From the Institut für Pharmakologie, Universität Gesamthochschule Essen, D-45122 Essen, Germany
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ABSTRACT |
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Sustained stimulation of muscarinic acetylcholine receptors (mAChRs) and other G protein-coupled receptors usually leads to a loss of receptor binding sites from the plasma membrane, referred to as receptor sequestration. Receptor sequestration can occur via endocytosis of clathrin-coated vesicles that bud from the plasma membrane into the cell but may also be accomplished by other, as yet ill-defined, mechanisms. Previous work has indicated that the monomeric GTPase dynamin controls the endocytosis of plasma membrane receptors via clathrin-coated vesicles. To investigate whether mAChRs sequester in a receptor subtype-specific manner via dynamin-dependent clathrin-coated vesicles, we tested the effect of overexpressing the dominant-negative dynamin mutant K44A on m1, m2, m3, and m4 mAChR sequestration in HEK-293 cells. The m1, m2, m3, and m4 mAChRs sequestered rapidly in HEK-293 cells following agonist exposure but displayed dissimilar sequestration pathways. Overexpression of dynamin K44A mutant fully blocked m1 and m3 mAChR sequestration, whereas m2 mAChR sequestration was not affected. Also, m4 mAChRs, which like m2 mAChRs preferentially couple to pertussis toxin-sensitive G proteins, sequestered in a completely dynamin-dependent manner. Following agonist removal, sequestered m1 mAChRs fully reappeared on the cell surface, whereas sequestered m2 mAChRs did not. The distinct sequestration of m2 mAChRs was also apparent in COS-7 and Chinese hamster ovary cells. We conclude that the m2 mAChR displays unique subtype-specific sequestration that distinguishes this receptor from the m1, m3, and m4 subtypes. These results are the first to demonstrate that receptor sequestration represents a new type of receptor subtype-specific regulation within the family of mAChRs.
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INTRODUCTION |
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Muscarinic acetylcholine receptors
(mAChRs)1 belong to the
superfamily of plasma membrane receptors that regulate a large number of signal transduction pathways via activation of heterotrimeric GTP-binding proteins (G proteins). The mAChR family consists of five
subtypes of cloned mAChRs and can be subdivided into two functional
groups: the m1, m3, and m5 subtypes, which preferentially couple to the
Gq family of G proteins, and the m2 and m4 subtypes, which
effectively activate the Gi family of G proteins. The m1, m2, m3, and m4 receptors are widely expressed in the central nervous system and peripheral tissues, whereas m5 receptors are present in only
minute amounts in hippocampus and other areas of the brain (1).
Prolonged exposure of mAChRs and other G protein-coupled receptors to
agonists usually results in the attenuation of the cellular response.
One molecular mechanism of attenuation involves the phosphorylation of
the receptors by G protein-coupled receptor kinases (GRKs) and
increased binding of the inhibitory protein -arrestin to the
phosphorylated receptors, thereby inhibiting the coupling with G
proteins (2). Another regulatory mechanism is the sequestration of
receptors by which G protein-coupled receptors become inaccessible for
hydrophilic membrane-impermeable ligands, including agonists. The
sequestration of receptors may represent endocytosis of receptors via
clathrin-coated vesicles (3, 4), caveolae (5, 6), or noncoated vesicles
(7), or may be associated with conformational changes of the receptor
in the plasma membrane that make the receptor inaccessible to agonists (8). For some G protein-coupled receptors like the
2-adrenergic receptors, sequestration is required to
allow resensitization of desensitized receptors in a presumably
subcellular compartment from which they can recycle back to the plasma
membrane (9, 10). For other G protein-coupled receptors, including
mAChRs and secretin receptors, receptor sequestration is a
desensitization mechanism (11-13). For example, sequestration of m4
mAChRs in CHO cells delays receptor resensitization by more than 2 h, whereas nonsequestered receptors acquire full responsiveness within
10 min after agonist removal (12).
A number of monomeric GTPases has been implicated as key regulators of
various stages of endocytosis, including the 100-kDa GTPase dynamin.
