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

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic β-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the β2-adrenergic receptor have demonstrated that β-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 β-arrestin in mAChR sequestration, we determined the effect of overexpressing β-arrestin-1 and the dominant-negative inhibitor of β-arrestin-mediated receptor sequestration, β-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60–75% in cells overexpressing β-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of β-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 β-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.

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 dominantnegative 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 dynamindependent 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 G s family, mAChRs efficiently activate G proteins of the G i and G q family. The family of mAChRs consists of five mammalian subtypes, with m1, m3, and m5 mAChRs predominantly activating phospholipase C via G q proteins and m2 and m4 mAChRs efficiently inhibiting adenylyl cyclase by activation of G i 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.
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 KH 2 PO 4 , 6.5 mM Na 2 HPO 4 , 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 l 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 l 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 g/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 g/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 M carbachol or 1 M 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 g/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-lysinecoated 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 [ 3 H]NMS in 500 l of ice-cold phosphate-buffered saline with and without 30 M 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 Ϫ quotient of cell surface receptors of carbachol-treated and untreated cells) ϫ 100%. Untransfected HEK-293 tsA201 do not express detectable levels of mAChR.
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 ␤-arrestinand dynamin-dependent internalization pathway as utilized by ␤ 2adrenergic 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 phos-phorylated p42 and p44 MAP kinases in response to stimulation with 10 M 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-acetateinduced MAP kinase stimulation.
Role of Clathrin in mAChR Sequestration-Two recent stud- ies 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). 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 agonistbound phosphorylated GPCRs (9,23).
Overexpression of ␤-arrestin-1 V53D suppressed sequestra-tion 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 ␤-arrestindependent 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. ϳ0.75 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 ␤-arrestinand 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 ␤-arrestinand 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)(3)(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 clathrinmediated 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)(34)(35). This underscores the plasticity of the molecular mechanisms of receptor trafficking within the family of GPCRs.