Cells
Previous reports from our laboratory
demonstrated that agonist activation of the m2 mAChR can mediate
inhibition of a cAMP-responsive reporter gene when transiently
cotransfected into JEG-3 choriocarcinoma cells (36). This reporter
construct, 168 CRE-luciferase, is composed of a 168-base pair region
of the
-glycoprotein promoter region upstream of the coding region
of the firefly luciferase gene (40) and is extremely sensitive to
changes in intracellular cAMP levels. Because the m2 mAChR exhibited
agonist-dependent phosphorylation in cells transfected
without added GRK2 expression vector, we determined whether
agonist-dependent desensitization could be detected in
these cells and whether cotransfection with GRK2 and
-arrestin-1
would lead to enhanced desensitization.
In JEG-3 cells transiently cotransfected with 168 CRE-luciferase and
a construct encoding the FLAG-tagged m2 mAChR, treatment with carbachol
led to a dose-dependent decrease in forskolin-stimulated luciferase expression to a maximal inhibition of 65% at 10 µM carbachol (Fig. 2A) (36).
Similar results were seen in cells transfected with wild-type nontagged
m2 mAChRs, indicating that addition of the FLAG epitope did not alter
receptor-G protein coupling.2 Pretreatment
of cells with 1 mM carbachol for 15 min caused a modest
amount of receptor desensitization, which was demonstrated by a shift
in the carbachol dose-response curve to the right and a slight decrease
in the maximal extent of inhibition (Fig. 2A). At
concentrations of carbachol below 100 µM, m2 mAChR
signaling was attenuated, but at concentrations of agonist of 100 µM and higher, maximal inhibition of luciferase
expression was similar to that seen in the absence of pretreatment.
Longer periods of pretreatment for up to 30 min did not result in
additional receptor desensitization.2
To determine whether GRK2 and -arrestin-1 could regulate the
function of the m2 receptor, JEG-3 cells were transiently cotransfected with GRK2 and/or
-arrestin-1 together with FLAG-m2. In the absence of pretreatment, cotransfection of the GRK2 construct had relatively mild effects on m2 mAChR signaling: at concentrations of carbachol below 10 µM, signaling was identical to that seen in
cells transfected with FLAG-m2 alone, whereas signaling was slightly
attenuated at higher concentrations of agonist (Fig. 2A). In
contrast, pretreatment with 1 mM carbachol for 15 min led
to a more dramatic shift in the carbachol dose-response curve than that
seen in cells transfected with FLAG-m2 alone. For example, following
pretreatment of GRK2-transfected cells, a subsequent treatment with 1 µM carbachol led to an ~5% decrease in
forskolin-stimulated luciferase expression as compared with an ~30%
decrease in cells expressing the m2 receptor in the absence of GRK2. In
addition to causing a shift in the carbachol dose-response curve,
cotransfection of GRK2 led to a reduction in the maximal activation of
the m2 mAChR. Following pretreatment for 15 min, maximal activation of
m2 mAChRs in control cells led to an ~48% decrease in
forskolin-stimulated luciferase expression, whereas maximal activation
of m2 mAChRs in GRK2-cotransfected cells led to an ~32% decrease in
forskolin-stimulated luciferase expression. Again, longer periods of
pretreatment for up to 30 min did not result in additional receptor
desensitization.2
Expression of -arrestin-1 had very little effect on m2 receptor
signaling by itself, but appeared to potentiate the effects of GRK2
expression. Cotransfection of
-arrestin-1 in the absence of GRK2 had
no measurable effect on m2 mAChR signaling. Indeed, carbachol
dose-response curves in the absence or presence of pretreatment for up
to 30 min with 1 mM carbachol were identical to that seen with the m2 mAChR alone (Fig. 2, compare A and
B). Cotransfection of
-arrestin-1 with GRK2 resulted in
greater attenuation of m2 receptor signaling than was seen with GRK2
alone. In the absence of pretreatment, signaling was similar to that
seen in cells transfected either with the m2 mAChR alone or with the m2
mAChR and
-arrestin-1 below 1 µM carbachol, but was
attenuated above 1 µM carbachol. Pretreatment of cells
cotransfected with GRK2 and
-arrestin-1 with 1 mM
carbachol for 15 min had more profound effects than that of cells
expressing only GRK2, resulting in a total blockade of m2 receptor
signaling. Taken together, these results suggest that phosphorylation
by GRK2 and subsequent binding of
-arrestin-1 are able to promote
desensitization of the m2 mAChR in an agonist-dependent and
synergistic fashion. This is the first report of synergistic regulation
of muscarinic receptor function in the intact cell by a specific
receptor kinase and
-arrestin.
