From the Department of Neurochemistry, Faculty of
Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan, the § Department of Occupational Diseases, National
Institute of Industrial Health, 6-21-1 Nagao, Tama-ku, Kawasaki,
Kanagawa, 214, Japan, and the
School of Pharmacy, University of
California, San Francisco, California 94143-0446
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ABSTRACT |
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Internalization and down-regulation
of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a
hm2 receptor mutant lacking a central part of the third intracellular
loop (I3-del m2 receptor) were examined in Chinese hamster ovary
(CHO-K1) cells stably expressing these receptors and G protein-coupled
receptor kinase 2 (GRK2). Agonist-induced internalization of up to
80-90% of hm2 receptors was demonstrated by measuring loss of
[3H]N-methylscopolamine binding sites
from the cell surface, and transfer of [3H]quinuclidinyl
benzilate binding sites from the plasma membrane into the light-vesicle
fractions separated by sucrose density gradient centrifugation.
Additionally, translocation of hm2 receptors with endocytic vesicles
were visualized by immunofluorescence confocal microscopy.
Agonist-induced down-regulation of up to 60-70% of hm2 receptors was
demonstrated by determining the loss of [3H]quinuclidinyl
benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in
the presence of 104 M carbamylcholine was
estimated to be 9.5 min and 2.3 h, respectively. The rates of both
internalization and down-regulation of hm2 receptors in the presence of
10
6 M or lower concentrations of
carbamylcholine were markedly increased by coexpression of GRK2.
Agonist-induced internalization of I3-del m2 receptors was barely
detectable upon incubation of cells for 1 h, but agonist-induced
down-regulation of up to 40-50% of I3-del m2 receptors occurred upon
incubation with 10
4 M carbamylcholine for
16 h. However, the rate of down-regulation was lower compared with
wild type receptors (t1/2 = 9.9 versus
2.3 h). These results indicate that rapid internalization of hm2
receptors is facilitated by their phosphorylation with GRK2 and does
not occur in the absence of the third intracellular loop, but
down-regulation of hm2 receptors may occur through both
GRK2-facilitating pathway and third intracellular loop-independent
pathways.
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INTRODUCTION |
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Loss of the cell's response to agonist acting at G
protein-coupled receptors can occur in three phases: uncoupling from G proteins, sequestration/internalization, and down-regulation of the
receptors. Many G protein-coupled receptors are phosphorylated by G
protein-coupled receptor kinases
(GRKs)1 in an
agonist-dependent manner as a major mechanism in receptor regulation (for review, see Refs. 1 and 2). Muscarinic acetylcholine receptor m2 subtypes have also been shown to be phosphorylated by GRK2
(-adrenergic receptor kinase 1) (3, 4) and other GRKs (5, 6). In
addition, phosphorylation of
2-adrenergic receptors by
GRK2 may be involved in the uncoupling of
2-adrenergic receptors from G protein Gs (1, 2). The phosphorylated
2-adrenergic receptors were reported to be uncoupled
from G proteins because of their interaction with
-arrestin (1, 2,
7). Indeed, phosphorylation by GRKs has been reported to correlate with
uncoupling for several G protein-coupled receptors including muscarinic
m2 (8),
1B-adrenergic (9),
2-adrenergic
(10), thrombin (11), dopamine D1A (12), and thyrotropin receptors (13).
-Arrestin and arrestin 3 (
-arrestin 2) were shown to interact with m2 muscarinic as well as with
2-adrenergic
receptors (14, 15).
Sequestration/internalization of 2-adrenergic receptors
seemed to be independent of phosphorylation by GRK2 on the basis of
results with
2-adrenergic receptor mutants lacking
phosphorylation sites or GRK-specific inhibitors (16-20). On the other
hand, the agonist-induced sequestration of hm2 receptors expressed in
HEK293 cells is hampered by deletion of the third intracellular loop (I3-loop) which includes the GRK2 phosphorylation sites (21, 22).
