Regulation of Muscarinic Acetylcholine Receptor Sequestration and
Function by
-Arrestin*
Oliver
Vögler,
Bettina
Nolte,
Matthias
Voss,
Martina
Schmidt,
Karl H.
Jakobs, and
Chris J.
van Koppen
From the Institut für Pharmakologie,
Universitätsklinikum Essen, D-45122 Essen, Germany
 |
ABSTRACT |
After activation, agonist-occupied G
protein-coupled receptors are phosphorylated by G protein-coupled
receptor kinases and bind cytosolic
-arrestins, which uncouple the
receptors from their cognate G proteins. Recent studies on the
2-adrenergic receptor have demonstrated that
-arrestin also targets the receptors to clathrin-coated pits for
subsequent internalization and activation of mitogen-activated protein
kinases. We and others have previously shown that muscarinic
acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require
functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2
mAChRs sequester in a dynamin-independent manner. To investigate the
role of
-arrestin in mAChR sequestration, we determined the effect
of overexpressing
-arrestin-1 and the dominant-negative inhibitor of
-arrestin-mediated receptor sequestration,
-arrestin-1 V53D, on
mAChR sequestration and function. Sequestration of m1, m3, and m4
mAChRs was suppressed by 60-75% in cells overexpressing
-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by
less than 10%. In addition, overexpression of
-arrestin-1 V53D as
well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we
investigated whether mAChRs sequester into clathrin-coated vesicles by
overexpressing Hub, a dominant-negative clathrin mutant. Although
sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2
mAChR sequestration was suppressed by less than 10%. We conclude that
m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into
clathrin-coated vesicles in a
-arrestin- and
dynamin-dependent manner, whereas sequestration of m2
mAChRs in these cells is largely independent of these proteins.
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INTRODUCTION |
Exposure of many G protein-coupled receptors
(GPCRs)1 to their agonists
results within seconds to minutes in attenuation of receptor
responsiveness. An important step in this process of receptor
desensitization is the rapid phosphorylation of agonist-bound receptors
by G protein-coupled receptor kinases (1). These kinases phosphorylate
serine and threonine residues located in the third cytoplasmic loop
(i.e. m1 and m2 muscarinic acetylcholine receptors (mAChRs))
(2, 3) or in the cytoplasmic carboxyl-terminal tail of the receptors
(for example,
2-adrenergic receptors) (4). After
phosphorylation, cytosolic
-arrestins bind with increased affinity
to the receptors and sterically inhibit further coupling of the
receptors with G proteins (1). To date, two
-arrestin isoforms,
-arrestin-1 (arrestin 2) and
-arrestin-2 (arrestin 3) have been
identified, each undergoing alternative splicing (1). Both isoforms are
ubiquitously expressed, with
-arrestin-1 being the major
-arrestin expressed in many tissues (1). Recent evidence indicates
that
-arrestins do not only bind to GPCRs but also associate with
nanomolar affinity with clathrin heavy chains and target
-arrestin-bound GPCRs to the clathrin-coated pits, leading to
receptor internalization (5, 6). This process has been particularly
well studied for the
2-adrenergic receptors. Overexpression of
-arrestin-1 or
-arrestin-2 in the presence of
sufficient G protein-coupled receptor kinases augments internalization of
2-adrenergic receptors, whereas overexpression of the
dominant-negative
-arrestin-1 V53D mutant, which binds with high
affinity to clathrin cages but is significantly impaired in its ability
to interact with
2-adrenergic receptors, suppresses
2-adrenergic receptor internalization (5, 7-9).
The budding of clathrin-coated vesicles from the plasma membrane into
the cytosol is catalyzed by the monomeric G protein dynamin. This
protein oligomerizes at the neck of the invaginated clathrin-coated
pits and pinches off the pits from the plasma membrane (10).