The third intracellular loop of the m2
mAChR has been shown to be important in agonist-induced receptor
desensitization (20) as well as internalization (41) and has been
reported to contain the putative GRK phosphorylation sites (16, 20,
42). To verify that the effects of GRK2 and -arrestin-1 coexpression on m2 mAChR signaling were occurring at the level of the receptor, we
deleted a portion of the third intracellular loop, encoding amino acids
227-380, from the FLAG-m2 coding sequence. This deletion mutant
(termed FLAG-m2
3) is similar but not identical to the mutant
receptor engineered by Kameyama et al. (16), which lacks amino acids 233-380, but which has expression and ligand binding properties similar to wild-type m2 mAChR. When the construct encoding the deletion mutant m2 receptor (FLAG-m2
3) was transiently
transfected into JEG-3 cells, treatment with carbachol led to a
dose-dependent inhibition of forskolin-stimulated
luciferase expression that was comparable in magnitude to that seen
with the full-length m2 mAChR (Fig. 3A). In
contrast to full-length receptors, pretreatment for up to 30 min with 1 mM carbachol did not lead to a shift in the dose-response
curve. In addition, coexpression of GRK2 and
-arrestin-1 either
singly2 or in combination (Fig. 3B) had no
effect on the ability of the FLAG-m2
3 receptor to signal in either
the absence or presence of pretreatment with 1 mM carbachol
for up to 30 min. In separate experiments, the FLAG-m2
3 receptor was
found to be expressed at levels comparable to full-length receptors
and, as expected from the data of Pals-Rylaarsdam et al.
(20) and Nakata et al. (42), was not phosphorylated
following exposure to agonist in either the absence or presence of
added GRK2 expression vector.2 Together, these data
demonstrate that the effects of GRK2 and
-arrestin-1 on m2 mAChR
signaling are dependent on the presence of the third intracellular loop
and suggest that they are acting at the level of the receptor and not
at a site downstream in the signaling pathway.
Titration of GRK2 cDNA in the Presence of
To further examine the specificity of the GRK2 effects
on m2 mAChR signaling, JEG-3 cells were transiently cotransfected with constant amounts of FLAG-m2 and -arrestin-1 and varying amounts of
GRK2. In the absence of pretreatment, increasing amounts of GRK2
expression vector led to a dose-dependent decrease in the ability of the m2 receptor to signal, primarily at concentrations of
carbachol above 1 µM (Fig. 4A).
Inhibition of forskolin-stimulated luciferase expression at carbachol
concentrations up to 1 µM was independent of the presence
of any amount of transfected GRK2. This experiment suggests that the
biphasic nature of the carbachol dose-response relationship in the
presence of both GRK2 and
-arrestin-1 is largely due to rapid
desensitization of the transfected receptors at the higher
concentrations of carbachol and not to increased coupling to a
stimulatory G protein such as Gs.
To measure the effect of increasing GRK2 expression on agonist-induced
desensitization, JEG-3 cells were transfected with varying amounts of
GRK2 in the presence of constant amounts of FLAG-m2 and -arrestin-1,
but then incubated in the presence or absence of carbachol for 15 or 30 min prior to treatment with medium containing forskolin without
(control) or with 10 µM carbachol. As seen in Fig.
4A, increasing concentrations of GRK2 expression vector in
the transfection mixture decreased the ability of the activated m2
mAChR to signal at this concentration of carbachol in the absence of
pretreatment (Fig. 4B). When cells were first pretreated for
15 min, signaling by the m2 receptor was further attenuated in a manner
that correlated with increasing amounts of transfected GRK2 expression
vector (Fig. 4B). Enhanced agonist-induced desensitization
was apparent at all levels of GRK2 expression vector tested, and in all
cases, maximal levels of desensitization were reached by 15 min.
Together, these data suggest that GRK2 is able to promote
desensitization of the m2 mAChR in a dose-dependent manner.
We next attempted to determine
the dose dependence of agonist pretreatment in promoting
desensitization of the m2 mAChR in JEG-3 cells transiently transfected
either alone or with various combinations of GRK2 and -arrestin-1.