Moreover, agonist-dependent phosphorylation and
sequestration of m2 receptors expressed in COS-7 cells are facilitated
by coexpression of GRK2 and attenuated by coexpression of a
dominant-negative mutant of GRK2 (DN-GRK2) that lacks kinase activity
(23). Recently, Ferguson et al. have reexamined the
relationship between the phosphorylation by GRK2 and sequestration of
2-adrenergic receptors, demonstrating that
phosphorylation by GRK2 (24) or other GRKs (25) facilitates sequestration of
2-adrenergic receptors. Phosphorylation
facilitates
-arrestin binding to
2-adrenergic
receptors (26) and thereby appears to enhance sequestration, possibly
interacting with clathrin (27), a major protein of coated pits.
Pals-Rylaarsdam et al. (8, 28) have provided results showing
that the phosphorylation by GRK2 of m2 receptors is involved in their
internalization as well as in their uncoupling from G proteins in
HEK293 cells. These results suggest that the phosphorylation by GRK2 of
m2 muscarinic and
2-adrenergic receptors may be involved
in both internalization and uncoupling through facilitation of their
interaction with
-arrestin/arrestin 3.
No studies have been carried out on the relation between
down-regulation and phosphorylation of G protein-coupled receptors, except that down-regulation of 2-adrenergic receptors
has been reported to be independent of their phosphorylation by GRK2
(16, 18). It is also unclear whether the cellular pathway leading to
down-regulation is distinct from that of internalization. If a portion
of receptors in clathrin-coated vesicles translocates into lysosomes
and is down-regulated, their phosphorylation with GRKs or the deletion
of I3-loop should also affect down-regulation. However, if
down-regulation occurs by a distinct pathway, receptor phosphorylation
may not play a role. Alternatively, both phosphorylated and
non-phosphorylated receptors may enter the
clathrin-dependent internalization pathway, albeit at
different rates. Finally, receptor phosphorylation could affect the
rate of translocation between endosomes and lysosomes, or recycling to
the cell surface.
Here, we provide evidence that down-regulation as well as internalization of hm2 receptors are facilitated by coexpression of GRK2. Moreover, deletion of I3-loop, which contains the GRK2 phosphorylation sites (22), suppressed rapid internalization and markedly reduced the rate of down-regulation.
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EXPERIMENTAL PROCEDURES |
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Materials-- [3H]NMS (specific activity of 71.3 Ci/mmol) and [3H]QNB (specific activity of 36.4 Ci/mmol) were purchased from NEN Life Science Products; restriction enzymes were from Toyobo Corp. and Takara Shuzo Co., Ltd.; Cy3-conjugated goat anti-mouse IgG antibody was from Jackson Laboratories. cDNA of GRK2 was kindly donated by Dr. R. J. Lefkowitz, mammalian expression vector for hygromycin-resistant gene (pSV-hygro) was from Dr. H. Okayama, and mammalian expression vector with neomycin-resistant gene (pEF-neo) and mammalian expression vector pEF-BOS were from Drs. S. Nagata and T. Shimizu. Hybridoma cells expressing 9E10 were obtained from the American Type Culture Collection; Chinese hamster ovary CHO-K1 cells were from the Japanese Cancer Research Resources Bank.