Overexpression of the dominant-negative dynamin mutant K44A, which is
not able to bind guanine nucleotides, effectively blocks
2-adrenergic receptor internalization, indicating that
2-adrenergic receptors sequester into clathrin-coated
vesicles in an arrestin- and dynamin-dependent manner (8).
The primary function of internalization of the
2-adrenergic receptors is to allow resensitization of
desensitized receptors in endosomes before their return to the plasma
membrane (11, 12). Interestingly, receptor internalization via
clathrin-coated vesicles has recently been reported to be essential for
2-adrenergic receptor-induced activation of the
mitogen-activated protein (MAP) kinase pathway (13).
The mAChRs have been subject of a large number of studies on the
regulation of GPCRs by G protein-coupled receptor kinases and
-arrestins as well (1). In contrast to
2-adrenergic
receptors, which couple predominantly to G proteins of the
Gs family, mAChRs efficiently activate G proteins of the
Gi and Gq family. The family of mAChRs consists
of five mammalian subtypes, with m1, m3, and m5 mAChRs predominantly
activating phospholipase C via Gq proteins and m2 and m4
mAChRs efficiently inhibiting adenylyl cyclase by activation of
Gi proteins. We and others have recently shown that the
monomeric GTPase dynamin is essential for internalization of m1, m3,
and m4 mAChRs in HEK-293 cells, whereas internalization of the m2
mAChRs is dynamin-independent (14, 15). These results indicate that m1,
m3, and m4 mAChRs sequester by a dynamin-dependent trafficking pathway, probably similar as used by
2-adrenergic receptors. Previous studies have shown that
-arrestins can interact with peptide sequences derived from the
third intracellular loop of the m2 and m3 mAChRs in vitro
(16, 17). By analogy on the regulation of internalization of
2-adrenergic receptors, we hypothesized that
-arrestins are essential for the internalization of mAChR subtypes
as well. In this study, we overexpressed the dominant-negative
-arrestin-1 mutant V53D in HEK-293 tsA201 cells and examined the
effect on m1, m2, m3, and m4 mAChR sequestration. In addition, we
investigated, using
-arrestin-1 V53D and dynamin K44A as inhibitors of clathrin-mediated endocytosis, whether receptor sequestration is
required for m1 mAChR-mediated MAP kinase activation.
 |
EXPERIMENTAL PROCEDURES |
Materials--
N-[3H]Methylscopolamine
([3H]NMS, specific activity 84 Ci/mmol) was purchased
from NEN Life Science Products. DNA encoding mouse m1 mAChR (18),
porcine m2 mAChR (19), human m3 mAChR (20), and mouse m4 mAChR (21)
were subcloned into pCD-PS expression vector. The cDNAs encoding
bovine
-arrestin-1 wild type in pBC (22) and rat
-arrestin-1 V53D
in pcDNA-1 Amp (5) were generously provided by Drs. M. J. Lohse and R. J. Lefkowitz, respectively. (Bovine)
-arrestin-1
(319-418) was generated by polymerase chain reaction amplification
using the sense and antisense primers as described by Krupnick et
al. (23). The PCR product was purified, digested with
HindIII and BamHI, purified, and then ligated
into HindIII-BamHI-cut pcDNA3 (Invitrogen).
The authenticity of the mutant was confirmed by DNA sequencing of both
strands (Sequence Laboratories Göttingen, Germany). The cDNA
encoding the T7 epitope-tagged Hub fragment in pCDM8 (24) was provided
by Dr. F. M. Brodsky. Mouse anti-arrestin monoclonal antibody F4C1
was a gift of Dr. L. A. Donoso (Wills Eye Hospital, Philadelphia,
PA). Rabbit anti-ERK1 antibody (C-16), mouse anti-T7 Tag antibody, and
rabbit anti-phosphospecific p44/p42 MAP kinase antibody were purchased
from Santa Cruz Biotechnology, Novagen, and New England Biolabs,
respectively. The goat peroxidase-conjugated anti-mouse and goat
peroxidase-conjugated anti-rabbit antibodies were obtained from Dianova
(Hamburg) and Sigma, respectively.