Prior to stimulation with forskolin in the presence or absence of 1 µM carbachol, transfected cells were pretreated with
various concentrations of carbachol or control medium for 15 min. As
seen previously with this concentration of carbachol, inhibition of
forskolin-stimulated luciferase expression in the absence of
pretreatment was only mildly attenuated in cells transfected either
with GRK2 or with GRK2 and
-arrestin-1 together (Fig.
5). The ability of the m2 mAChR to inhibit
forskolin-stimulated CRE-luciferase expression following pretreatment
with 1 µM carbachol was almost identical to what was seen
in the absence of pretreatment. Furthermore, signaling was independent
of the presence of transfected GRK2 and/or
-arrestin-1, which
suggests that the levels of desensitization at this concentration of
carbachol are relatively mild and below the limits of our detection.
Strikingly, increasing the concentration of carbachol used for
pretreatment to 5 µM promoted significant (p < 0.02) levels of desensitization only in cells
transfected either with GRK2 or with GRK2 and
-arrestin-1 together,
reducing the amount of inhibition seen in these cells from 45 ± 6 to 23 ± 2% and from 46 ± 1 to 21 ± 5%,
respectively. In contrast, signaling in cells transfected with the m2
mAChR either alone or in combination with
-arrestin-1 was not
significantly affected by a similar pretreatment. Of the concentrations
tested for their ability to promote desensitization, only 1 mM carbachol was sufficient to cause significant
attenuation of signaling in cells transfected either with FLAG-m2 alone
(p < 0.02) or with FLAG-m2 and
-arrestin-1 together
(p < 0.002). Taken together, these data demonstrate
that the extent of m2 mAChR desensitization is proportional to the concentration of agonist regardless of the presence of cotransfected GRK2, but that desensitization occurs at much lower agonist
concentrations in cells cotransfected with GRK2. In addition, they
suggest that desensitization of the m2 mAChR, when cotransfected with
GRK2, occurs primarily at concentrations of carbachol (i.e.
>1 µM) at which signaling becomes attenuated in the
absence of pretreatment.
Agonist-induced Sequestration: Regulation by GRK2 and
Phosphorylation of the 2-adrenergic
receptor by GRK2, GRK3, and GRK5 (21, 22) and
-arrestin binding (23,
24) have recently been found to promote agonist-induced sequestration
of this receptor. Evidence for a role of GRK phosphorylation in
agonist-induced sequestration of m2 mAChRs is contradictory (20, 28),
whereas no data exist for the role of
-arrestin-1 in the
sequestration of m2 mAChRs. Because coexpression of GRK2 and
-arrestin-1 was able to regulate the m2 mAChR in a functional assay,
we tested the effects of these molecules on agonist-induced
sequestration in transiently transfected JEG-3 cells.
Agonist-induced sequestration was measured using the
membrane-impermeable muscarinic ligand [3H]NMS, which
detects only cell-surface receptors. In transiently transfected JEG-3
cells, m2 mAChRs underwent agonist-induced sequestration that was both
time- and dose-dependent (Fig. 6,
A and B, respectively) (43). Cotransfection with
GRK2 led to an increase in both the rate and extent of m2 mAChR
sequestration over the entire time course tested (Fig. 6A).
In contrast to previous experiments in which coexpression of
-arrestin-1 in the absence of GRK2 did not alter m2 mAChR
desensitization, coexpression of
-arrestin-1 led to an increase in
the rate and extent of agonist-induced sequestration that was similar
but not identical to that seen with GRK2 coexpression (Fig.
6A). Cotransfection of both
-arrestin-1 and GRK2
expression vectors led to an enhancement of the extent of m2 mAChR
sequestration that was slightly greater than what would be expected if
the effects of each were added together (Fig. 6A). In
addition, the apparent rate of m2 mAChR sequestration as determined
from the first-order decay curve was increased by ~130% in cells
transfected with both GRK2 and
-arrestin-1 as compared with ~36 or
~45% in cells transfected with either GRK2 or
-arrestin-1,
respectively (Fig. 6A, inset). This greater than
additive effect implies that the two molecules are acting in concert,
or synergistically, to enhance agonist-induced sequestration of the m2
mAChR.