Construction of Stable Transfectant Expressing hm2 Receptors and GRK2-- The construction of mammalian expression vectors for c-Myc epitope-tagged hm2 receptor (pEF-Myc-hm2) and GRK2 (pEF-GRK2) was described previously (23). CHO-K1 cells (5 × 104 cells) were transfected with 18 µg of expression vectors of pEF-Myc-hm2 and 2 µg of pEF-neo by the calcium phosphate precipitation method (29). Stable transfectants were selected in the presence of 400 µg/ml Geneticin (Life Technologies, Inc.) and were subcloned by limiting dilution. Expression of receptors was detected by [3H]QNB binding. The [3H]QNB binding sites in these cells were estimated to be 165 fmol/mg of protein in total homogenate. The transfectants were cultured in F-12 nutrient mixture (Ham's) (Life Technologies Inc.) supplemented with 10% fetal bovine serum (Cansera International Inc.), 40 units/ml penicillin G (Meiji Seika, Kaisha Ltd.), 40 mg/ml streptomycin sulfate (Meiji Seika, Kaisha Ltd.), and 100 µg/ml Geneticin at 37 °C in 95% air and 5% CO2. One of the CHO cell clones expressing hm2 receptors was transfected with 18 µg of pEF-GRK2 and 2 µg of pSV-hygro, and stable transfectants were selected in the presence of 300 µg/ml hygromycin B (Boehringer Mannheim) and subcloned by limiting dilution. Expression of GRK2 was detected with use of Western blotting as described previously (23). The [3H]QNB binding sites of these cells were estimated to be 330 fmol/mg of protein in total homogenate, and expressed amounts of GRK2 were estimated to be 300-600 fmol/mg of protein in the supernatant by immunostaining with anti-GRK2 antibodies. The transfectants were cultured in F-12 nutrient mixture (Ham's) supplemented with 10% fetal bovine serum, 40 units/ml penicillin G, 40 mg/ml streptomycin sulfate, and 100 µg/ml hygromycin B at 37 °C in 95% air and 5% CO2. A mammalian expression vector for a m2 receptor mutant that lacks a central part of the third intracellular loop (I3-del m2 receptor) was constructed by inserting the NheI-XhoI fragment of the pSG5/Hm2(d234-381) (21) into the NheI/XhoI site of pEF-Myc-hm2. I3-del m2 receptors were stably expressed in CHO-K1 cells as described above, and the [3H]QNB binding sites of these cells were estimated to be 260 fmol/mg of protein in total homogenate.
Sucrose Density Gradient Centrifugation Experiments--
Sucrose
density gradient centrifugation was carried out as described by Harden
et al. (30). Semiconfluent CHO cells cultured in a 15-cm
diameter dish were treated with 105 M
carbamylcholine for 20 min and then washed three times with 10 ml of
ice-cold, phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4,
1.5 mM KH2PO4, pH 7.5). Washed
cells were incubated with 10 ml of serum-free F-12 medium containing 50 µg/ml concanavalin A for 20 min on ice, then washed with 10 ml of
lysis buffer (1 mM Tris, 2 mM EDTA, pH 7.4),
and hypotonically lysed by incubation in 10 ml of lysis buffer for 20 min on ice. After removing the lysis buffer, cells were collected in a
small volume of lysis buffer with rubber policeman. The lysate (1 ml) was layered on top of a sucrose density gradient comprising 55, 50, 47.5, 45, 42.5, 40, 37.5, 35, 32.5, and 30% of sucrose solution (0.9 ml each) buffered with 2 mM Tris-HCl (pH 8.0). The gradient was centrifuged in a Hitachi SW 27 rotor at 25,000 rpm for 2.5 h
at 4 °C. Twenty 0.5-ml fractions were collected from bottom and then
subjected to [3H]QNB binding assay. A portion of each
fraction (0.1 ml) was mixed with 0.9 ml of HEN buffer (20 mM Hepes/KOH, 1 mM EDTA, 160 mM NaCl, pH 7.4) containing 0.36 nM [3H]QNB, and
incubated at 30 °C for 1 h. After incubation, membranes were
trapped on a Whatman GF-B glass fiber filter, washed with 1 ml of 20 mM potassium phosphate buffer (pH 7) four times, and radioactivity determined by scintillation counting.
[3H]NMS and [3H]QNB Binding Assay-- CHO cells (1 × 104 cells/well) were plated onto 12-well culture dishes. Forty to forty-eight hours after plating, various concentrations of carbamylcholine were added to culture media. After incubation with carbamylcholine for 15 min to 16 h, cells were washed three times with 1 ml of ice-cold PBS/well and incubated with 1.2-1.6 nM [3H]NMS or [3H]QNB in Hepes-buffered saline (25 mM Hepes, 113 mM NaCl, 6 mM glucose, 3 mM CaCl2, 3 mM KCl, 2 mM MgSO4, and 1 mM NaH2PO4, pH 7.4; 0.5 ml/well) at 4 °C for 4 h. After incubation, cells were washed three times with 1 ml ice-cold PBS/well. After washing, cells were dissolved in 0.3 ml of 1% Triton X-100 (w/v), mixed with 4.5 ml of Triton-toluene mixture containing 0.4% 2,5-diphenyloxazole and 0.01% 1,4-bis-2-(methyl-5-phenyloxazolyl)benzene, and the radioactivity measured. Quadruplicate samples were assayed for each point. In some experiments, cells were treated with carbamylcholine in the hypertonic medium containing 0.32 M sucrose besides normal constituents. Down-regulation in the hypertonic medium was examined for cells treated with carbamylcholine for 1-4 h, because the incubation for longer than 4 h in the hypertonic medium caused CHO cells to deteriorate.