Dynamin is an essential, early acting component of the endocytic
pathway that operates via clathrin-coated vesicles. This GTPase
controls the formation of clathrin-coated vesicles by regulating the
constriction and budding of clathrin-coated pits from the plasma
membrane (14, 15). Overexpression of a dominant-negative dynamin mutant
(dynamin K44A), which displays reduced GTP binding affinity and
hydrolysis, potently inhibits the sequestration of several plasma
membrane receptors, including receptors for transferrin and epidermal
growth factor. Recently, Zhang et al. (16) showed that
functional dynamin is required for the internalization of
2-adrenergic receptors in HEK-293 cells via
clathrin-coated vesicles. In contrast, angiotensin II AT type 1A
receptors, which are structurally and functionally distinct from
2-adrenergic receptors, sequester in a
dynamin-independent manner (16). Thus, G protein-coupled receptors are
able to sequester via dynamin-dependent and
dynamin-independent sequestration pathways.
Previous radioligand binding studies have suggested that mAChRs may
sequester in a receptor subtype-specific manner. For example, whereas
both m1 and m2 mAChRs rapidly sequester in Y1 adrenal cells following
agonist exposure, only m1 mAChRs sequester upon addition of phorbol
ester (17). Likewise, in JEG-3 cells, agonist-treatment induces m2 but
not m1 mAChR sequestration (18). Similarly, immunocytochemical studies
on the localization of 2-adrenergic receptor subtypes in
HEK-293 cells have shown that following agonist binding,
2A-adrenergic receptors do not sequester, whereas
2B-adrenergic receptors are able to undergo
agonist-induced sequestration (19). Thus, within a subfamily of
structurally related G protein-coupled receptors, receptors can also
differ in their capacity to sequester. The goal of the present work was
to analyze the functional role of the GTPase dynamin in mAChR
sequestration, and to investigate whether sequestration of the m1, m2,
m3, and m4 mAChR subtypes is differentially regulated by dynamin. Here
we demonstrate that whereas m1, m3, and m4 mAChR sequestration is
regulated by dynamin, m2 mAChRs sequester in a dynamin-independent
manner. These results provide strong evidence that receptor
sequestration represents a novel type of receptor subtype-specific
regulation of mAChRs.
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EXPERIMENTAL PROCEDURES |
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Materials-- [3H]Quinuclidinyl benzilate ([3H]QNB, specific activity 43 Ci/mmol) and N-[3H]methylscopolamine ([3H]NMS, specific activity 84 Ci/mmol) were purchased from New England Nuclear. Mouse anti-dynamin monoclonal antibody and peroxidase-conjugated goat anti-mouse antibody were obtained from Transduction Laboratories and Dianova, respectively.
Plasmid Construction-- All recombinant DNA procedures were carried out following standard protocols. DNA encoding mouse m1 mAChR (20), porcine m2 mAChR (21), human m3 mAChR (22), and mouse m4 mAChR (23) were subcloned into pCD-PS expression vector. The cDNAs encoding hemagglutinin-tagged dynamin wild-type and K44A (14) were subcloned into pRK5 expression vector.
Cell Culture and Transfection--
HEK-293 tsA201 cells stably
expressing simian virus 40 large T antigen (24) and COS-7 cells
(American Type Culture Collection) were grown in Dulbecco's
modified Eagle's/F-12 medium supplemented with 10% fetal calf
serum, penicillin G (100 units/ml), and streptomycin (100 µg/ml) in
an atmosphere of 5% CO2. CHO cells (American Type Culture
Collection) were grown in medium containing 10% fetal calf serum,
penicillin G (100 units/ml), and streptomycin (100 µg/ml). Cell media
were from Life Technologies, Inc. Cells on 150-mm plates were
transfected with either 12.5 or 25 µg pCD-PS containing m1 or m2
mAChR DNA, respectively, together with either (unless indicated
otherwise) 50 µg of pRK5 dynamin wild-type, pRK5 dynamin K44A, or
empty pRK5, using the calcium phosphate method (25). Transfection
efficiency of HEK-293 tsA201 cells was determined by in situ
staining for
-galactosidase activity of the cells cotransfected with
the constitutively active pSV
-gal (Promega).