Dose and time dependence of agonist-induced
sequestration of FLAG-tagged m2 mAChRs coexpressed with various
combinations of GRK2 and/or -arrestin-1. A and
B, JEG-3 cells were transfected with FLAG-m2 alone
(squares) or in combination with
-arrestin-1 (
ARR1; triangles), GRK2 (inverted
triangles), or both GRK2 and
-arrestin-1 (circles)
and then treated with 1 mM carbachol for the times
indicated (A) or for 15 min with the concentrations of
carbachol indicated (B). The inset shows the data
from A fit to an exponential decay curve; the apparent
first-order rate constants in cells transfected with the various
constructs are as follows: m2 mAChR alone, 2.2 × 10
2 min
1; m2 mAChR +
-arrestin-1,
3.2 × 10
2 min
1; m2 mAChR + GRK2,
3.0 × 10
2 min
1; and m2 mAChR +
-arrestin-1/GRK2, 5.1 × 10
2 min
1.
Cell-surface receptors were measured using the membrane-impermeable muscarinic ligand [3H]NMS as described under
"Experimental Procedures." Data are expressed as the percent of cell-surface receptors measured in untreated cells and represent
the means ± S.E. of three or four independent experiments, each
performed in quadruplicate. C, receptors expressed on the
surface of cells transfected with FLAG-m2 alone or in combination with
GRK2,
-arrestin-1, or both GRK2 and
-arrestin-1 as indicated were
measured using [3H]NMS as described under "Experimental
Procedures." Data are expressed as the percent of cell-surface
receptors measured in cells transfected with FLAG-m2 alone and
represent the means ± S.E. of four independent experiments, each
performed in quadruplicate.
The synergistic effect was even more evident upon examination of the
dose dependence of sequestration. At the lowest concentrations of
agonist (i.e. 1 and 10 µM), the presence of
-arrestin-1 did not lead to enhanced sequestration in cells
cotransfected with only the m2 mAChR, but did promote sequestration in
cells also cotransfected with GRK2 (Fig. 6B). Interestingly,
cotransfection of GRK2 and
-arrestin-1 together led to a significant
(p < 0.05) enhancement of sequestration at
concentrations of carbachol as low as 1 µM (Fig.
6B), a concentration at which transiently transfected m2
mAChRs failed to undergo measurable desensitization even when cotransfected with GRK2 and/or
-arrestin-1 (Fig. 5). It is unlikely that the effects of GRK2 and/or
-arrestin-1 cotransfection were due
to alterations in receptor expression because basal levels of
cell-surface m2 mAChR expression were fairly similar for each of the
conditions tested (Fig. 6C). Taken together, these data agree with the work of Tsuga et al. (28), who suggested that GRK2 can promote agonist-induced sequestration of the m2 mAChR. In
addition, we show for the first time that
-arrestin-1 is able to
promote m2 mAChR sequestration in a way that appears to be synergistic
with GRK2.
Numerous studies have demonstrated phosphorylation of the m2 mAChR
by a variety of GRKs using in vitro assay systems, but it
has proven difficult to determine the functional consequences of this
modification within an intact cell. Pals-Rylaarsdam et al.
(20) demonstrated that coexpression of kinase-inactive GRK2 in a
human embryonic kidney cell line not only attenuates phosphorylation of
the m2 mAChR following exposure to agonist, but dramatically inhibits
the associated desensitization. The primary drawback to this type of
study is that while it demonstrates that a molecule that interacts with
the kinase-inactive mutant is involved in receptor desensitization, it
does not prove that the GRK itself is actually involved. In addition,
this type of method cannot distinguish between different functional
effects potentially caused by the actions of different GRK isoforms.
For example, Tiberi et al. (44) found that whereas GRK2,
GRK3, and GRK5 were able to phosphorylate the dopamine D1A receptor to
similar extents, the desensitization associated with GRK5
phosphorylation was significantly more profound than that seen with
either GRK2 or GRK3. We describe here a system that allows
reconstitution of the desensitization machinery in intact cells to test
the effects of different components on the functional responsiveness of
the m2 mAChR using a cAMP-regulated reporter gene. Our laboratory has
previously demonstrated the utility of such an approach, showing that
the m2 and m4 mAChRs differentially couple to members of the
Gi and Go class of G proteins in transiently
transfected JEG-3 cells (36), as well as the effects of different
mutations on the activity of Gi-2 and Go
(45).
Initially, using whole cell phosphorylation studies, we demonstrated
that the m2 mAChR is phosphorylated following exposure to agonist in
JEG-3 cells in the absence of added GRK2 expression vector (Fig. 1).