Immunofluorescence Confocal Microscopy of hm2 Receptors-- CHO cells expressing human c-Myc-tagged m2 receptors were grown overnight on plastic chamber slides (Nunc Inc.). Treatment with various concentrations of carbamylcholine was carried out at 37 °C for 10 min. At the end of drug treatment, cells were washed twice with PBS, fixed for 10 min at room temperature with 3.7% paraformaldehyde in PBS, permeabilized in PBS containing 0.25% fish gelatin, 0.04% saponin, and 0.05% NaN3. After permeabilization, cells were labeled with anti-Myc monoclonal antibody (9E10) (31) for 1 h, washed four times with PBS, incubated with Cy3 (indocarbocyamine)-conjugated goat anti-mouse secondary antibody, and then washed four times with PBS and once with water. Slides were mounted using Fluoromount G (Fisher Scientific) containing a trace amount of phenylenediamine and stored at 4 °C. Samples were visualized using laser scanning confocal microscopy with a krypton-argon laser coupled with a Bio-Rad MRC-600 confocal head attached to an Optiphot II Nikon microscope with a Plan Apo 60 × 1.4 NA objective lens with 1.4 numeric aperture. Cy3 emission was detected with a yellow high sensitivity filter block.
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RESULTS |
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Sequestration of hm2 Receptors as Assessed by Loss of
[3H]NMS Binding Sites from the Cell Surface--
CHO
cells expressing hm2 receptors with or without GRK2 were treated with
carbamylcholine for various times, and then [3H]NMS
binding activity of intact cells was measured. Fig.
1 summarizes the effects of incubation
time and concentrations of carbamylcholine on the sequestration of hm2
receptors, as assessed by loss of [3H]NMS binding
activity from the cell surfaces. In the presence of 105
M or higher concentrations of carbamylcholine, 70-80% of
[3H]NMS binding sites were sequestered with a half-life
of approximately 10 min. In the presence of 10
6
M carbamylcholine, the rate of sequestration was slower,
and less than 20% of [3H]NMS binding sites were
sequestered in 60 min. Coexpression of GRK2 markedly increased the rate
of sequestration in the presence of 10
6 M
carbamylcholine but only slightly increased the extent of sequestration in the presence of 10
5 M or higher
concentrations of carbamylcholine (Fig. 1, A-C). Fig.
1D shows the [3H]NMS binding sites remaining
on the surface of cells pretreated with carbamylcholine for 60 min.
Without GRK2 cotransfection, 10
6 M or higher
carbamylcholine concentrations were required to elicit sequestration,
whereas, with GRK2 cotransfection, 10
7 M
carbamylcholine was sufficient to detect sequestration. Apparent EC50 values of carbamylcholine were estimated to be 0.37 and 2.4 µM for cells with or without coexpression of
GRK2. Similar results were obtained in COS-7 cells expressing m2
receptors, where GRK2 stimulated the sequestration of
[3H]NMS binding sites in the presence of low, but not
high, concentrations of carbamylcholine. In m2 receptors expressed in
BHK-21 cells, the effect of GRK2 was observed in the presence of high
concentrations of carbamylcholine, but the effect in the presence of
low concentration of carbamylcholine was greater (21). It should be
noted that the portion of sequestered m2 receptors was higher for CHO
cells (80%) than for COS-7 cells (40%) or BHK-21 cells
(20-25%).
