Agonist mediated internalization of M2 mAChR is β-arrestin-dependent
© Jones et al. 2006
Received: 17 May 2006
Accepted: 05 December 2006
Published: 05 December 2006
Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2).
In wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells.
In summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.
Muscarinic acetylcholine receptors belong to the superfamily of G-protein coupled receptors (GPCRs) that are commonly expressed in a variety of tissues and are classified into five known subtypes (M1 -M5 mAChR). M1, M3, and M5 mAChRs are selectively coupled to Gq proteins while M2 and M4 mAChRs are linked to Gi/G0 proteins [1, 2]. M2 mAChRs are the primary muscarinic subtype in the heart where their stimulation leads to the regulation of myocardial contractility . As with other GPCRs, M2 mAChR activity is tightly regulated by desensitization and internalization. These regulatory mechanisms are typically associated with receptor phosphorylation followed by either recycling or down-regulation [4–9].
Desensitization is a complex process that involves agonist-dependent phosphorylation at specific serine/threonine residues by G-protein-coupled receptor kinases (GRKs) followed by β-arrestin binding. Two widely expressed isoforms of β-arrestin (1 and 2) are known to be involved in uncoupling receptors from their cognate G-proteins thereby attenuating receptor signalling [10, 11]. Typically, agonist-induced phosphorylation facilitates receptor internalization, which serves to either resensitize or down-regulate desensitized receptors . β-arrestins have been shown to facilitate internalization by directly interacting with the β2 subunit of the clathrin-AP2 (adaptor protein 2) complex and clathrin itself [11, 13]. Thus, β-arrestins can induce receptor sequestration by directly interacting with the endocytic machinery. Many receptors such as the prototypic β2-adrenergic receptor (β2AR) internalize in a clathrin and β-arrestin dependent fashion. Hence, β-arrestin facilitates clathrin-mediated endocytosis [11, 13].
In addition to desensitization and internalization, β-arrestins are known to play a role in other cellular processes that include intracellular trafficking and signalling . Association of β-arrestin with agonist-occupied receptors has been shown to initiate intracellular signalling by functioning as an assembly site for signalling components such as Src, JNK3, and ERK1/2 [14–17]. Therefore, β-arrestin-receptor complexes can lead to cytosolic retention and activation of signalling molecules following receptor-mediated signalling at the cell surface. The physiological roles of this process include decreasing cell proliferation and regulating cytoskeletal rearrangements by spatially restricting ERK activation to the cytosol [16, 18]. Recent reports have also suggested that β-arrestins can function at post-endocytic stages to regulate receptor sorting. It has been shown that receptors exhibit differential affinities for β-arrestin and therefore are classified into two groups . Class A receptors (including β2AR and dopamine receptors) are thought to interact with β-arrestin at the plasma membrane but immediately disassociate following localization to clathrin-coated pits. Hence receptors enter early endosomes devoid of β-arrestin and are typically resensitized and rapidly recycled . In contrast, Class B receptors (vasopressin-V2R, angiotensin-AT1AR, and neurotensin receptors) stably associate with β-arrestin so that β-arrestin/receptor complexes remain intact and are internalized into juxtanuclear endosomal compartments . This interaction can persist for prolonged periods of time. This stable association may dictate the kinetics of receptor recycling since AT1AR and V2R recycle very slowly [20, 21]. A functional consequence of β-arrestin association may also be to facilitate receptor down-regulation.
The role of β-arrestins in regulating the trafficking of M2 mAChRs has been contradictory and unclear. Reports have demonstrated that phosphorylation by GRK2 on serine/threonine residues in the third intracellular loop of M2 mAChRs recruits β-arrestin and leads to receptor desensitization and subsequent internalization .