Coexpression of GRK2 with the m2 mAChR led to a slight enhancement of
basal phosphorylation, but the predominant effect was an increase in
agonist-induced phosphorylation, which indicates that the m2 mAChR
serves as a substrate for GRK2 in JEG-3 cells. This is in agreement
with previous studies that demonstrated that the m2 receptor is an
excellent substrate for both GRK2 and GRK3 in vitro (14).
Increased basal phosphorylation of other receptors has been seen in
similar studies in which transient transfection has been used to
overexpress GRK isoforms. For example, Ménard et al.
(22) reported that overexpression of GRK4 and GRK6 enhanced primarily
basal phosphorylation of the 2-adrenergic receptor and
that GRK5 enhanced both basal and agonist-induced phosphorylation.
Interestingly, coexpression of
-arrestin-1 led to a modest reduction
of agonist-induced phosphorylation in cells cotransfected with GRK2
(Fig. 1). This might be explained by recent work demonstrating that
-arrestin-1 is involved in coupling activated receptors to the
internalization machinery (23-25). It has been suggested that one
function of receptor internalization might be dephosphorylation of
desensitized receptors in preparation for their return to the cell
surface (26, 27). Indeed, a phosphatase with activity toward
GRK-phosphorylated G protein-coupled receptors has been identified and
found to be exclusively associated with the particulate fraction
(46).
Using a heterologous expression system in which receptor activation is
coupled to inhibition of CRE-luciferase expression, we demonstrated
that transiently transfected m2 mAChRs become functionally desensitized
when exposed to agonist (Fig. 2A). Cotransfection of GRK2
led to a modest attenuation of receptor signaling in the absence of
pretreatment and, more significantly, enhanced functional desensitization following exposure to agonist (Fig. 2A).
This agonist-induced desensitization in the presence of GRK2 was
manifested by a shift in the dose-response curve for inhibition of
forskolin-stimulated luciferase expression as well as a marked decrease
in the maximal extent of inhibition. Coexpression of -arrestin-1 led
to an increase in both components of the desensitization seen in cells
transfected with GRK2: signaling at high concentrations of agonist in
the absence of pretreatment was decreased, and agonist-induced
desensitization was enhanced (Fig. 2B). The effects of
-arrestin-1 on m2 mAChR desensitization were dependent on the
presence of cotransfected GRK2 cDNA; cotransfection of
-arrestin-1 in the absence of GRK2 had no effect on m2 mAChR
signaling even though transiently expressed m2 mAChRs are
phosphorylated when expressed alone in JEG-3 cells (Fig. 1). The lack
of effect of
-arrestin-1 (in the absence of GRK2) on m2 mAChR
signaling could be explained by a number of possibilities. First,
phosphorylation of the m2 mAChR by the endogenous kinase(s) might be
rate-limiting and the endogenous arrestin(s) present in excess, so
additional
-arrestin-1 is unable to promote additional
desensitization. Alternatively, the endogenous m2 mAChR kinase activity
could be distinct from GRK2, so phosphorylation by this kinase does not
promote the association of transiently expressed
-arrestin-1 in
these cells in a way that promotes functional desensitization. Whereas
different GRKs can promote qualitatively different forms of
desensitization (44), the question of whether different GRKs can
promote the association of different
-arrestins or differentially
regulate the functional effects of
-arrestin binding remains to be
examined.
Titration of GRK2 cDNA in the presence of -arrestin-1 caused a
dose-dependent decrease in the ability of the m2 mAChR to signal in the absence of pretreatment, primarily at higher
concentrations of agonist (Fig. 4A). Signaling at carbachol
concentrations up to 1 µM was independent of the presence
of exogenously expressed GRK2, suggesting that desensitization is
negligible at these lower concentrations of carbachol. Alteration of
the concentration of carbachol with which transiently transfected cells
were pretreated allowed us to demonstrate that desensitization in JEG-3
cells coexpressing the m2 mAChR and either GRK2 or GRK2 in combination with
-arrestin-1 was negligible at concentrations of carbachol below
5 µM (Fig. 5). In contrast, pretreatment of these cells with 5 µM carbachol caused an ~50% decrease in
signaling. This is similar to the EC50 for carbachol to
induce phosphorylation of exogenously expressed m2 mAChRs in Sf9 cell
membranes (11.5 µM) (17) and of the endogenous muscarinic
receptors found in chick heart (20 µM) (47), which are
predominately the m2 subtype (48-50). Together, these data suggest
that phosphorylation of the m2 mAChR by GRK2 is responsible for the
decreased ability of the receptor to signal at higher concentrations of
carbachol.