Cell Culture and Transfection--
HEK-293 tsA201 cells (25)
were grown in Dulbecco's modified Eagle's medium/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.
Cell media were from Life Technologies, Inc. Cells on 150-mm plates
were transfected with either 12.5 µg (m1, m3) or 25 µg (m2, m4) of
pCD-PS containing mAChR DNA, together with 15 µg of
pBC/
-arrestin-1, pcDNA-1 Amp/
-arrestin-1 V53D,
pcDNA3/
-arrestin-1 (319-418), or control vector (pRK5) using
the calcium phosphate method. In some experiments, cells were
transfected with 150 µg instead of 25 µg of pCD-PS/m2 mAChR to
increase m2 mAChR expression from ~200 to ~800 fmol/mg of protein.
Immunoblot Analysis of
-Arrestin and Hub
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). One hundred 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 10% 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-arrestin monoclonal antibody (diluted 1: 2000) or mouse anti-T7
Tag monoclonal antibody (0.1 µg/ml) in blocking buffer for 1 h.
After three washes for 5 min, the blot was incubated with
peroxidase-conjugated goat anti-mouse antibody (0.16 µg/ml) at room
temperature. After 1 h, the blot was washed again, and
immunoreactivity was visualized by enhanced chemiluminescence (Amersham
Pharmacia Biotech).
MAP Kinase Assay--
Fourty-eight h after transfection, HEK-293
tsA201 cells on 100-mm plates were serum-starved overnight in
Dulbecco's modified Eagle's medium/F12 medium before stimulation with
10 µM carbachol or 1 µM phorbol
12-myristate 13-acetate. After stimulation for 5 min at 37 °C, cells
were lysed in 0.5 ml of lysis buffer and processed as described above.
After SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide
gels, phosphorylated MAP kinases on nitrocellulose filters were
detected using a rabbit anti-phosphospecific MAP kinase antibody
(diluted 1: 1000) and goat peroxidase-conjugated anti-rabbit
antibody (diluted 1:5000). Expression of the total amount of MAP
kinases was detected by incubation of nitrocellulose blots with
rabbit anti-ERK1 antibody (0.1 µg/ml), which recognizes p44 and p42
MAP kinases, and goat peroxidase-conjugated anti-rabbit antibody
(diluted 1:5000). Immunoreactivity was visualized by enhanced chemiluminescence.
mAChR Sequestration Assay--
As described before (14), 24 h after transfection, cells from 150-mm plates were replated on
poly-L-lysine-coated 24-well plates and allowed to reattach
and grow for another 24 h. The cells were then incubated with and
without carbachol for 0-60 min in 25 mM HEPES-buffered
Dulbecco's modified Eagle's medium/F-12 medium. For each
manipulation, 6 wells of cells were taken. After washing with ice-cold
phosphate-buffered saline, cells were incubated with 2 nM
[3H]NMS in 500 µl of ice-cold phosphate-buffered saline
with and without 30 µM atropine to measure total and
nonspecific binding, respectively. 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
before radioactivity counting. Sequestration is expressed as (1
quotient of cell surface receptors of carbachol-treated and untreated
cells) × 100%. Untransfected HEK-293 tsA201 do not express detectable
levels of mAChR.
 |
RESULTS |
Effect of
-Arrestin-1 on m1 mAChR Sequestration--
Western
blot analysis demonstrated equal overexpression of
-arrestin-1 and
-arrestin-1 V53D in HEK-293 tsA201 cells transiently transfected
with the expression vector encoding either
-arrestin (Fig.