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Assessment of Internalization of hm2 Receptors by Sucrose Density
Gradient Centrifugation and Confocal Microscopy--
Sequestration of
muscarinic receptors as assessed by the loss of [3H]NMS
binding sites from the cell surface is generally thought to represent
internalization of receptors in the form of endocytosed vesicles. We
confirmed internalization of hm2 receptors expressed in CHO cells with
two different methods: sucrose density gradient centrifugation and
confocal microscopy. Sucrose density gradient centrifugation was
carried out as described by Harden et al. (30). The
carbamylcholine-treated cells were incubated with concanavalin A,
hypotonically lysed, and then subjected to the centrifugation, which
resulted in the separation of two fractions: a heavy membrane fraction
containing cell surface membranes and a light fraction containing
intracellular vesicles (endosomes). As shown in Fig. 2, the peak of [3H]QNB
binding sites shifted from the heavy to light fraction by treatment of
cells with 105 M carbamylcholine for 20 min.
This result is consistent with the interpretation that the sequestered
[3H]NMS binding sites corresponding to approximately 50%
of total hm2 receptors were transferred from cell membranes to light
vesicle fractions.
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Down-regulation of hm2 Receptors as Assessed by the Decrease in
[3H]QNB Binding Sites--
The down-regulation of hm2
receptors was assessed as the agonist-induced decrease in
[3H]QNB binding sites, as the tertiary amine
[3H]QNB can penetrate the cell membranes and label total
hm2 receptors, whereas the quaternary amine [3H]NMS
cannot penetrate the cell membrane and labels only surface hm2
receptors. As shown in Fig. 4, the
[3H]QNB binding sites decreased with slower rates
compared with the decrease in [3H]NMS binding sites from
the cell surface, and the rate was dependent on the carbamylcholine
concentration. In cells expressing hm2 receptors alone,
[3H]QNB binding sites decreased by 60% upon incubation
with 105 M or higher concentration of
carbamylcholine for 16 h. In cells expressing both hm2 receptors
and GRK2, the loss of [3H]QNB binding site was similar
initially, and enhanced down-regulation by 70% appeared only at
16 h pretreatment (Fig. 4B). In contrast, down-regulation was undetectable in the presence of 10
6
M carbamylcholine without GRK2, whereas significant
down-regulation occurred with GRK2 coexpression (Fig. 4A).
Apparent EC50 values of carbamylcholine for the
down-regulation of hm2 receptors after 16 h of treatment were
estimated to be 0.7 and 6 µM for cells with or without
coexpression of GRK2 (Fig. 4D). These results provide the
first evidence that the down-regulation of G protein-coupled receptors
is facilitated by coexpression of GRK2 and suggest that phosphorylation
by GRK2 of hm2 receptors is directly or indirectly linked to their
down-regulation.
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Sequestration and Down-regulation of I3-del m2 Receptors-- We have stably expressed I3-del m2 receptors in CHO cells and examined changes in [3H]NMS and [3H]QNB binding sites on cells treated with different concentrations of carbamylcholine for various times. Phosphorylation sites by GRK2 in hm2 receptors are known to be in the I3-loop (22), and I3-del m2 receptors are not phosphorylated by GRK2 (4). I3-del m2 receptors transiently expressed in HEK293 cells have been shown to sequester much less than hm2 receptors (21). Similarly, I3-del m2 receptors in CHO cells failed to sequester significantly upon treatment with carbamylcholine for 1 h (Fig. 5A). The [3H]NMS binding sites were gradually decreased upon prolonged incubation with carbamylcholine, but the rate of loss was much lower for I3-del m2 receptors (t1/2 = 8.4 h) than for wild type hm2 receptors (t1/2 = 9.5 min) (Fig. 5C).
