- Research article
- Open Access
Differential regulation of muscarinic M1 receptors by orthosteric and allosteric ligands
© Davis et al; licensee BioMed Central Ltd. 2009
Received: 7 May 2009
Accepted: 2 December 2009
Published: 2 December 2009
Activation of muscarinic M1 receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists.
The orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M1 receptors and stimulated interaction of the receptors with β-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles.
These results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.
Muscarinic M1 receptors are the predominant muscarinic receptor subtype expressed in the brain with high levels of expression in the cerebral cortex and hippocampus. Muscarinic M1 receptors are believed to mediate many important central processes such as cognition and memory [1, 2] and thus are targeted for the development of drugs to treat various neurological disorders, such as Alzheimer's disease. However, discovery of selective M1 agonists has been challenging due to the highly conserved orthosteric acetylcholine binding site of muscarinic receptors. Recent structure-function studies have led to the identification of functionally selective muscarinic M1 agonists [3–8]. Mutagenesis studies have shown that mutations that abolish activation of M1 receptors by orthosteric ligands do not affect, and in some cases even enhance, activation by these selective ligands [4, 9]. These novel selective ligands have been named allosteric M1 agonists because their mode of interaction with M1 receptor is different from that of orthosteric agonists [4, 10–12].
While allosteric M1 agonists appear to bind to a receptor domain different from that of the orthosteric ligands, in many cases they have been shown to induce similar signaling pathways as the orthosteric M1 agonists. Moreover, allosteric M1 agonists can displace conventional orthosteric M1 muscarinic radioligands such as [3H]-NMS [4, 12–14]. Based on these data, it has been suggested that there is some degree of overlap between the allosteric and orthosteric binding sites on the M1 receptor for these ligands .
A recent study  has demonstrated that some allosteric muscarinic M1 agonists differentially activate downstream signaling pathways for M1receptors, suggesting "stimulus trafficking" with respect to receptor signaling events. However, presently little is known about the potential differences between orthosteric and allosteric muscarinic M1 ligands in modulating receptor regulatory pathways such as internalization, down-regulation or recycling. The present study was carried out to assess these cellular regulatory events in muscarinic M1 receptors in response to a number of allosteric and orthosteric M1 agonists.
In order to understand the mode of interaction of a ligand with the receptor, it is important to characterize receptor-ligand interaction in multiple assays and in multiple systems  and also to correlate it with the induction of downstream signaling and regulatory pathways. Receptor regulatory events include receptor processes such as desensitization (rapid uncoupling of receptors from signaling molecules), internalization/endocytosis (translocation of receptors from the cell surface into intracellular vesicles) and down-regulation (translocation of receptors into lysosomes where receptors are degraded).
In the present study, several different assays were employed in order to comprehensively evaluate various intracellular signaling and regulatory processes. The results demonstrate that allosteric ligands differ from orthosteric ligands and amongst each other in induction of internalization, down-regulation, recycling and signaling of M1 muscarinic receptors.
A preliminary report of the present work was presented at the meetings for Society for Neuroscience, 2007 and at Recent Advances in Muscarinic Receptor Pharmacology and Therapeutics Colloquium, 2008.
Allosteric and orthosteric agonists induce signaling of human muscarinic M1 receptors
Activation of muscarinic M1 receptors as measured by R-SAT® and PI assays
M1 Agonist RSAT
6.2 ± 0.3
100 ± 10
6 ± 0.1
100 ± 3
7.4 ± 0.3
105 ± 13
7.0 ± 0.1
106 ± 7
5.7 ± 0.2
93 ± 15
6.2 ± 0.9
97 ± 31
7.4 ± 0.5
120 ± 21
8.4 ± 0.2
99 ± 12
7.4 ± 0.2
76 ± 13
7.1 ± 0.4
67 ± 23
6.7 ± 0.3
62 ± 14
6.5 ± 0.5
79 ± 14
7.6 ± 0.4
102 ± 21
7.7 ± 0.3
90 ± 17
Allosteric M1 agonists, AC-260584 and xanomeline, show distinctive effects on receptor endocytosis
Hypertonic sucrose treatment blocks M1 receptor internalization
Lack of M1 receptor recycling following treatment with allosteric agonists
Recycling of muscarinic receptors after treatment with various ligands
3 h recycle
48 ± 4*
102 ± 10
60 ± 2*
97 ± 1
65 ± 5*
69 ± 7*
63 ± 3*
69 ± 1*
59 ± 2*
64 ± 3*
Allosteric and orthosteric M1 agonists induce down-regulation of M1 receptors in CHO-M1 cells
Allosteric and orthosteric M1 agonists induce recruitment of β-arrestin-1 to M1 receptors
Activation of muscarinic M1 receptors as measured by BRET-2
4.6 ± 0
100 ± 0
5.6 ± 0.2
100 ± 12
5.4 ± 0.3
16 ± 3*
5.4 ± 0.6
12 ± 0.7*
6.2 ± 0.14
26 ± 6*
7.2 ± 0.3
16 ± 6*
M1 receptor endocytosis is distinctively affected by AC-260977 an AC-260584 related ligand
Activation of muscarinic M1 receptors by analogues of AC-260584 as measured by R-SAT
M1 Agonist RSAT
6.6 ± 0.2
66 ± 16
7.2 ± 0
107 ± 16
7.7 ± 0.1
107 ± 10
AC-260584 and xanomeline induce persistent activation of M1 receptors
The present study was designed to characterize various receptor regulatory pathways initiated after treatment with allosteric or orthosteric M1agonists. We demonstrate that specific regulatory pathways initiated after activation of M1 receptors with different ligands are unique to each ligand and not due to the allosteric or orthosteric nature of receptor-ligand interaction.