1A). In the absence of
overexpressed
-arrestins, agonist stimulation led to significant
internalization of m1 mAChRs (Fig. 1B). A 10-min incubation
with 1 mM carbachol reduced cell surface receptor number by
21 ± 4%, with 35 ± 2 and 38 ± 3% of receptors internalized after 30 min and 60 min of incubation, respectively. Overexpression of
-arrestin-1 modestly increased the extent of m1
mAChR sequestration. After 10 min of incubation with 1 mM
carbachol, m1 mAChRs were sequestered by 27 ± 4%, and after 60 min of incubation, by 45 ± 3%. An increase in m1 mAChR
sequestration of similar magnitude was observed at a lower carbachol
concentration of 10 µM carbachol (Fig. 1C).
Overexpression of
-arrestin-1, however, did not appear to decrease
the EC50 value of carbachol for inducing m1 mAChR internalization. In contrast to wild-type
-arrestin-1,
-arrestin-1 V53D significantly suppressed m1 mAChR sequestration.
Receptor sequestration in
-arrestin-1 V53D-overexpressing cells was
only 19 ± 3% after 60 min of incubation with 1 mM
carbachol. Furthermore, inhibition of m1 mAChR sequestration was
evident at lower concentrations of carbachol as well. Incubation of
control cells with 10 µM carbachol for 60 min led to a
23 ± 3% loss of cell surface receptor number, whereas receptor
sequestration in
-arrestin-1 V53D-overexpressing cells was only
6 ± 2%.

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Fig. 1.
Overexpression of
-arrestin-1 wild-type and V53D mutant in HEK-293
tsA201 cells. Effects on m1 mAChR sequestration. A,
detection of -arrestins in total lysates of HEK-293 tsA201 cells
grown on 150-mm plates and transiently transfected with pRK5
(pRK5), pBC/ -arrestin-1 (WT), or pcDNA-1
Amp/ -arrestin-1 V53D (V53D) by immunoblotting. Equal
amounts of cell lysates (15 µg of protein/lane) were
subjected to SDS-polyacrylamide gel electrophoresis, and -arrestins
were detected using the anti-arrestin monoclonal antibody F4C1.
B, and C, HEK-293 tsA201 cells transiently
transfected with pCD-PS/m1 mAChR together with pBC/ -arrestin-1
(WT), pcDNA-1 Amp/ -arrestin-1 V53D (V53D),
or empty pRK5 (pRK5) were incubated in the absence and
presence of 1 mM carbachol for the indicated periods of
time (B) or with the indicated concentrations of carbachol
for 60 min (C) at 37 °C. Sequestration was assessed by
[3H]NMS binding to intact cells at 4 °C. Data are the
mean ±S.E. of six sets of experiments each. Specific
[3H]NMS binding to untreated cells transfected with pRK5,
pBC/ -arrestin-1, and pcDNA-1 Amp/ -arrestin-1 V53D was
290 ± 35, 229 ± 44, and 226 ± 28 fmol/mg of protein,
respectively.
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Role of Receptor Internalization in m1 mAChR-induced MAP Kinase
Stimulation--
Daaka et al. (13) recently demonstrated
that overexpression of
-arrestin-1 V53D or dynamin K44A in HEK-293
cells inhibits activation of MAP kinases by
2-adrenergic
receptors and lysophosphatidic acid receptors, suggesting that receptor
internalization into clathrin-coated vesicles is required for
receptor-mediated activation of MAP kinase. As m1 mAChRs appear to
internalize by the same
-arrestin- and dynamin-dependent
internalization pathway as utilized by
2-adrenergic
receptors, we examined the effect of overexpressing
-arrestin-1 V53D
and dynamin K44A on m1 mAChR-mediated activation of MAP kinase. As
shown in Fig. 2, pCD-PS/m1
mAChR-transfected cells showed a significant increase in phosphorylated
p42 and p44 MAP kinases in response to stimulation with 10 µM carbachol for 5 min (left panel).
