Regulation of Muscarinic Acetylcholine Receptor Sequestration and Function by beta -Arrestin*

Oliver Vögler, Bettina Nolte, Matthias Voss, Martina Schmidt, Karl H. Jakobs, and Chris J. van KoppenDagger

From the Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta -arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta 2-adrenergic receptor have demonstrated that beta -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 beta -arrestin in mAChR sequestration, we determined the effect of overexpressing beta -arrestin-1 and the dominant-negative inhibitor of beta -arrestin-mediated receptor sequestration, beta -arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta -arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta -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 beta -arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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, beta 2-adrenergic receptors) (4). After phosphorylation, cytosolic beta -arrestins bind with increased affinity to the receptors and sterically inhibit further coupling of the receptors with G proteins (1). To date, two beta -arrestin isoforms, beta -arrestin-1 (arrestin 2) and beta -arrestin-2 (arrestin 3) have been identified, each undergoing alternative splicing (1). Both isoforms are ubiquitously expressed, with beta -arrestin-1 being the major beta -arrestin expressed in many tissues (1). Recent evidence indicates that beta -arrestins do not only bind to GPCRs but also associate with nanomolar affinity with clathrin heavy chains and target beta -arrestin-bound GPCRs to the clathrin-coated pits, leading to receptor internalization (5, 6). This process has been particularly well studied for the beta 2-adrenergic receptors. Overexpression of beta -arrestin-1 or beta -arrestin-2 in the presence of sufficient G protein-coupled receptor kinases augments internalization of beta 2-adrenergic receptors, whereas overexpression of the dominant-negative beta -arrestin-1 V53D mutant, which binds with high affinity to clathrin cages but is significantly impaired in its ability to interact with beta 2-adrenergic receptors, suppresses beta 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 beta 2-adrenergic receptor internalization, indicating that beta 2-adrenergic receptors sequester into clathrin-coated vesicles in an arrestin- and dynamin-dependent manner (8). The primary function of internalization of the beta 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 beta 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 beta -arrestins as well (1). In contrast to beta 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 beta 2-adrenergic receptors. Previous studies have shown that beta -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 beta 2-adrenergic receptors, we hypothesized that beta -arrestins are essential for the internalization of mAChR subtypes as well. In this study, we overexpressed the dominant-negative beta -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 beta -arrestin-1 V53D and dynamin K44A as inhibitors of clathrin-mediated endocytosis, whether receptor sequestration is required for m1 mAChR-mediated MAP kinase activation.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta -arrestin-1 wild type in pBC (22) and rat beta -arrestin-1 V53D in pcDNA-1 Amp (5) were generously provided by Drs. M. J. Lohse and R. J. Lefkowitz, respectively. (Bovine) beta -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/beta -arrestin-1, pcDNA-1 Amp/beta -arrestin-1 V53D, pcDNA3/beta -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 beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Effect of beta -Arrestin-1 on m1 mAChR Sequestration-- Western blot analysis demonstrated equal overexpression of beta -arrestin-1 and beta -arrestin-1 V53D in HEK-293 tsA201 cells transiently transfected with the expression vector encoding either beta -arrestin (Fig. 1A). In the absence of overexpressed beta -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 beta -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 beta -arrestin-1, however, did not appear to decrease the EC50 value of carbachol for inducing m1 mAChR internalization. In contrast to wild-type beta -arrestin-1, beta -arrestin-1 V53D significantly suppressed m1 mAChR sequestration. Receptor sequestration in beta -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 beta -arrestin-1 V53D-overexpressing cells was only 6 ± 2%.


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Fig. 1.   Overexpression of beta -arrestin-1 wild-type and V53D mutant in HEK-293 tsA201 cells. Effects on m1 mAChR sequestration. A, detection of beta -arrestins in total lysates of HEK-293 tsA201 cells grown on 150-mm plates and transiently transfected with pRK5 (pRK5), pBC/beta -arrestin-1 (WT), or pcDNA-1 Amp/beta -arrestin-1 V53D (V53D) by immunoblotting. Equal amounts of cell lysates (15 µg of protein/lane) were subjected to SDS-polyacrylamide gel electrophoresis, and beta -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/beta -arrestin-1 (WT), pcDNA-1 Amp/beta -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/beta -arrestin-1, and pcDNA-1 Amp/beta -arrestin-1 V53D was 290 ± 35, 229 ± 44, and 226 ± 28 fmol/mg of protein, respectively.