Whether β-arrestin is involved directly in agonist-promoted endocytosis of M2 mAChRs remains unclear. Indeed over-expression of β-arrestin has been reported to increase agonist-promoted internalization of M2 mAChRs but not of M1 or M3 mAChRs . Furthermore, Claing et al. have shown that M2 mAChRs internalize in a dynamin- and β-arrestin-insensitive manner when expressed in HEK293 cells . Others have reported that the Arf6 GTPase (ADP-ribosylation factor 6) facilitates M2 mAChR entry into primary vesicles, which fuse with clathrin-derived early endosomes [24, 25]. These data do not necessarily rule out β-arrestin as a regulator in agonist-promoted endocytosis of M2 mAChRs. Therefore, to clarify whether agonist-promoted internalization of M2 mAChRs is arrestin dependent, we utilized mouse embryonic fibroblasts (MEFs) derived from β-arrestin null mice that lack expression of one or both isoforms (β-arrestin 1 and 2) and their wild type littermates as control cells . Here we report that agonist-promoted internalization of M2 mAChRs is β-arrestin dependent and M2 mAChRs form stable complexes with β-arrestin at the early endosome. Furthermore, we demonstrate that agonist-promoted internalization of M2 mAChRs is clathrin-dependent. These results suggest that β-arrestin plays an important role in regulating M2 mAChR activity.
Recent studies by Santini and co-workers  showed that agonist-mediated activation of the β2AR was still capable of inducing recruitment into clathrin coated pits in cells expressing mutant arrestin proteins that were defective in binding with clathrin or AP-2, albeit to a reduced degree. Expression of the truncated COOH-terminal region of β-arrestin 1 (319–418), which contains a clathrin binding site but lacks receptor binding, completely inhibited the β2AR mediated clustering of clathrin coated pits . Therefore, we conducted experiments with the truncated β-arrestin 1 (319–418) to determine whether agonist-promoted internalization of the M2 mAChR in MEFs would be affected. Transient expression of the truncated β-arrestin 1 (319–418) completely inhibited the agonist-promoted internalization of the M2 mAChR in MEF wild type cells (Fig. 6B). Thus, it could be argued that the agonist-promoted internalization of M2 mAChR involved a clathrin-dependent pathway. However, as shown previously, expression of an arrestin 2 mutant that was defective in interaction with both clathrin and AP-2 only moderately antagonized the agonist-promoted internalization of M2 mAChR in MEF KO1/2 cells (Fig. 6A). It could be argued that this arrestin mutant, defective in clathrin/AP-2 binding, was still capable of interacting with clathrin/AP-2, albeit to a significantly reduced degree. Thus, it is reasonable to conclude that the agonist-promoted internalization of M2 mAChRs was clathrin-dependent.
In the present study, we investigated the role of β-arrestin in agonist-promoted internalization of the M2 mAChR, which has previously been reported to be β-arrestin independent. In previous studies, heterologous over-expression of wild type and dominant-negative forms of arrestins was used to assess the function of these proteins [22, 32]. Unfortunately, such studies are difficult to interpret because of the complications associated with endogenous proteins. In an attempt to alleviate these complications, we utilized mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockouts in which endogenously expressed β-arrestin 1 and 2 have been genetically eliminated . These cells provide us a unique opportunity to assess whether β-arrestin proteins are involved in the process of agonist-promoted internalization of M2 mAChRs. Herein, we show that agonist-promoted endocytosis of the M2 mAChR is β-arrestin- and clathrin-dependent.
Both β-arrestin 1 and 2 isoforms were reported to form high affinity complexes with the agonist-activated M2 mAChR , suggesting that either isoform is capable of mediating agonist-promoted internalization of the receptor. In corroboration with these findings, we observed no selectivity between β-arrestin isoforms in mediating agonist-promoted internalization of M2 mAChRs. Perhaps, this lack of selectivity between β-arrestin 1 and 2 may explain why using over-expression of a single mutant form of β-arrestin fails to completely block the agonist-promoted internalization of M2 mAChRs. Interestingly, our studies further revealed that β-arrestin remained stably associated with the M2 mAChR in juxtanuclear endosomes for prolonged periods of time following agonist exposure. Given that MEF cells do not endogenously express mAChRs, we compared our observations in a physiologically relevant cell line (RASMCs) and two model cell lines (HeLa and COS-7). Similar findings were also observed in these cells. Closer examination of β-arrestin post-endocytic trafficking revealed that M2 mAChR stimulation led to arrestin redistribution into Tfn and EEA-1 positive compartments, markers of the early endosome. In accordance with our findings, Delaney et al. have reported that stimulated M2 mAChRs internalize in a manner that quickly merges with clathrin-derived early endosomes .