Immunoblot Analysis of Dynamin Expression-- Cells on 150-mm plates were washed twice with phosphate-buffered saline (150 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 6.5 mM Na2HPO4, pH 7.4) and lysed by the addition of 1.0 ml of lysis buffer (1% SDS, 10 mM Tris-HCl, pH 7.4). Lysate was transferred to a microcentrifuge tube and boiled for 5 min. After 5 passages through a 25-gauge needle, samples were centrifuged for 5 min to remove insoluble material and diluted with lysis buffer to an equal amount of protein as measured by the BCA method (Pierce). 100 µl of electrophoresis sample buffer (250 mM Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.006% bromphenol blue, 2% 2-mercaptoethanol) were added to 100 µl of the diluted samples and boiled for another 5 min. After SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels, protein was blotted onto nitrocellulose. Nitrocellulose was then blocked with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% bovine serum albumin (Fraction V, Sigma). After washing three times for 5 min in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, the blot was incubated with mouse anti-dynamin monoclonal antibody (diluted 1:1000; 0.25 µg/ml) in blocking buffer for 1 h. Following three washes for 5 min, the blot was incubated with peroxidase-conjugated goat anti-mouse antibody (diluted 1:5000; 0.16 µg/ml) at room temperature. After 1 h, the blot was washed again and immunoreactivity was visualized by enhanced chemiluminescence (Amersham). Densitometric analysis of the immunoblots was performed using a Shimadzu CS-9000 dual wavelength spot scanner.
mAChR Sequestration Assay--
Briefly, 24 h after
transfection, cells from 150-mm plates were replated on
poly-L-lysine-coated 24-well plates and allow to reattach
and grow for another 24 h. The cells were then incubated with or
without carbachol for 0-60 min in 25 mM HEPES-buffered Dulbecco's modified Eagles's/F-12 medium. For each manipulation, 6 wells of cells were taken. After washing with ice-cold
phosphate-buffered saline, 4 wells were incubated with 2 nM
[3H]NMS in 500 µl of ice-cold phosphate-buffered
saline, and 2 wells received atropine also (final concentration of 3 µM) to measure nonspecific binding amounting to maximally
10% of total binding. After 4 h of incubation at 4 °C, cells
were washed with ice-cold phosphate-buffered saline, solubilized in 1%
Triton X-100, scraped, and transferred into scintillation vials, which
received 3.5 ml of scintillation fluid prior to radioactivity counting.
Sequestration is expressed as (1 quotient of cell surface receptors
of carbachol-treated and untreated cells) × 100%. Total receptor
number was determined in crude cell homogenates by binding of the
membrane-permeable muscarinic radioligand [3H]QNB at
37 °C as described previously (25). Untransfected HEK-293 tsA201,
COS-7, and CHO cells do not express detectable levels of mAChR as
determined by [3H]QNB binding to total cell
homogenates.
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RESULTS |
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Effect of Overexpression of Dynamin Wild-type and K44A Mutant on m1
mAChR Sequestration--
To determine whether mAChR sequestration is
regulated in a receptor subtype-selective manner, we first examined the
effect of overexpressing dynamin wild-type and K44A mutant on m1 and m2
mAChR sequestration in HEK-293 tsA201 cells. Transfection of HEK-293
tsA201 cells with 50 µg of DNA encoding the dynamin wild-type and
K44A mutant resulted in 15-30-fold overexpression of the corresponding protein over the endogenous dynamin expression level as measured by
laser densitometry and corrected for an average transfection efficiency
of 10-20% (Fig. 1A).
Stimulation of m1 mAChR-expressing HEK-293 tsA201 cells with a
receptor-saturating concentration of 1 mM carbachol for a
period of 60 min resulted in a 47 ± 5% loss in cell surface
receptor number, as measured with the membrane-impermeable muscarinic
antagonist [3H]NMS (Fig. 1B). There was no
detectable change in total mAChR number as measured by binding of the
membrane-permeable muscarinic antagonist [3H]QNB to
homogenates prepared from transfected HEK-293 cells (data not shown).