Role of Receptor Internalization in m1 mAChR-induced MAP Kinase Stimulation-- Daaka et al. (13) recently demonstrated that overexpression of beta -arrestin-1 V53D or dynamin K44A in HEK-293 cells inhibits activation of MAP kinases by beta 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 beta -arrestin- and dynamin-dependent internalization pathway as utilized by beta 2-adrenergic receptors, we examined the effect of overexpressing beta -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 beta -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 beta -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 beta -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/beta -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/beta -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.

Effect of beta -Arrestin-1 on m2, m3, and m4 mAChR Sequestration-- As shown in Fig. 3, overexpression of beta -arrestin-1 V53D only marginally inhibited m2 mAChR sequestration, whereas m2 mAChR sequestration was only modestly stimulated by overexpression of wild-type beta -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 beta -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 beta -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 beta -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 beta -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/beta -arrestin-1 (WT), pcDNA-1 Amp/beta -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/beta -arrestin-1, and pcDNA-1 Amp/beta -arrestin-1 V53D was 252 ± 60, 112 ± 22, and 183 ± 68 fmol/mg protein, respectively.

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 beta -arrestin-dependent. As depicted in Fig. 4, overexpression of beta -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 beta -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 beta -arrestin-1 V53D, we also examined the influence of another dominant-negative inhibitor of beta -arrestin-mediated receptor sequestration, beta -arrestin-1 (319-418). Overexpression of beta -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 beta -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/beta -arrestin-1 (WT), pcDNA-1 Amp/beta -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/beta -arrestin-1, and pcDNA-1 Amp/beta -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/beta -arrestin-1, and pcDNA-1 Amp/beta -arrestin-1 V53D was 346 ± 93, 242 ± 63, and 154 ± 60 fmol/mg of protein, respectively.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we determined the role of beta -arrestin in mAChR sequestration and function using beta -arrestin-1 wild type and the dominant-negative inhibitor of beta -arrestin-mediated receptor sequestration, beta -arrestin-1 V53D. This beta -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 beta -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 beta -arrestin-dependent manner. On the other hand, overexpression of beta -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 beta -arrestin is hardly able to promote receptor internalization further. In line with our observation that m1, m3, and m4 mAChR sequester in a beta -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 beta 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 beta -arrestin-1, beta -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 beta -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 beta -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 beta -arrestin now supports sequestration of m2 mAChRs by the other, less efficient beta -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 beta -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 beta 2-adrenergic receptors was significantly suppressed. This beta -arrestin mutant, which encodes the last 100 amino acids of beta -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, beta -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 beta -arrestin-1 (319-418) was insufficient to block interaction of mAChR-bound beta -arrestin-1 to clathrin, whereas beta 2-adrenergic receptor internalization was effectively inhibited. In this respect, it is important to note that the degree of competition between beta -arrestin-1 and beta -arrestin-1 (319-418) for binding clathrin is related to the difference in binding affinity of the beta -arrestins for clathrin. Because the G protein-coupled receptor kinase phosphorylation sites on the mAChRs and beta 2-adrenergic receptor are located differently (2-4), the binding affinity of beta -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 beta -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 beta -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 beta 2-adrenergic receptors (1). It has been proposed that the agonist-occupied, beta -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 beta -arrestin (or a beta -arrestin-like protein) in the internalization of m1, m3, and m4 mAChRs in HEK-293 tsA201 cells. The lack of effect of beta -arrestin V53D overexpression on the internalization of m2 mAChRs suggests that desensitization of m2 mAChRs can be beta -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 beta -arrestin binding site (16) does not bind beta -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 beta -arrestins, perhaps there are other cytosolic proteins that preferentially bind to the m2 mAChR and effectively compete with beta -arrestin. Like the beta -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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Krupnick, J. G., and Benovic, J. L. (1998) Annu. Rev. Pharmacol. Toxicol. 38, 289-319[CrossRef][Medline] [Order article via Infotrieve]