M2 mAChRs follow the general pattern utilized by most GPCRs in that they are internalized via a β-arrestin-dependent mechanism. Additionally, the stable binding of β-arrestin with activated M2 mAChRs within microcompartments follows the paradigm of other Class B GPCRs. Implications of these findings are that β-arrestin may dictate the intracellular trafficking and/or signalling of the M2 mAchRs. Since β-arrestin has emerged as a versatile adaptor and scaffolding protein, its role in regulating M2 mAChR-dependent cellular activity may be significant. It has been shown that β-arrestins interact with trafficking machinery such as Arf6, RhoA, NSF, and a variety of signalling proteins such as ASK1, JNK3, and ERK1/2 . Stable β-arrestin/receptor complexes, as exhibited by Class B receptors, appear to redirect signalling complexes to the cytoplasm thereby activating cytoplasmic targets while preventing ERK translocation to the nucleus [15, 16, 35]. The physiological role of this process may be to participate in actin cytoskeleton reorganization and chemotaxis [18, 36]. With regard to intracellular trafficking, patterns of β-arrestin binding to activate receptors appear to modulate receptor recycling and/or degradation . Class A receptors are typically resensitized and subsequently recycled while Class B receptors undergo slow recycling and/or down-regulation. M2 mAChRs have been shown to undergo slow recycling back to the plasma membrane upon agonist removal . What role or roles β-arrestin plays in M2 mAChR recycling and/or degradation is currently unknown. The functional consequence of stable β-arrestin/M2 mAChR complexes remains to be determined.
Previous studies have suggested that M2 mAChR internalization does not proceed through a β-arrestin/clathrin mediated pathway [22, 23, 28]. For example, Delaney and co-workers  previously reported that M2 mAChRs internalized by a clathrin-independent pathway based upon the use of a dominant-negative K44A dynamin-1 mutant. However, expression of a N-terminal deletion dynamin-1 mutant N272 that lacks the complete GTP-binding domain, unlike K44A dynamin, strongly inhibited agonist-promoted M2 mAChR internalization . Therefore, we conducted experiments with arrestin mutants that were selectively deficient in interaction with clathrin, AP-2, or both clathrin and AP-2, to determine whether agonist mediated internalization of M2 mAChRs was clathrin-dependent. Expression of arrestin mutants defective in interaction with either clathrin (β-arrestin 2-ΔLIELD) or AP-2 (β-arrestin 2-F391A) failed to antagonize M2 mAChR internalization. Moreover, over-expression of a dominant-negative arrestin mutant that was defective in interaction with both clathrin and AP-2 only modestly antagonized M2 mAChR internalization in MEF KO1/2 cells. Thus, it is reasonable to conclude that these data corroborate previous studies indicating that M2 mAChR internalization is clathrin-independent. However, Santini and co-workers  have reported that arrestin mutants with impaired binding to clathrin or AP-2 were still capable of displaying recruitment of β2AR to clathrin-coated pits, albeit to a reduced degree. Therefore, it may be premature to conclude that M2 mAChR internalization is β-arrestin-dependent but clathrin/AP-2-independent. Expression of the truncated carboxy-terminal region of β-arrestin 1, which contained the clathrin interaction site, has been shown to completely abrogate β2AR mediated clustering of clathrin coated pits . Exogenous expression of this mutant completely block agonist-promoted internalization of M2 mAChRs in wild type MEFs. Collectively, these results indicate that agonist-promoted internalization of M2 mAChRs is β-arrestin-dependent and most likely clathrin/AP-2-dependent. However, we cannot rule out that the C-terminal region of arrestin 1 is interacting with another factor, independent from clathrin/AP-2 that may be responsible for mediating internalization of the M2 mAChR. Indeed previous studies have shown that the Arf6 GTPase regulates agonist-promoted endocytosis of the M2 mAChR [24, 25]. It has been shown that β2AR stimulation leads to activation of Arf6 GTPase, which facilitates receptor endocytosis . It is feasible that sequestration of M2 mAChR requires activation of Arf6 GTPase by a β-arrestin-mediated pathway, which may be an important component of agonist-promoted internalization of the M2 mAChR. This would corroborate previous studies, which indicate a critical role for Arf6 GTPase in mediating agonist-promoted M2 mAChR internalization .