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DISCUSSION |
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In previous studies (23), we have shown that sequestration of m2 receptors transiently expressed in COS-7 and BHK-21 cells was facilitated by coexpression of GRK2, an effect of which was evident only at low concentrations of carbamylcholine. In the present study, a similar effect of coexpression of GRK2 was observed for the sequestration of hm2 receptors stably expressed in CHO cells. Furthermore, the sequestration assessed as the loss of [3H]NMS binding sites from the cell surface was confirmed to represent the internalization of hm2 receptors from plasma membranes into cytoplasmic vesicles by analyses involving sucrose density gradient centrifugation of membrane fractions and confocal microscopy. The fact that a similar effect was observed in three different cell lines suggests that facilitation by GRK of the internalization of hm2 receptors is a general phenomenon independent of cell species. On the other hand, Pals-Rylaarsdam et al. (8) have argued against the involvement of GRK2 in the internalization of hm2 receptors, based on the finding that the level of sequestration was not affected by coexpression of GRK2 or a DN-GRK2 in a clone of HEK293. They measured the sequestration of hm2 receptors in cells treated with only a high concentration of carbamylcholine (1 mM), and therefore could have missed the effect of GRK2 coexpression. Very recently, these authors have shown that a hm2 receptor mutant with alanine residues in the place of serine/threonine residues in the GRK2 phosphorylation sites was sequestered by a lower extent compared with the wild type receptor, and concluded that sequestration of hm2 receptors was promoted by their phosphorylation (28). As for the effect of coexpression of DN-GRK2, we have also failed to detect any effect on the sequestration of m2 receptors in CHO cells and BHK-21 cells (23), although the sequestration of m2 receptors expressed in COS-7 cells was significantly attenuated by coexpression of DN-GRK2. At present, we have not identified the species of endogenous GRKs or related kinases in these cells and do not know the reason why the expression of DN-GRK2 affect the sequestration of m2 receptors in some cells but not in other cells.
In contrast to wild type hm2 receptors, I3-del m2 receptors (deletion
234-381), which lack phosphorylation sites by GRK2, failed to
internalize rapidly. The simplest interpretation for this finding is
that phosphorylation by GRK2 of serine or threonine residues in the
I3-loop is a necessary step for rapid internalization. We cannot
exclude, however, the possibility that the I3-loop may have other
functions. Pals-Rylaasdam et al. reported that a hm2 mutant
with a deletion (252-327) in the I3-loop was not phosphorylated by
GRK2; yet 50% of the mutant stably expressed in HEK293 cells were
sequestered by treatment with 103 M
carbamylcholine for 2 h, although the sequestration of the mutant
was less in its extent and slower in its rate as compared with the
sequestration of wild type hm2 receptors. Possibly, internalization depends on phosphorylation-independent sites which were deleted from
our mutant but not from the 252-327 deletion mutant. Ferguson et
al. (24) have shown that overexpression of
-arrestin rescues sequestration of
2-adrenergic receptor mutant lacking
phosphorylation sites, and proposed that the interaction between
-arrestin and receptors is essential for internalization and that
the internalization is facilitated by but does not require the
phosphorylation by GRK2. Both phosphorylation sites and
phosphorylation-independent sites in the I3-loop might be involved
in the interaction with
-arrestin which accelerates
internalization.
We have found in the present study that the coexpression of GRK2
facilitates the down-regulation of hm2 receptors by reducing the
effective concentrations of carbamylcholine. As the effects of GRK2
coexpression on internalization and down-regulation of hm2 receptors
were similar to each other, it is tempting to speculate that both
internalization and down-regulation involve the same event,
e.g. the phosphorylation by GRKs of agonist-bound receptors. To our knowledge, a positive relationship between down-regulation and
phosphorylation by GRK2 has not been reported for any G protein-coupled receptors. As for 2-adrenergic receptors, receptor
mutants lacking phosphorylation sites for GRK2 have been shown to
down-regulate normally (16, 18). It should be noted, however, that
these authors have not examined the effect of different concentrations of agonist, and therefore, the ability of GRK2 to reduce the effective concentration might not have been noticed.
When hm2 receptor-expressing cells were treated with 104
M of carbamylcholine, hm2 receptors were rapidly
internalized with t1/2 of 9.5 min and slowly
down-regulated with t1/2 of 2.3 h. Thus, approximately 60% of receptors were down-regulated, 30% were in an
internalized form, and 10% remained in the cell surface after a 16-h
incubation (see Fig. 5C). In contrast, I3-del m2 receptors were lost from the cell surface and down-regulated with slower rates of
t1/2 = 8.4 and 9.9 h, respectively, so that
approximately 60% of receptors were down-regulated, no appreciable
receptors were detectable in an internalized form, and 40% remained in
the cell surface after a prolonged incubation (see Fig. 5C).