Among the M1 agonists in the present study, AC-42 and AC-260584 have been characterized as allosteric agonists [4, 12, 15]. Other compounds, NDMC and xanomeline, were reported to have an allosteric component in their interaction with M1 receptors [4, 9, 13, 16]. Carbachol, oxotremorine-M and pilocarpine are classical orthosteric agonists.
All agonists in this study were characterized as either full agonists or high efficacy partial agonists at M1 receptors as measured by RSAT and PI assays. No correlation could be made between efficacy or potency of each agonist and its efficacy in activation of various receptor regulatory processes. Moreover, none of the agonists studied induced receptor desensitization, as measured by the lack of a shift in carbachol dose response curve or a change in maximal response in PI hydrolysis assay (data not shown). Still, the possibility of desensitization of other signaling pathways cannot be ruled out.
To assess the differences in receptor regulatory processes induced by each agonist, the extent of loss of cell surface receptors and total receptors were evaluated. All ligands were capable of inducing a loss of receptor binding. These results indicate that the processes of receptor internalization and down-regulation are induced after treatment with all these ligands.
To further assess receptor internalization/endocytosis, confocal microscopy was used to visualize receptor localization. After treatment with orthosteric ligands, carbachol, pilocarpine and oxotremorine-M, M1 receptors could be visualized in endosomes. However, after treatment of the cells with the allosteric agonist AC-260584, no receptors could be observed in the endosomes. To test the hypothesis that allosteric interaction with the receptors was responsible for this difference, we tested several other allosteric agonists for their propensity to induce receptor endocytosis into endosomes. The allosteric ligands AC-42 and NDMC were able to induce receptor internalization/endocytosis, while xanomeline was not. To determine whether lack of endocytosis was associated with the specific structure of AC-260584, three close analogues of this compound were studied. Two of these compounds induced internalization/endocytosis of M1 receptor while one did not. These results indicate that allosteric nature of the ligand-receptor interaction is not sufficient explanation for lack of ligand-induced endocytosis.
It was puzzling that no intracellular vesicles could be seen after treatment with AC-260584 and xanomeline while binding to cell surface receptors was reduced after treatment with these ligands. One possible reason for this observation could be induction of different endocytic pathways by these ligands. It has been previously demonstrated that carbachol-induced internalization of M1 receptors is mediated via clathrin-coated pits . Formation of clathrin-coated vesicles can be blocked by treatment with hypertonic sucrose solution [18–21]. In order to investigate whether AC-260584 and xanomeline induce similar endocytic pathways as carbachol, cells were pretreated with hypertonic sucrose solution to block formation of clathrin coated vesicles. Hypertonic sucrose treatment blocked receptor internalization induced by all the agonists, indicating that formation of clathrin coated vesicles is necessary for the loss of cell surface receptor binding after treatment with all these ligands. This observation suggests that divergence of receptor regulatory processes induced by the different ligands occur down-stream from the formation of clathrin-coated pits.