Co-expression of
-arrestin-1 V53D (middle panel) or
dynamin K44A (right panel) significantly reduced
carbachol-induced phosphorylation of the MAP kinases. In accordance
with the study of Daaka et al. (13), overexpression of
-arrestin-1 V53D or dynamin K44A did not alter phorbol 12-myristate
13-acetate-induced MAP kinase stimulation.

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Fig. 2.
Effects of
-arrestin-1 V53D and dynamin K44A on m1
mAChR-mediated activation of MAP kinases. HEK-293 tsA201 cells
transiently transfected with pCD-PS/m1 mAChR together with either empty
pRK5 (left panel), pcDNA-1 Amp/ -arrestin-1 V53D
(middle panel), or pRK5/dynamin K44A (right
panel) were serum-starved overnight before a 5-min incubation with
10 µM carbachol (CARB) or 1 µM
phorbol 12-myristate 13-acetate (PMA) at 37 °C in 25 mM HEPES-buffered Dulbecco's modified Eagle's medium/F12
medium. Cells were lysed, and cellular protein (75 µg of
protein/lane) was subjected to SDS-polyacrylamide gel
electrophoresis and blotted onto nitrocellulose. Phosphorylation of MAP
kinases was determined by immunoblotting with a phosphospecific MAP
kinase antibody. Representative Western blots of 3 (K44A) or 6 (V53D)
sets of independent experiments are shown. Expression of m1 mAChRs in
untreated cells transfected with pRK5, pcDNA-1 Amp/ -arrestin-1
V53D, and pRK5/dynamin K44A was 275 ± 50, 244 ± 52, and
559 ± 62 fmol/mg of protein, respectively. Treatment of HEK-293
tsA201 cells, which were transfected with empty pRK5 only, did not show
phosphorylation of MAP kinase in response to 1 mM carbachol
(n = 3 independent experiments). NS,
nonstimulated.
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Effect of
-Arrestin-1 on m2, m3, and m4 mAChR
Sequestration--
As shown in Fig. 3,
overexpression of
-arrestin-1 V53D only marginally inhibited m2
mAChR sequestration, whereas m2 mAChR sequestration was only modestly
stimulated by overexpression of wild-type
-arrestin-1. Comparison of
the extent of receptor sequestration after 60 min of incubation with 10 µM and 0.1 µM carbachol showed that
overexpression of either
-arrestin was without any significant effect on m2 mAChR sequestration at lower carbachol concentrations either (results not shown). However, under conditions of higher m2
mAChR expression (i.e. 737 ± 79 versus
112 ± 22 fmol/mg of protein), overexpression of wild-type
-arrestin-1 significantly augmented m2 mAChR internalization. After
60 min of incubation with 10 µM and 1 mM
carbachol, m2 mAChR internalization in control cells was 24 ± 9 and 22 ± 8%, whereas in
-arrestin-1-transfected cells, m2
mAChR internalized by 43 ± 9 and 51 ± 3%, respectively (mean ±S.E., n = 6-9 independent experiments).

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Fig. 3.
Effects of
-arrestin-1 wild type and V53D mutant on
sequestration of m2 mAChRs in HEK-293 tsA201 cells. HEK-293 tsA201
cells transiently transfected with pCD-PS/m2 mAChR together with
pBC/ -arrestin-1 (WT), pcDNA-1 Amp/ -arrestin-1 V53D
(V53D), or empty pRK5 (pRK5) were incubated in
the absence and presence of 1 mM carbachol for the
indicated periods of time at 37 °C. Sequestration was assessed by
[3H]NMS binding to intact cells. Data are the mean ±S.E.
from six sets of experiments. Specific [3H]NMS binding to
untreated cells transfected with pRK5, pBC/ -arrestin-1, and
pcDNA-1 Amp/ -arrestin-1 V53D was 252 ± 60, 112 ± 22, and 183 ± 68 fmol/mg protein, respectively.