  2. Haga, K., Kameyama, K., Haga, T., Kikkawa, U., Shiozaki, K., and Uchiyama, H. (1996) J. Biol. Chem. 271, 2776-2782[Abstract/Free Full Text]
  3. Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158[Abstract/Free Full Text]
  4. Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796-13803[Abstract/Free Full Text]
  5. Ferguson, S. S. G., Downey, W. E., Colapietro, A.-M., Barak, L. S., Ménard, L., and Caron, M. G. (1996) Science 271, 363-366[Abstract]
  6. Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450[CrossRef][Medline] [Order article via Infotrieve]
  7. Ménard, L., Ferguson, S. S. G., Zhang, J., Lin, F.-T., Lefkowitz, R. J., Caron, M. G., and Barak, L. S. (1997) Mol. Pharmacol. 51, 800-808[Abstract/Free Full Text]
  8. Zhang, J., Ferguson, S. S. G., Barak, L. S., Ménard, L., and Caron, M. G. (1996) J. Biol. Chem. 271, 18302-18305[Abstract/Free Full Text]
  9. Zhang, J., Barak, L. S., Winkler, K. E., Caron, M. G., and Ferguson, S. S. G. (1997) J. Biol. Chem. 272, 27005-27014[Abstract/Free Full Text]
  10. Van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowitz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553-563[Abstract]
  11. Yu, S. S., Lefkowitz, R. J., and Hausdorff, W. P. (1993) J. Biol. Chem. 268, 337-341[Abstract/Free Full Text]
  12. Pippig, S., Andexinger, S., and Lohse, M. J. (1995) Mol. Pharmacol. 47, 666-676[Abstract]
  13. Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S. G., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688[Abstract/Free Full Text]
  14. Vögler, O., Bogatkewitsch, G. S., Wriske, C., Krummenerl, P., Jakobs, K. H., and Van Koppen, C. J. (1998) J. Biol. Chem. 273, 12155-12160[Abstract/Free Full Text]
  15. Lee, K. B., Pals-Rylaarsdam, R., Benovic, J. L., and Hosey, M. M. (1998) J. Biol. Chem. 273, 12967-12972[Abstract/Free Full Text]
  16. Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689[Abstract/Free Full Text]
  17. Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842[Abstract/Free Full Text]
  18. Shapiro, R. A., Scherer, N. M., Habecker, B. A., Subers, E. M., and Nathanson, N. M. (1988) J. Biol. Chem. 263, 18397-18403[Abstract/Free Full Text]
  19. Peralta, E. G., Winslow, J. G., Peterson, G. L., Smith, D. H., Ashkenazi, A., Ramachandran, J., Schimerlik, M. I., and Capon, D. J. (1987) Science 236, 600-605[Medline] [Order article via Infotrieve]
  20. Bonner, T. I., Buckley, N. J., Young, A. C., and Brann, M. R. (1987) Science 237, 527-532[Medline] [Order article via Infotrieve]
  21. Van Koppen, C. J., Lenz, W., and Nathanson, N. M. (1993) Biochim. Biophys. Acta 1173, 342-344[Medline] [Order article via Infotrieve]
  22. Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G., and Lefkowitz, R. J. (1990) Science 248, 1547-1550[Medline] [Order article via Infotrieve]
  23. Krupnick, J. G., Santini, F., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 32507-32512[Abstract/Free Full Text]
  24. Liu, S.-H., Marks, M. S., and Brodsky, F. M. (1998) J. Cell Biol. 140, 1023-1037[Abstract/Free Full Text]
  25. Margolskee, R. F., McHendry-Rinde, B., and Horn, R. (1993) Biotechniques 15, 906-911[Medline] [Order article via Infotrieve]
  26. Henley, J. R., Krueger, E. W. A., Oswald, B. J., and McNiven, M. A. (1998) J. Cell Biol. 141, 85-99[Abstract/Free Full Text]
  27. Oh, P., McIntosh, D. P., and Schnitzer, J. E. (1998) J. Cell Biol. 141, 101-114[Abstract/Free Full Text]
  28. Tolbert, L. M., and Lameh, J. (1996) J. Biol. Chem. 271, 17335-17342[Abstract/Free Full Text]
  29. Slowiejko, D. M., McEwen, E. L., Ernst, S. A., and Fisher, S. K. (1996) J. Neurochem. 66, 186-196[Medline] [Order article via Infotrieve]
  30. Bogatkewitsch, G. S., Lenz, W., Jakobs, K. H., and Van Koppen, C. J. (1996) Mol. Pharmacol. 50, 424-429[Abstract]
  31. Yang, J., Williams, J. A., Yule, D. I., and Logsdon, C. D. (1995) Mol. Pharmacol. 48, 477-485[Abstract]
  32. Szekeres, P. G., Koenig, J. A., and Edwardson, J. M. (1998) J. Neurochem. 70, 1694-1703[Medline] [Order article via Infotrieve]
  33. Tsuga, H., Kameyama, K., Haga, T., Kurose, H., and Nagao, T. (1994) J. Biol. Chem. 269, 32522-32527[Abstract/Free Full Text]
  34. Pals-Rylaarsdam, R., Xu, Y., Witt-Enderby, P., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270, 29004-29011[Abstract/Free Full Text]
  35. Schlador, M. L., and Nathanson, N. M. (1997) J. Biol. Chem. 272, 18882-18890[Abstract/Free Full Text]


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