In summary, the data presented in this study demonstrate that the agonist-promoted endocytosis of the M2 mAChR subtype occurs via an arrestin dependent pathway in MEF cells. Exogenously expressed β-arrestin proteins remained stably associated with the M2 mAChR upon entry into early endosomal compartments. The lack of stable β-arrestin interaction with other mAChR subtypes suggests a unique role of β-arrestin in regulating activity of the M2 mAChR subtype.
[3H]-N-methylscopolamine (NMS) (81–84 Ci/mmol) was purchased from Amersham Corp. (Buckinghamshire, England). Dulbecco's Modified Eagle's Medium (DMEM), F-10, penicillin/streptomycin, fetal bovine serum, restriction enzymes and LipofectAMINE 2000 were purchased from Invitrogen (Carlsbad, CA) EX-GEN was purchased from Fermentas (Hanover, MD). The anti-FLAG M2 monoclonal antibody and mouse anti-M1 FLAG antibody were purchased from Sigma-Aldrich (St. Louis, MO); mouse antibodies against β-arrestin 1 and 2 were purchased from Santa Cruz (Santa Cruz, CA). The anti-HA.11 monoclonal antibody was purchased from Covance Research Product (Berkley, California) Secondary HRP-conjugated antibodies were purchased from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). Carbachol, atropine and all other reagents were purchased from Sigma-Aldrich. Dr. Neil Nathanson (University of Washington) kindly provided the construct expressing the porcine FLAG-tagged M2 mAChR . HA-tagged M1, M3, M4, and M5 mAChRs were purchased from UMR cDNA Resource Center (University of Missouri). Arrestin mutants, β-arrestin 2-ΔLIELD, β-arrestin 2-F391A, β-arrestin 2 ΔLIELD/F391A, and truncated carboxyl-terminal region of β-arrestin 1 (319–418) were kindly provided by Dr. Jeffrey Benovic (Thomas Jefferson University) [30, 31]. The MEF wild type, β-arrestin 1 and 2 single knockouts, β-arrestin 1 and 2 double knockout cells, and constructs for FLAG-tagged β-arrestin 1 and 2 were kindly provided by Dr. Robert Lefkowitz (Duke University Medical Center) . Constructs encoding β-arrestin 2-GFP and β-arrestin 1-GFP were generous gifts from Dr. Stefano Marullo and have been previously described .
Cell Culture and Transient Transfection
HeLa, MEF wild-type, MEF single and double β-arrestin knockout, RASMCs, and COS-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 I.U./ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO2. For immunocytochemistry, HeLa cells were grown on glass coverslips at a density of 120,000 cells/well in six-well dishes and transfected with EX-GEN or LipofectAMINE 2000 according to the manufacturer's protocol using 1 μg of DNA/well. For ligand binding assays, MEF cells were plated at 80,000 cells/well in 24 well plates and transfected with EX-GEN or LipofectAMINE 2000 according to the manufacturer's protocol using 1 μg of DNA/well.
