The M1 Receptor Is Required for Muscarinic Activation of Mitogen-activated Protein (MAP) Kinase in Murine Cerebral Cortical Neurons*

Susan E. Hamilton and Neil M. NathansonDagger

From the Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195-7750

Received for publication, December 21, 2000, and in revised form, February 14, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Muscarinic acetylcholine receptors (mAChR) in the central nervous system are involved in learning and memory, epileptic seizures, and processing the amyloid precursor protein. The M1 receptor is the predominant mAChR subtype in the cortex and hippocampus. Although the five mAChR fall into two broad functional groups, all five subtypes, when expressed in recombinant systems, can activate the mitogen-activated protein kinase (MAPK) pathway. The MAPK pathway has been implicated in learning and memory, amyloid protein processing, and neuronal plasticity. We used M1 knock-out mice to determine the role of this receptor subtype in signal transduction in the mouse forebrain. In primary cortical cultures from mice lacking the M1 mAChR, agonist-stimulated phosphoinositide hydrolysis was reduced by more than 60% compared with cultures from wild type mice. Although muscarinic agonists induced robust activation of MAPK in cortical cultures from wild type mice, mAChR-mediated activation of MAPK was virtually absent in cultures from M1-deficient mice. These results indicate that the M1 mAChR is the major subtype that mediates activation of phospholipase C and MAPK in mouse forebrain.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Muscarinic acetylcholine receptors (mAChR)1 are the predominant cholinergic receptors in the central nervous system where they are involved in learning and memory (1), in epileptic seizures (2), and in processing the amyloid precursor protein (3, 4). Of the five subtypes of these seven transmembrane, G protein-coupled receptors, M1 is found in greatest abundance in the cortex and hippocampus where it constitutes 40-50% of the total mAChR (5). The M1, M3, and M5 subtypes preferentially couple via the Gq/11 protein family to activate phospholipase C (PLC), stimulating phosphoinositide (PI) hydrolysis (6). The resulting products, diacylglycerol and inositol trisphosphate, activate PKC and trigger a release of intracellular calcium, respectively. These events initiate an overlapping network of signals, including the activation of mitogen-activated protein kinase (MAPK) (7). M2 and M4 preferentially couple via Gi to inhibit adenylyl cyclase activity but in some cell types can also activate PLC (8, 9). These subtypes can also activate MAPK by direct G protein beta gamma subunit-mediated mechanisms (10) and potentially also via activation of PKC.

The MAP kinases ERK1 and ERK2 (p44 and p42) are known to play a pivotal role in signaling events in the brain where they are involved in neuronal plasticity (11), neuronal survival (12), and processing the amyloid precursor protein (13, 14). They play a crucial role in learning and memory, including long term potentiation (15, 16) and long term spatial memory (17). In addition to activation by G protein-coupled receptors, the ERKs can be stimulated by growth factors signaling through receptor tyrosine kinases. Thus these kinases are poised at a critical position allowing cross-talk between a variety of signal transduction pathways.