Overexpression of dynamin K44A inhibited m1 mAChR sequestration in a
dose-dependent manner. Transfection of cells with 5 µg of
pRK5 dynamin K44A per 150-mm tissue culture plate inhibited m1 mAChR
sequestration by about 60%, whereas sequestration was blocked by about
80 and 90% following transfection with 50 and 150 µg of pRK5 dynamin
K44A DNA, respectively (Fig. 1B). In subsequent experiments,
cells were transfected with 50 µg of pRK5 dynamin wild-type or K44A
mutant per 150-mm tissue culture plate. The inhibition of receptor
sequestration was due to a decrease in the extent rather than the rate
of mAChR sequestration (Fig. 2A). Sequestration in both
control cells and cells overexpressing dynamin K44A was essentially
complete within 10-30 min of exposure to 1 mM carbachol.
Inhibition of m1 mAChR sequestration was apparent at low concentrations
of carbachol as well (Fig. 2B). Whereas incubation with
105 M carbachol reduced cell surface number
on control cells by 22 ± 3%, receptor sequestration in dynamin
K44A-overexpressing cells was only 7 ± 3%. In contrast to
dynamin K44A, overexpression of dynamin wild-type did not change the
time course of m1 mAChR sequestration or the potency of carbachol to
induce receptor sequestration, indicating that endogenous expression
levels of dynamin are not rate-limiting (Fig. 2, A and
B). Taken together, our data provide strong evidence that m1
mAChR sequestration in HEK-293 tsA201 cells is regulated by
dynamin.
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Sequestration of m2 mAChRs in HEK-293 tsA201 Cells-- We next determined whether m2 mAChR sequestration in HEK-293 cells is regulated by dynamin as well. As shown in Fig. 3A, m2 mAChRs rapidly sequestered, and cell surface receptor number was reduced by 60-80% within 10-30 min of incubation with 1 mM carbachol at 37 °C. The loss of cell surface receptor number took place without a measurable change in total receptor number as determined by [3H]QNB binding to cell homogenates (data not shown). Overexpression of dynamin wild-type or dynamin K44A did not alter m2 mAChR sequestration in HEK-293 tsA201 cells. In all three cell systems, m2 mAChRs sequestered with a similar time course, and the potency of carbachol in inducing receptor sequestration was indistinguishable (Fig. 3B). These results strongly suggest that in HEK-293 tsA201 cells m2 mAChRs sequester by a dynamin-independent pathway.
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Reappearance of Sequestered m1 and m2 mAChRs in HEK-293 tsA201 Cells-- The dissimilar sequestration characteristics of m1 and m2 mAChRs in HEK-293 tsA201 cells prompted us to investigate whether m1 and m2 mAChRs also display different reappearance kinetics following agonist-induced receptor sequestration. For this, m1 and m2 mAChR-expressing cells were incubated with 1 mM carbachol for 60 min at 37 °C, and then washed to remove ligand. As shown in Fig. 4, cell surface mAChR numbers were reduced by 48 ± 5% and 62 ± 5% in carbachol-treated m1 and m2 mAChR-expressing cells. Within 1-3 h, almost all sequestered [3H]NMS binding sites on m1 mAChR-expressing HEK-293 cells reappeared. In contrast, reappearance of sequestered [3H]NMS binding sites on m2 mAChR-expressing cells was only very small. 3 h after carbachol removal, only 10-20% of initial binding had reappeared, and in the next 3 h, there was no further increase. These data are consistent with the observation that m1 and m2 mAChRs utilize distinct pathways of sequestration in HEK-293 cells.