Despite having no direct effect on agonist-induced desensitization of
the m2 mAChR, expression of -arrestin-1 in JEG-3 cells in the
absence of exogenous GRK2 expression enhanced both the rate and extent
of agonist-induced m2 mAChR sequestration (Fig. 6A). These
effects were qualitatively similar to those seen with GRK2 expression,
although the enhancement of sequestration by
-arrestin-1 at the
earlier time points was slightly less than that seen with GRK2. The
enhancement of sequestration seen with GRK2 and
-arrestin-1
coexpression was slightly greater than if the effects of each were
simply added together, implying that the two molecules are acting
synergistically. This synergism between GRK2 and
-arrestin-1 is
similar to that seen with receptor desensitization (Fig. 2) and
demonstrates for the first time that agonist-induced internalization of
the m2 mAChR can be regulated by
-arrestin-1 and GRK2 together.
Regulation of m2 mAChR sequestration by GRK2 has been reported
previously (28). It was found that overexpression of GRK2 in COS-7 and
BHK-21 cells enhanced sequestration of the m2 mAChR, whereas
overexpression of a kinase-inactive GRK2 allele attenuated sequestration. In contrast, Pals-Rylaarsdam et al. (20)
performed similar experiments in a human embryonic kidney cell line and found that coexpression of either GRK2 or kinase-inactive GRK2 had no
effect on transiently expressed m2 mAChR internalization. The data
presented in this report demonstrate that GRK2, in addition to
-arrestin-1, can indeed promote m2 mAChR internalization when expressed in JEG-3 cells. These data do not, however, prove that GRK2
and
-arrestin-1 are absolutely required for sequestration to occur.
Indeed, at least two sequestration pathways appear to exist in HEK-293
cells, one that is dependent on
-arrestin and dynamin and one that
is independent of these molecules (24), and it is likely that multiple
sequestration pathways exist in other cell types as well. In HEK-293
cells, sequestration of the angiotensin II type 1A receptor was found
to be unaffected by a dominant-negative allele of dynamin unless
recruited to this pathway by overexpression of
-arrestin-1. One
explanation for the results of Pals-Rylaarsdam et al. (20)
is that the dynamin-independent pathway may be the primary pathway by
which m2 mAChRs undergo agonist-induced sequestration in human
embryonic kidney cells and that this pathway may be insensitive to the
effects of added GRK2 or kinase-inactive GRK2.
In summary, we have demonstrated that the m2 mAChR can be regulated
synergistically by coexpression of GRK2 and -arrestin-1 in JEG-3
cells. In addition to being the predominant muscarinic receptor in the
heart, the m2 mAChR is found in a variety of peripheral tissues and
throughout the brain (51). In neuronal tissues, the m2 mAChR is found
associated with cholinergic as well as non-cholinergic neurons, which
suggests that it is found presynaptically and may be found
postsynaptically as well (52, 53). This is similar to the distribution
of both GRK2 and GRK3, which are found throughout the brain associated
with both postsynaptic densities and axon terminals (54). In addition,
-arrestin-1 is found to be highly expressed in the brain (13),
although its ultrastructural localization has not been examined.
Together, these observations suggest that both GRK2 and
-arrestin-1
may colocalize with the m2 mAChR in the brain, making them ideally
situated to regulate m2 receptor function. We have shown here that GRK2
coexpression in the intact cell enhanced phosphorylation of the m2
mAChR in an agonist-dependent manner, resulting in
increased desensitization and sequestration.
-Arrestin-1
coexpression had relatively mild effects on m2 mAChR function by
itself, but significantly enhanced the effects of GRK2, suggesting that
the two molecules can act synergistically and showing that
-arrestin-1 can regulate the function of a muscarinic receptor in
the intact cell. These results provide new and useful information that
helps to explain the intricate mechanisms utilized by the cell to
regulate m2 mAChR signaling.
We thank Dr. Geetha Kumar for work in the generation of the FLAG epitope-tagged m2 mAChR and Drs. William P. Schieman and Marc L. Rosoff for helpful discussions during the course of this study and critical reading of the manuscript.