Internalization of G-protein coupled receptors has been shown to involve interaction with β-arrestin [22–27]. Moreover, it has been suggested that endocytosis and signaling of muscarinic M1 receptors involve interaction with β-arrestin [23, 24, 26]. In order to determine if absence of interaction with β-arrestin attributed towards the differences observed in vesicle formation, interaction of M1 receptors with β-arrestin-1 was assessed using BRET-2 assays. All ligands studied were able to stimulate recruitment of β-arrestin-1 to M1 receptors. While carbachol and oxotremorine-M were full agonists in BRET-2 assay, all other ligands, including pilocarpine, were partial agonists. Other studies have also described pilocarpine as a partial agonist at M1 receptors [28, 29]. Although AC-260584 and xanomeline stimulated an increase in the BRET-2 signal with lower efficacy compared to carbachol and oxotremorine-M, the lower efficacy in this interaction could not explain the absence of vesicle formation in response to these ligands. This conclusion is based on the fact that while both AC-42 and pilocarpine have similar potency and efficacy in the BRET-2 assay compared to AC-260584 and xanomeline, both compounds can induce receptor endocytosis into endosomes.
Another difference observed between the receptor regulatory processes induced by the different agonists was the difference seen in the recycling of the receptors back to the cell surface. While M1 receptors recycled back to the cell surface after treatment with orthosteric ligands (carbachol and oxotremorine-M) following treatment with allosteric ligands (AC-260584, AC-42, xanomeline or NDMC) and ligand removal, binding to cell surface receptors remained low. These results suggest that after removal from the cell surface, receptors sequestered following treatment with the latter set of compounds do not return to the cell surface. We have shown that after a brief treatment with some of these ligands, M1 receptors remain active and produce higher basal levels of inositol phosphates. Thus, it is likely that receptors treated with these ligands remain in a sequestered state and continue to signal via G-proteins (AC-260584 and xanomeline) or other signaling proteins. Following this continued signaling event, the receptors are then processed through various degradative processes and subsequently destroyed.
The results from the present study indicate that M1 signaling and regulatory pathways induced by different allosteric M1 agonists are ligand specific. Allosteric agonists differ from orthosteric agonists and amongst each other in their ability to induce different regulatory processes for M1 receptors. Moreover, it is important to note that although there are differences in how multiple types of agonists can regulate the activity of M1 receptors, activation with allosteric ligands, like activation with orthosteric ligands, ultimately results in receptor internalization/down-regulation. Thus, it is expected that the normal physiological limits on receptor signaling will occur regardless of which agonist is used.
Human embryonic kidney (HEK-293) cells were purchased from American Tissue Culture Collection. Chinese hamster ovary (CHO) cells stably expressing human muscarinic M1 receptors (CHO-M1) were established as described previously . [3H]-NMS and [3H]-QNB were purchased from GE Healthcare (Piscataway, NJ). Carbachol, atropine, oxotremorine-M, pilocarpine and poly-D-lysine were purchased from Sigma-Aldrich (St. Louis, MO). Xanomeline, N- desmethylclozapine (NDMC), AC-260584 (4-[3-(4-butylpiperidin-1-yl)propyl]-7-fluoro-2H- benzo[b][1, 4]oxazin-3-one)), AC-42 (4-(4-butylpiperdin-1-yl)-1-(2-methylphenyl)butan-1-one), AC-260515 (4-[3-(4-butyl-piperidin-1-yl)-propyl]-7-chloro-2H- benzo[b][1, 4]oxazin-3-one), AC-262469 (4-[3-(4-cyclopropylmethyloxypiperidin-1-yl)propyl]-7-fluoro-2H- benzo[b][1, 4]oxazin-3-one) and AC-260977 ((R)-4-[3-(4-butylpiperidin-1-yl)propyl]-7-fluoro-2H- benzo[b][1, 4]oxazin-3-one) were synthesized at ACADIA Pharmaceuticals LLC. The EE-hM1 antibody was prepared as described previously . All other reagents were obtained from common vendors.
Receptor binding assays
Two different systems were employed to assess receptor internalization and down-regulation in response to various ligands. This selection was based on previous data identifying each system as the best choice for the specific study.
Previous studies have shown that transiently transfected HEK-293 cells efficiently internalize M1 muscarinic receptors [34, 35] while CHO cells do not . To quantify receptor internalization, agonist-induced decreases in cell surface receptor binding were measured in HEK-293 cells transiently transfected with hM1 receptor by using non cell-permeating quaternary amine, [3H]-NMS. In addition, in each case total receptor binding was measured by the membrane permeating tertiary amine, [3H]-QNB. In each case, the highest concentration of agonist that did not result in a reduction of total receptor binding after 2 hours, as measured by [3H]-QNB, was selected for the assay. Using this process for the selection of agonist concentration ensured that all residual ligand had been completely removed and could not contribute to reduction of receptor binding.