Although the functional properties of the individual mAChR subtypes have been well characterized in recombinant systems, the analysis of endogenous mAChR function in cells and tissues expressing multiple subtypes of mAChR is complicated by the lack of subtype-specific agonists and overlapping selectivity of antagonists. In addition, the apparent affinity of a putative subtype-selective antagonist can vary dramatically depending on membrane composition and lipid environment (18), so that the same receptor polypeptide can potentially exhibit different ligand affinities in different cell types. We have previously shown that in mice with a targeted deletion of the gene encoding the M1 receptor, mAChR-mediated suppression of the M current potassium channel is completely lost in sympathetic neurons (19), whereas mAChR-mediated suppression of the M current in hippocampal neurons is unaltered (20). Thus, a given subtype of mAChR may not couple to the same functional pathway in different cell types. In this paper, we use mice lacking the M1 receptor to demonstrate that M1 is the main receptor subtype responsible for activation of PLC and MAPK in cultured cerebral cortical neurons.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Neurobasal medium, B-27 supplement, and penicillin-streptomycin were obtained from Life Technologies, Inc. Carbamylcholine chloride (carbachol), cytosine-beta -D-arabinoside, phorbol 12-myristate 13-acetate (PMA), tetrodotoxin, atropine sulfate, and papain came from Sigma. Research Biochemicals International was the source of oxotremorine methiodide (oxo), (+)-2-amino-5-phosphonopentanoic acid (APV), nimodipine, and trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD). ICN Biomedicals Inc. supplied 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Myo-[2-3H]inositol and ECL reagents came from Amersham Pharmacia Biotech, poly-D-lysine hydrobromide (high molecular weight) came from Collaborative Biomedical Products, phospho-p44/42 MAPK antibody came from New England Biolabs, monoclonal anti-MAPK (ERK1 + ERK2) antibody came from Zymed Laboratories Inc., Immobilon-P membrane came from Millipore, and ion exchange resin AG 1-X8 100-200 mesh (formate form) came from Bio-Rad.

Immunoblot Analysis for Detection of M1 mAChR-- Membranes were prepared from mouse heart, cerebellum, and forebrain as described by Luetje et al. (21). Dissected mouse tissues were placed in phosphate-buffered saline (PBS) containing protease inhibitors, homogenized on ice 25 times in a glass-glass homogenizer, and centrifuged at 36 × g for 15 min to remove debris. The supernatant was centrifuged at 8000 × g for 15 min to pellet membranes. After determination of protein concentration, 55 µg of membrane protein was solubilized in an SDS sample buffer containing M urea and loaded onto a 3.5% stacking SDS-polyacrylamide gel and a 7% separating SDS-polyacrylamide gel, each containing 4 M urea. Proteins were transferred to Immobilon-P membrane, and immunoblot analysis was carried out as described by McKinnon and Nathanson (22) using affinity-purified polyclonal anti-M1 antibody directed against the third cytoplasmic loop of the mouse M1 (19).

Primary Cortical Cell Culture-- Primary cortical cultures were prepared as described by Chan et al. (23). Briefly, the cerebral cortices of newborn mouse brains were digested with papain for 20 min at 37 °C. The rinsed tissue was then triturated 15 times and allowed to settle. Supernatants were saved, and the trituration was repeated on remaining tissue. Supernatants were combined, and cortical neurons were seeded on plates treated with polylysine for a minimum of 2 h. Neurons for PI assays and determination of MAPK activation were plated in 24-well (0.15 × 106 cells/well) and 6-well (1 × 106 cells/well) plates, respectively. Cells were grown in neurobasal medium containing B-27 supplement, glutamine (1 mM), penicillin (0.05 units/ml), and streptomycin (0.05 µg/ml) and maintained at 37 °C in 5% CO2. Medium was changed one and three days after plating and contained 3.6 µM cytosine-beta -D-arabinoside to retard glial growth.

Quantitative Immunoprecipitation Analysis of M3 Expression-- The expression of the M3 mAChR was determined by immunoprecipitation analysis using a polyclonal antibody specific for the M3 mAChR as described (19).

Phosphoinositide Hydrolysis Assays-- After six to eight days in culture, PI turnover was measured as described (24, 25). In brief, cells were incubated overnight in culture medium containing 1 µCi/ml myo-[2-3H]inositol (18.3 Ci/mmol). Cells were washed and incubated 30 min at 37 °C with physiological saline solution (118 mM NaCl, 4.7 mM KCl, 3 mM CaCl2, 1.2 mM KH2PO4, 10 mM glucose, 0.5 mM EDTA, 20 mM HEPES, pH 7.4) containing 10 mM LiCl. Carbachol or ACPD was added for 15 min before the reaction was terminated with ice-cold methanol. Total inositol phosphates were quantified using ion exchange chromatography. Each drug condition was performed in quadruplicate for each of four cultures/genotype.