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In contrast to the m2 mAChRs, sequestration of m3 and m4 mAChRs, which
like m1 mAChRs sequester in a dynamin-dependent manner (14,
15), was
-arrestin-dependent. As depicted in Fig.
4, overexpression of
-arrestin-1 V53D
inhibited m3 and m4 mAChR sequestration by 68 and 64%, respectively,
after 60 min of incubation with 1 mM carbachol.
Overexpression of wild-type
-arrestin-1 had no (significant)
stimulatory effect on the maximal extent of sequestration of either
receptor subtype. In addition to investigating the effect of
overexpressing
-arrestin-1 V53D, we also examined the influence of
another dominant-negative inhibitor of
-arrestin-mediated receptor
sequestration,
-arrestin-1 (319-418). Overexpression of
-arrestin-1 (319-418) reduced sequestration of m1, m3, and m4
mAChRs from 43 ± 4, 40 ± 2, and 47 ± 3% to 6 ± 4, 13 ± 2, and 12 ± 2%, respectively, following 60 min of
incubation with 1 mM carbachol (mean ±S.E.,
n = 6-8 independent experiments).

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Fig. 4.
Effects of
-arrestin-1 wild-type and V53D mutant on
sequestration of m3 and m4 mAChRs in HEK-293 tsA201 cells. HEK-293
tsA201 cells transiently transfected with pCD-PS/m3 or pCD-PS/m4 mAChR
together with pBC/ -arrestin-1 (WT), pcDNA-1
Amp/ -arrestin-1 V53D (V53D), or empty pRK5
(pRK5) were incubated with 1 mM carbachol for 60 min at 37 °C. Sequestration was assessed by [3H]NMS
binding to intact cells. Data are the mean ±S.E. from five sets of
experiments each. Expression of m3 mAChRs in cells transfected with
pRK5, pBC/ -arrestin-1, and pcDNA-1 Amp/ -arrestin-1 V53D was
234 ± 75, 308 ± 69, and 186 ± 57 fmol/mg of protein,
respectively. Expression of m4 mAChRs in cells transfected with pRK5,
pBC/ -arrestin-1, and pcDNA-1 Amp/ -arrestin-1 V53D was
346 ± 93, 242 ± 63, and 154 ± 60 fmol/mg of protein,
respectively.
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Role of Clathrin in mAChR Sequestration--
Two recent studies
have indicated that dynamin not only catalyzes the budding of
clathrin-coated vesicles but of caveolae as well (26, 27). To directly
test whether m1, m3, and m4 mAChRs sequester into clathrin-coated
vesicles, we took advantage of the recent availability of a
dominant-negative form of clathrin, termed Hub (24). Hub comprises the
carboxyl-terminal third of the clathrin heavy chain (residues
1073-1675) and specifically blocks clathrin-mediated endocytosis by
depletion of clathrin light chains, causing clathrin-coated pits to be
frozen at the plasma membrane. Transfection of HEK-293 tsA201 cells
with pCDM8 containing T7 epitope-tagged Hub led to a large expression
of Hub (Fig. 5A). Expression
of Hub caused a 50-70% inhibition of m1, m3, and m4 mAChR
sequestration. In contrast, sequestration of m2 mAChRs was not affected
(Fig. 5B).

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Fig. 5.
Effect of Hub on sequestration of mAChR
subtypes in HEK-293 tsA201 cells. A, detection of T7
Hub in total lysates of HEK-293 tsA201 cells transiently transfected
with pCDM8-T7 Hub (Hub) or empty pRK5 (pRK5) by
immunoblotting with a T7 epitope-tag-specific monoclonal antibody (75 µg of protein/lane). B, HEK-293 tsA201 cells
transiently transfected with pCD-PS containing mAChR DNA together with
pCDM8-T7 Hub or empty pRK5 were incubated for 60 min with 1 mM carbachol. Specific [3H]NMS binding to
intact untreated pRK5-transfected cells expressing m1, m2, m3, and m4
mAChRs was 268 ± 78, 169 ± 34, 262 ± 34, and 341 ± 31 fmol/mg of protein, respectively. Specific [3H]NMS
binding to intact untreated T7 Hub-transfected cells expressing m1, m2,
m3, and m4 mAChRs was 342 ± 115, 130 ± 54, 253 ± 76, and 516 ± 63 fmol/mg of protein, respectively. Data are the mean
±S.E. of 3-6 independent experiments.