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Sequestration of mAChR Subtypes in HEK-293 tsA201, COS-7, and CHO Cells-- To test whether the alternative sequestration pathway of m2 mAChRs in HEK-293 cells is a property unique to m2 mAChRs, we investigated whether other members of the mAChR family share this ability. To do this, HEK-293 tsA201 cells were cotransfected with either m3 or m4 mAChR DNA along with dynamin K44A DNA. As shown in Table I, overexpression of dynamin K44A completely blocked the sequestration of m3 mAChRs in HEK-293 tsA201 cells, whereas sequestration of m4 mAChRs was inhibited by 80%. This suggests that the dynamin-independent sequestration of m2 mAChRs is not correlated with a preferential coupling profile to pertussis toxin-sensitive G proteins. This finding was confirmed by sequestration studies of m2 and m4 mAChRs in COS-7 and CHO cells. In COS-7 cells, overexpression of dynamin K44A inhibited m2 mAChR sequestration by 55%, whereas m4 mAChR sequestration was completely abolished. Similarly, sequestration of m4 mAChRs in CHO cells was blocked by 80-90% by overexpression of dynamin K44A, whereas m2 mAChR sequestration was not altered at all.
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DISCUSSION |
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In view of the pivotal role of dynamin in regulating clathrin-coated vesicle endocytosis, we investigated whether dynamin is involved in mAChR sequestration, and in particular, whether the sequestration of the mAChR subtypes m1, m2, m3, and m4 is regulated differentially by this GTPase. Our study is the first systematic analysis of the regulation of receptor sequestration by dynamin within a particular family of G protein-coupled receptors. Whereas all four mAChR subtypes in HEK-293 cells sequestered rapidly and to a large extent, m2 mAChR sequestration was distinct from that of the other mAChR subtypes. Overexpression of the dominant-negative dynamin K44A mutant fully blocked m1, m3, and m4 mAChR sequestration, whereas m2 mAChR sequestration was not affected. The distinct sequestration of m2 mAChRs in HEK-293 cells was underscored by the finding that m2 mAChR sequestration was hardly reversible following agonist removal, whereas sequestered m1 mAChRs reappeared almost completely within 1-3 h. The dissimilarity in sequestration between m1 and m2 mAChRs was not related to their differential coupling properties. Also, the m4 mAChR subtype, which like the m2 mAChR subtype preferentially couples to pertussis toxin-sensitive G proteins, sequestered essentially in a dynamin-dependent manner in HEK-293 cells. The distinct sequestration of m2 mAChR was not confined to HEK-293 cells but also apparent in transiently transfected COS-7 and CHO cells. Overexpression of dynamin K44A in COS-7 cells blocked m4 mAChR sequestration completely, whereas m2 mAChR sequestration was inhibited by 55%. Similarly, while sequestration of m4 mAChRs in CHO cells could essentially be fully blocked by overexpressing dynamin K44A, m2 mAChR sequestration was completely dynamin-independent. During the processing of this manuscript, Hosey and co-workers (26) also showed that m2 mAChRs sequester in a dynamin-independent manner in HEK-293 cells. However, these investigators did not investigate the seminal question whether the other mAChR subtypes also utilize the dynamin-independent sequestration pathway.
Of particular interest was the observation that although sequestration
of m2 mAChRs in HEK-293 cells was fully dynamin-independent, m2 mAChR
sequestration in COS-7 cells was accomplished via both dynamin-dependent and dynamin-independent pathways. These
findings may offer a molecular explanation for two contradictory
studies on the regulation of m2 mAChR sequestration by GRK2 in COS-7
and HEK-293 cells (27, 28). In COS-7 cells, overexpression of a
dominant-negative mutant of GRK2 was found to block phosphorylation and
sequestration by about 50%, whereas in HEK-293 cells, overexpression inhibited m2 mAChR phosphorylation but not mAChR sequestration. On the
basis of our study, it is therefore possible that these findings are
related to the dissimilar sequestration pathways of m2 mAChRs in
HEK-293 and COS-7 cells. Benovic and co-workers (29) and Caron and
co-workers (16) have recently proposed the following early steps in the
sequestration of 2-adrenergic receptors, which like m1
mAChRs sequester via dynamin-dependent clathrin-coated
vesicles. Following agonist stimulation, receptors are phosphorylated
by GRKs in an agonist-dependent manner and the subsequent
binding of
-arrestin to the phosphorylated receptor uncouples the
receptor from its G protein. In turn, receptor-bound
-arrestin may
function as a high affinity clathrin adaptor, and on encountering a
coated pit,
-arrestin binds with high affinity to the assembled
clathrin lattice, resulting in receptor internalization. Although this
model is very attractive, it fails to explain why m2 mAChRs in HEK-293
cells do not sequester via dynamin-dependent clathrin-coated vesicles, yet are being phosphorylated by GRKs in
HEK-293 cells (28) and are able to bind
-arrestin efficiently (30).