These results indicate that down-regulation may occur without the
I3-loop. However, the I3-loop is necessary for rapid internalization
and accumulation of internalized receptors.
In Fig. 6, we have presented a tentative schema for the relationship
between internalization and down-regulation of hm2 receptors. We assume
in this schema that agonist-bound receptors are rapidly internalized
and that internalized receptors are slowly down-regulated. This schema
explains the present findings that both rapid internalization and
down-regulation in the presence of low concentrations of
carbamylcholine are accelerated in parallel by coexpression of GRK2;
this explanation is based on the assumption that the amounts of
phosphorylated hm2 receptors are increased by coexpression of GRK2, the
rate of internalization is limited by the concentration of
phosphorylated receptors, and the rate of down-regulation is limited by
concentrations of internalized receptors. The finding that both
sequestration and down-regulation are inhibited in the hypertonic
medium supports the scheme and suggests that the rapid internalization
occurs via coated vesicles. We, however, cannot exclude the possibility that the down-regulation occurs through multiple pathways. In contrast
to hm2 receptors, the I3-del m2 receptors are lost from the cell
surface and down-regulated with similar slow rates (see Fig.
5C), indicating that no appreciable amounts of receptors exist in an internalized form. It is possible that hm2 receptors may
down-regulate via two independent pathways, the I3-loop-requiring and I3-loop-independent pathways, which do and do not involve the rapid
internalization, respectively. The I3-loop-requiring and
I3-loop-independent pathways may represent the coated vesicle-mediated and coated vesicle-independent pathways, respectively. This
interpretation is consistent with the results that the internalization
of hm2 receptors caused by 104 M
carbamylcholine was completely suppressed in the hypertonic medium but
the down-regulation was only partly suppressed, and that the
proportions of down-regulated hm2 receptors in the hypertonic medium
were similar to those of down-regulated I3-del hm2 receptors in normal
medium (compare Figs. 4E and 5C). Another
interpretation is that down-regulation of hm2 receptors occurs through
a single step involving internalized vesicles and that the
internalization step proceeds rapidly for hm2 receptors with intact
I3-loop but greatly slows down and becomes the rate-limiting step for
down-regulation for I3-del hm2 receptors. At present, the question
remains open whether down-regulation of hm2 receptors occurs through a
single route via internalized receptors or through multiple independent pathways.
In the present study, we have shown that both internalization and down-regulation of hm2 receptors are facilitated by coexpression of GRK2, and that the I3-loop is necessary for rapid internalization but not necessary for down-regulation, although the rate of down-regulation is reduced in its absence.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. J. Lefkowitz for cDNA of GRK2, Dr. S. Nagata for the mammalian expression vector pEF-BOS and pEF-neo, Dr. H. Okayama for pSV-hygro, Health Science Research Resources Bank for CHO-K1 cells, and Dr. M. Itokawa and E. Okuno for help in experiments.
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FOOTNOTES |
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* This work was supported in part by grants from the Japan Society for the Promotion of Science, the Ministry of Education, Science, and Culture of Japan, and the Japan Health Science Foundation; by National Institute of Health Grants GM43102, DA04166, and MH00996; and by National Science Foundation Grant INT-9418769.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: Dept. of Occupational Diseases, National Institute of Industrial Health, 6-21-1 Nagao, Tama-ku, Kawasaki, Kanagawa 214, Japan. Tel.: 81-44-865-6111; Fax: 81-44-865-6116; E-mail address: tsuga{at}m.u-tokyo.ac.jp.
1 The abbreviations used are: GRK, G protein-coupled receptor kinase; hm2 receptor, human muscarinic acetylcholine receptor m2 subtype; DN-GRK2, dominant negative form of GRK2; I3-loop, the third intracellular loop; I3-del m2 receptor, a hm2 receptor mutant that lacks a central part of the third intracellular loop; G protein, guanine nucleotide-binding regulatory protein; NMS, N-methylscopolamine; QNB, quinuclidinyl benzilate; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary.
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REFERENCES |
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