Previous studies have demonstrated that after chronic treatment with the muscarinic agonist carbachol, hM1 receptors stably expressed in HEK-293 cells do not efficiently go through receptor down-regulation process  while CHO cells are a good model to study this regulatory pathway [21, 36]. Thus, in order to assess receptor down-regulation, loss of total receptor binding was measured in stably transfected CHO cells using [3H]-QNB. CHO-M1 cells were grown in F-12 medium supplemented with 10% fetal bovine serum.
HEK-293 cells were grown in DMEM medium supplemented with 10% fetal bovine serum. For studies using transiently transfected cells, exponentially growing HEK-293 cells (6 × 106 cells) were seeded onto 10 cm tissue culture dish and transfected with hM1 in PSI vector using FuGene HD transfection reagent (Roche Applied Sciences, Indianapolis, IN) following manufacturer's instructions. One day after transfection, the cells were harvested using phosphate buffered saline (PBS) containing EDTA and seeded onto 24-well tissue culture plates as described below.
For receptor binding studies, cells were seeded onto 24-well tissue culture plates (poly D-lysine coated for HEK-293 cells), allowed to attach overnight, and then treated with various concentrations of the compounds in serum-free medium at 37°C for 2 hrs (internalization studies, HEK-M1) or 24 hrs (down-regulation studies, CHO-M1). Cells were placed on ice, washed three times with ice-cold PBS, and incubated with PBS containing saturating concentration of [3H]-NMS (2 nM, internalization assay) or [3H]-QNB (1 nM, down-regulation assays) at 12°C for 90 min [21, 34]. These conditions were established previously and were sufficient for achieving equilibrium binding [21, 34]. Binding assays were carried out at 12°C to ensure no receptor recycling occurred. Cells were placed on ice, scraped, harvested by filtration (24-well GF/B plates, Perkin Elmer, Waltham, Massachusetts), and washed three times with ice-cold PBS. The radioactivity on the filters was quantitated by liquid scintillation counting using Top Count (Perkin Elmer, Waltham, Massachusetts). Average hM1 receptor expression in untreated transiently transfected HEK cells was 325,000 sites/cell as measured by [3H]-NMS and 360,000 site/cell as measured by [3H]-QNB. Average receptor expression in untreated CHO-M1 cells was 280,000 sites/cells as measured by [3H]-QNB. Thus receptor expression levels were roughly similar in both these cells lines.
HEK-293 cells transiently transfected with hM1 receptors were treated with various ligands for 30 minutes at 37°C. Following treatment with the ligands, control (non-recycle) plates were washed with ice-cold PBS and 2 nM [3H]-NMS in PBS was added for 90 min at 12°C. Recycling plates were washed 3× with room temperature PBS. Serum free media was added and cells were incubated for 3 hrs at 37°C. After this incubation period, presence of receptors at the cell surface, as measured by binding to the membrane impermeable tracer [3H]-NMS, was determined as described above.
Hypertonic sucrose treatment
HEK-293 cells transfected with hM1 receptors were plated onto poly-D-lysine coated 24-well plates on the day after transfection. The cells were incubated for 30 minutes in the presence or absence of hypertonic sucrose (0.4 M) prior to addition of the ligands and an additional incubation for 30 min with the ligands. Cells were then washed with room temperature (RT) PBS, ice cold PBS containing 2 nM [3H]-NMS was added and binding carried out for 90 min at 12°C. Cells were removed from the plates, harvested onto 24-well filter plates and washed with cold PBS. Radioactivity was quantitated by scintillation counting using a Top Count scintillation counter.
Immunofluorescence confocal microscopy
Construction of hM1 gene containing the EYMPME (EE) epitope tag (EE-hM1) and generation of mouse monoclonal antibody to EE tag were previously described [17, 33]. Internalization of this EE-hM1 receptor has been well characterized and it has been shown that the N-terminal tag does not interfere with normal trafficking and signaling of the M1receptors [17, 33].