These studies lead us to conclude that GRK2-mediated phosphorylation of
m2 mAChRs does not dictate whether mAChRs sequester via either dynamin-dependent or dynamin-independent pathways.
At present, the identity of the m2 mAChR sequestration pathway is
unclear. A possible m2 mAChR sequestration pathway may be the
endocytosis via caveolae. Caveolae are noncoated plasma membrane invaginations, which seem to have the ability to either internalize into the cell interior or to persist in the proximity of the plasma membrane (31). Previous data have suggested that cholecystokinin receptors in CHO cells as well as 2-adrenergic receptors
in A431 cells may sequester via caveolae (5, 6). Very recently, Feron
et al. (32) reported that m2 mAChRs in cardiac myocytes are
targeted to caveolae following agonist binding. This observation led
the authors to postulate that this receptor trafficking is required to
cluster m2 mAChRs within the caveolae to initiate specific downstream
signaling cascades, including activation of the resident caveolar
isoform of nitric oxide synthase. Our study suggests that in the
caveolae of cardiac myocytes, agonist-activated m2 mAChRs evidently
also sequester and lose their ability to bind hydrophilic ligands.
Further studies should be performed to test whether m2 mAChRs indeed
sequester into caveolae in HEK-293 cells and cardiac myocytes.
Our study is the first demonstration of a receptor subtype-specific sequestration pathway within the family of mAChRs. In this context, it is interesting to note that, since receptor sequestration and recycling regulate receptor function (9-13), the m2 subtype-specific sequestration (and recycling) pathway may serve a regulatory mechanism of receptor responsiveness, distinct from that of the m1, m3, and m4 subtypes. At present, it is unknown whether activated m2 mAChRs are excluded from the dynamin-dependent clathrin-coated vesicle sequestration pathway, or are "specifically" recognized by a repertoire of as yet unidentified regulatory elements, which direct the activated m2 mAChRs into the dynamin-independent sequestration pathway. The observation that the m4 subtype, which has a striking homology in amino acid sequence and functional characteristics with the m2 subtype (1), as well as the less homologous m1 and m3 subtypes, sequester via the dynamin-dependent pathway, lends support to the latter possibility. The characterization of the molecular mechanisms that govern the distinct dynamin-independent m2 mAChR sequestration pathway in HEK-293 and other cells will provide new insights into the regulation of G protein-coupled receptors.
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ACKNOWLEDGEMENTS |
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We thank Riccarda Krudewig, Barbara Langer, and Nicole Markschies for expert technical assistance. We are indebted to Dr. S. Schmid for the gift of the human dynamin wild-type and dynamin K44A cDNA, Dr. A. Ullrich for pRK5, Dr. T. Bonner for pCD-PS and the human m3 mAChR cDNA, Dr. N. M. Nathanson for the mouse m1 mAChR DNA, Dr. D. J. Capon for the porcine m2 mAChR cDNA, and Dr. M. M. Hosey for providing HEK-293 tsA201 cells.
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FOOTNOTES |
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* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and the IFORES program of the Universitätsklinikum Essen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a fellowship of the Heinrich Hertz-Stiftung.
§ To whom correspondence should be addressed. Tel.: 49-201-723-3462; Fax: 49-201-723-5968; E-mail: van_koppen{at}uni-essen.de.
1 The abbreviations used are: mAChR, muscarinic acetylcholine receptor; G protein, guanine nucleotide-binding protein; GRK, G protein-coupled receptor kinase; NMS, N-methylscopolamine; QNB, quinuclidinylbenzilate; CHO, Chinese hamster ovary.
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REFERENCES |
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