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 |
DISCUSSION |
In the present study, we determined the role of
-arrestin in
mAChR sequestration and function using
-arrestin-1 wild type and the
dominant-negative inhibitor of
-arrestin-mediated receptor sequestration,
-arrestin-1 V53D. This
-arrestin-1 mutant was chosen for its increased ability to interact with clathrin and its
impaired capacity to bind to agonist-bound phosphorylated GPCRs (9,
23).
Overexpression of
-arrestin-1 V53D suppressed sequestration of m1,
m3, and m4 mAChRs in HEK-293 tsA201 cells by 60-75%, indicating that
these mAChR subtypes sequester predominantly in a
-arrestin-dependent manner. On the other hand,
overexpression of
-arrestin-1 wild type only slightly stimulated m1,
m3, and m4 mAChR internalization. The small magnitude of stimulation
may be related to the possibility that other downstream partners
involved in receptor internalization are rate-limiting, so additional
-arrestin is hardly able to promote receptor internalization
further. In line with our observation that m1, m3, and m4 mAChR
sequester in a
-arrestin-dependent manner,
overexpression of Hub, a dominant-negative clathrin mutant (24),
significantly blocked sequestration of m1, m3, and m4 mAChRs. These
data lend strong support to the hypothesis that m1, m3, and m4 mAChRs
utilize the same sequestration pathway as
2-adrenergic
receptors in HEK-293 cells. Our findings are consistent with
immunocytochemical and biochemical studies on the internalization of
m1, m3, and m4 mAChRs in a number of cells including HEK-293 cells. In
these studies, internalized m1 mAChRs were found to colocalize with
clathrin (28), or perturbation of clathrin distribution inhibited m3
and m4 mAChR internalization (29, 30). In contrast, m2 mAChR
sequestration was hardly affected by overexpression of wild-type
-arrestin-1,
-arrestin-1 V53D, or Hub under conditions of low m2
mAChR expression levels (i.e. 0.1-0.2 pmol/mg of protein). At higher levels of receptor expression (i.e. ~0.75
pmol/mg of protein), overexpression of wild-type
-arrestin-1
strongly stimulated m2 mAChR sequestration in HEK-293 tsA201 cells, in
accordance with a previous study by Pals-Rylaarsdam et al.
(16). These results suggest that at low m2 mAChR levels, the
endogeneous internalization components are in excess over the number of
m2 mAChRs, and the receptors sequester via the
-arrestin- and
dynamin-independent pathway. At higher levels of receptor expression,
the capacity of this internalization pathway becomes saturated and its
components become rate-limiting, so overexpressed
-arrestin now
supports sequestration of m2 mAChRs by the other, less efficient
-arrestin- and dynamin-dependent pathway in HEK-293
tsA201 cells (16).
During the course of this study, Lee et al. (15) reported
that overexpression of another
-arrestin-1 mutant, termed arrestin 2- (319-418), did not lead to inhibition of m1, m3, and m4 mAChR internalization in HEK-293 tsA201 cells, whereas internalization of
2-adrenergic receptors was significantly suppressed.