Transiently-transfected HEK-293 cells expressing EE-hM1 receptors were grown overnight on CC2 chamber slides (Nunc Inc., Napperville, IL). Treatment with various concentrations of compounds was carried out at 37°C for 2 hrs. After removing the ligands, cells were fixed for 10 min at RT with 3.7% paraformaldehyde in PBS, and permeabilized in PBS containing 0.25% fish gelatin and 0.04% saponin. Following fixation, cells were labeled with anti-EE monoclonal antibody for 1 h, washed three times with PBS, incubated with Alexa Fluor 488 goat anti-mouse secondary antibody (Invitrogen, Carlsbad, CA) for 30 min in dark, followed by three washes with PBS and one with water. Slides were mounted using Fluoromount G [17, 33]. Images were collected on a Delta Vision Optical Sectioning microscope consisting of an Olympus IX-70 microscope (Tokyo, Japan) and a photometrics CH 350 cooled CCD camera. An Olympus oil immersion 60× objective was used to collect the images.
Receptor selection and amplification (RSAT) assays
Receptor Selection and Amplification (RSAT) functional assays were carried out as described previously [4, 5]. Concentration response curves were generated using non-linear regression to fit the data to appropriate logistic equations using GraphPad Prism Software (Graph-Pad Software, Inc., San Diego, CA).
Phosphatidylinositol (PI) hydrolysis assays
PI hydrolysis assays were performed as described previously with some modifications [4, 5]. Exponentially growing CHO cells stably expressing hM1 muscarinic receptors (CHO-M1) were harvested, plated onto 96-well TC plates in media containing [3H]-myo-inositol (2 μCi/ml), and allowed to attach overnight. On the day of the assay, supernatant was removed and the cells washed three times. Fresh serum-free medium containing 10 mM LiCl and varying concentrations of each ligand was added to the cells. After 60 minutes at 37°C, supernatant was removed and 20 mM formic acid was added to the cells. Following 60 minutes at 0°C, a sample of the supernatant was removed and counted in the scintillation counter. Data were analyzed using non-linear regression analysis using GraphPad Prism Software.
Bioluminescence resonance energy transfer (BRET-2) assay
BRET-2 assays were performed as described  with the following modifications. cDNA encoding the human muscarinic M1 receptor was cloned into the plasmid pRLuc(h) (Perkin Elmer Life Sciences, Waltham, Massachusetts) to generate a DNA vector for expression of the fusion protein M1-Luc (M1 carboxyl-terminally tagged with Renilla luciferase). Beta-arrestin-1 cDNA was cloned into plasmid DNA pGFP2 (PerkinElmer Life Sciences, Waltham, Massachusetts) to generate a DNA vector for expression of the fusion protein GFP2-β-arrestin-1 (β-arrestin-1 amino-terminally tagged with GFP2). HEK-293T (2 × 106) cells were plated onto 10 cm2 tissue culture plates and cultured for two days. Cells were transiently co-transfected with 1 μg of the bioluminescence donor plasmid encoding M1-Luc and 40 μg of the fluorescence acceptor plasmid encoding GFP2-β-arrestin-1 using Polyfect (QIAGEN, Valencia, CA) following manufacturer's instructions. Two days after transfection, cells were harvested and re-suspended at a density of 2 × 106 - 4 × 106 cells/ml (depending on transfection efficiency) in PBS pH 7.5, containing glucose and sodium pyruvate. BRET-2 signals were calculated as the ratio between the Renilla luciferase emission and the GFP2 emission corrected by the background emissions of non-transfected cells.
Induction of persistent activation of muscarinic receptors
CHO-M1 cells were labeled with [3H]-myo-inositol overnight as described above for PI assays. On the day of the assay, the cells were washed three times with PBS and treated for 1 minute with serum free media alone, or medium containing carbachol, AC-260584, xanomeline, AC-42 or NDMC. Basal levels of PI turnover after this 1 minute of treatment with each ligand was measured by washing off the ligand, addition of assay buffer containing 10 mM LiCl2 and incubation for an additional 60 minutes at 37°C . Inositol phosphate levels were assessed as described above.
CND has published several manuscripts on the signaling of G protein coupled receptors.
JL has over 15 years of experience working with muscarinic receptors, more than 10 years investigating regulatory pathways of various receptors including muscarinic receptors and has published several manuscripts on signaling and regulation of muscarinic receptors.
SRB, JL and DWBspent several years investigating activities of various allosteric and orthosteric agonists of muscarinic receptors.
The authors would like to thank Abhishek Bajpai, Stephen Fuhs and Qing Lu and Thomas Son for their technical assistance, Dr. Ethan Burstein for critical review of this manuscript and Professor Tamas Bartfai (The Scripps Research Institute, La Jolla, CA) for use of the confocal microscope. This work was supported by ACADIA Pharmaceuticals Inc.
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