Radioligand Binding Assay
Receptor internalization was determined by measuring the binding of the membrane impermeable muscarinic antagonist [3H]-N-methylscopolamine ([3H]-NMS) to intact cells as previously described . Briefly, 24–42 h after transfection, MEF cells cultured in 24-well plates were treated or not treated with 1 mM carbachol for 60 min at 37°C. Cultures were washed twice with 1 ml of ice-cold PBS, and labelled with 720 fmol of [3H]-NMS in 1 ml PBS for 4 h at 4°C. Non-specific binding was determined as the bound radioactivity in the presence of 1 μM atropine. Labelled cells were washed two times with 1 ml of ice-cold PBS, solubilized in 0.5 ml of 1% Triton X-100 and combined with 3.5 ml of scintillation fluid followed by measurement of radioactivity. Receptor internalization is defined as percent of surface M2 mAChRs not accessible to [3H]-NMS at each time relative to non-carbachol-treated cells.
Western blot analysis was performed on cells cultured in 6-well plates. The cells were solubilized in 0.5 ml of lysis buffer containing: 50 mM HEPES (pH 7.5), 0.5% (v/v) Nonidet P-40, 250 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1 mM sodium orthovanadate, 1 mM sodium fluoride and 1 μg/ml of protease inhibitors leupeptin, aprotinin, pepstatin A, and 100 μM benzamidine. The protein concentration was determined using the Bradford assay method. Fifty μg of cell lysates were subjected to 4–20% SDS-PAGE. After transfer, the nitrocellulose membrane was blocked and then probed with anti-FLAG monoclonal antibody. Immunoreactive bands were visualized by enhanced chemiluminescence after adding HRP-conjugated anti-mouse antibody. After stripping with 0.1 M glycine (pH 2.5), the membrane was re-probed with anti-β-actin using a detection kit from Oncogene (Cambridge, MA).
24 h following transfection, cells were treated as described in the figure legends, fixed in 4% formaldehyde in PBS for 5 minutes, and rinsed with 10% fetal bovine serum and 0.02% azide in PBS (PBS/serum). Fixed cells were incubated with primary antibodies diluted in PBS/serum containing 0.2% saponin for 45 minutes, and then washed with PBS/serum (3 × 5 min.). The cells were then incubated with fluorescently labelled secondary antibodies in PBS-serum and 0.2% saponin for 45 minutes, washed with PBS/serum (3 × 5 min.) and once with PBS, and mounted on glass slides. Images were acquired using a Zeiss LSM 510 scanning confocal microscope or an Olympus BX40 epifluorescence microscope equipped with a 60× Plan pro lens, and photomicrographs were prepared using an Olympus MagnaFire SP digital camera (Olympus America, Inc.). All images were processed with Adobe Photoshop 7.0 software.
RNA Isolation and RT-PCR
Total cellular RNA from MEF cells, cortex and cerebellum of 2–3 week old Sprague Dawley rat pups was isolated using TriZol according to the manufacturer's instructions. A 50 μl reaction solution containing 1 μg total RNA was reverse-transcribed, and PCR was performed using gene-specific primers and the Qiagen One-step RT-PCR kit. Gene specific primers and amplification reactions were as follows: Rat M1 mAChR (175 bp amplified product): CCTCTGCTGCCGCTGTTG (sense) and GGTGGGTGCCTGTGCTTCA (antisense); Rat M2 mAChR (686 bp amplified product): CACGAAACCTC TGA CCTACCC (sense) and TCTGACCCGACGACCCAACTA (antisense); Rat M4 mAChR (587 bp amplified product): TGGGTCTTGTCCTTTGT GCTC (sense) and TTCATTGCCTGTCTGCTT TGTTA (antisense); Rat β-actin (764 bp amplified product): TTGTAACCAACTGGGACGATATGG (sense) and GATCTT GATCT TCATGGT GCTAGG (antisense). Cycling parameters were 30 minutes at 50°C for reverse transcription followed by 1 minute 95°C hot start followed by 28 cycles at 95°C for 1 minute, 62°C for 1 minute, and 72°C for 45 seconds and a final cycle at 72°C for 7 minutes.
We thank Dr. Nael McCarty and Dr. Harish Radhakrishna, Georgia Institute of Technology, for critical reading of the manuscript. The source of funding for this study was from the National Institutes of Health Grants NINDS NS044164, U54-NS 34194, NCRR P20 RR15583, and NCRR P20 RR17670.
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