This
-arrestin mutant, which encodes the last 100 amino acids of
-arrestin-1 as the major clathrin binding determinants, binds weakly
to phosphorylated agonist-activated GPCRs and blocks internalization
via clathrin-coated vesicles as well (6). In our study, however,
-arrestin-1 (319-418) significantly blocked sequestration of m1,
m3, and m4 mAChRs. A possible explanation for this discordance may be
that in the aforementioned study, expression of
-arrestin-1
(319-418) was insufficient to block interaction of mAChR-bound
-arrestin-1 to clathrin, whereas
2-adrenergic
receptor internalization was effectively inhibited. In this respect, it
is important to note that the degree of competition between
-arrestin-1 and
-arrestin-1 (319-418) for binding clathrin is
related to the difference in binding affinity of the
-arrestins for
clathrin. Because the G protein-coupled receptor kinase phosphorylation
sites on the mAChRs and
2-adrenergic receptor are
located differently (2-4), the binding affinity of
-arrestin for
clathrin may in part be determined by the receptor species as well. In
any event, our study definitively renews interest in the role of
-arrestin in mAChR internalization, which was called into question
by the report of Lee et al. (15).
In the present study, we observed that overexpression of
-arrestin-1
V53D and dynamin K44A blocks m1 mAChR-mediated activation of MAP kinase
in HEK-293 cells. These results further corroborate the idea that m1,
m3, and m4 mAChRs in HEK-293 cells sequester by the same
clathrin-mediated sequestration pathway as is used by
2-adrenergic receptors (1). It has been proposed that
the agonist-occupied,
-arrestin-bound GPCR is actually part of a multisignaling complex assembled at the plasma membrane, which includes
not only the receptor but various intermediates including active Raf
kinase and which is internalized by the clathrin-coated vesicle pathway
to activate cytosolic MAP kinase (13). Thus, receptor sequestration and
recycling is not only required to regulate mAChR responsiveness
(30-32) but also, at least in the case of m1 mAChRs, for activation of
the MAP kinase cascade in HEK-293 cells.
In summary, the present study demonstrates an important role for
-arrestin (or a
-arrestin-like protein) in the internalization of
m1, m3, and m4 mAChRs in HEK-293 tsA201 cells. The lack of effect of
-arrestin V53D overexpression on the internalization of m2 mAChRs
suggests that desensitization of m2 mAChRs can be
-arrestin-independent as well. Wu et al. (17) recently
showed that a peptide sequence derived from the third cytoplasmic loop of m2 mAChRs and containing the G protein-coupled receptor kinase phosphorylation sites and a putative
-arrestin binding site (16) does not bind
-arrestins derived from an enriched brain cytosol fraction, whereas a peptide sequence from the third cytoplasmic loop of
m3 mAChRs is able to do so (17). As the m2 receptor peptide sequence
was able to bind to purified
-arrestins, perhaps there are other
cytosolic proteins that preferentially bind to the m2 mAChR and
effectively compete with
-arrestin. Like the
-arrestins,
association of these unidentified proteins to the m2 mAChR might
uncouple the receptor from its cognate G proteins and target the m2
mAChR to the clathrin-independent internalization pathway. It is,
however, noteworthy, that m2 mAChRs (and the other mAChR subtypes
likely as well) can use alternative sequestration pathways, dependent
on the cell species involved (14, 33-35). This underscores the
plasticity of the molecular mechanisms of receptor trafficking within
the family of GPCRs.
 |
ACKNOWLEDGEMENTS |
We thank Riccarda Krudewig and Barbara Langer
for expert technical assistance. We are indebted to Drs. M. J. Lohse, R. J. Lefkowitz, and F. M. Brodsky for the gift of the
various DNA plasmids and to Dr. L. A. Donoso for providing mouse
anti-arrestin antibody F4C1.
 |
FOOTNOTES |
*
This work was supported by a grant of 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.
To whom correspondence should be addressed. Tel.: 49-201-723 3462;
Fax: 49-201-723 5968; E-mail: van_koppen{at}uni-essen.de.
 |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G
protein-coupled receptor;
mAChR, muscarinic acetylcholine receptor;
MAP, mitogen-activated protein;
NMS, N-methylscopolamine.
 |
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