Determination of MAPK Activation-- MAPK analysis was performed on cultures seven to 10 days after plating. Two h before stimulation, cultures were treated with tetrodotoxin (1 µM), CNQX (40 µM), APV (100 µM), and nimodipine (5 µM) to decrease endogenous synaptic activity (26). Cells were stimulated for the intervals indicated with oxo (100 µM) and PMA (0.1 µM) or pretreated with atropine (3 µM) for 5 min before a 5-min stimulation with oxo. Medium was aspirated, and cells were washed two times with ice-cold PBS and lysed with 100 µl 4× SDS sample buffer. Scraped cells were placed in 1.5-ml polypropylene tubes, sonicated 10 s, boiled 5 min, cooled, and centrifuged. Samples (25 µl) were run on 10% SDS-polyacrylamide gels and transferred to Immobilon-P membrane. Membranes were incubated with phospho-p44/42 MAPK antibody (1:2000) overnight, and bands were detected by ECL. Blots were stripped using the method supplied with Immobilon-P membrane and reprobed with monoclonal anti-MAPK antibody (1:2000) for 2 h, and bands were visualized by ECL. Autoradiograms were scanned (Scan Wizard PPC 3.0.7, Microtek) and processed (Adobe Photoshop 5.0), and the p42 phospho-ERK band intensities were determined (NIH Image 1.62). Results were internally normalized and applied to the intensity data obtained from the respective anti-phospho-MAPK blots to correct for differences in protein loading.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Functional M1 mAChR Protein Fragments Are Not Reconstituted in M1-deficient Mice-- Previously, we showed that the M1-KO mice do not express detectable M1 protein as determined by immunoprecipitation of radioligand-labeled, digitonin-solubilized receptors (19). Recent work has shown that fragments of the mAChR can interact to form a reconstituted functional receptor. The interaction between receptor fragments containing transmembrane domains I-V and fragments containing transmembrane VI-VII depends on the presence of the long third intracellular loop, and chimeric receptors containing these separate domains exhibited agonist-induced functional activity (27, 28, 29). Because our targeting construct used to generate M1-KO mice lacked the nucleotides coding for the first 55 amino acids of the M1 receptor, it would be possible for protein synthesis to initiate at a downstream methionine, resulting in the expression of a truncated polypeptide containing most of the transmembrane domains as well as the third cytoplasmic loop. If this truncated protein were indeed synthesized, it could interact with one of the remaining mAChR polypeptides to produce a protein exhibiting the functional coupling of the M1 receptor but would not necessarily be stable to detergent solubilization and subsequent detection by immunoprecipitation. To determine whether this possibility was occurring, we performed immunoblot analysis using an antiserum prepared against the third cytoplasmic loop of the M1 receptor to ensure that this portion of M1 was absent in our M1-KO mice. In addition to mouse tissues, protein from the mouse Y1 adrenal cell line and Y1 cells stably transfected with mouse M1 (Y1-M1) under an inducible promoter (24) were analyzed simultaneously. A strong signal was detected in the WT forebrain, which was significantly reduced in forebrain from heterozygote mice (Fig. 1). As expected from the negligible expression of the M1 receptor in cerebellum and heart, no signal was detected in these tissues. Immunoreactivity to M1 was not detectable in any of the M1-KO tissues. The Y1-M1 cells express two immunoreactive proteins, the lower corresponding to the band in the M1-HET and WT forebrain samples with an apparent molecular mass of 60 kDa. These results, combined with our previous data (19), indicate that a polypeptide containing the functionally vital third cytoplasmic loop of the M1 receptor is completely absent in the M1-KO mice and that there most likely is no ectopic translation of a truncated protein.


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Fig. 1.   Immunoblot analysis of M1-mAChR expression in WT, HET, and M1-KO mouse tissues. SDS-polyacrylamide gel electrophoresis and immunoblot analysis using an antibody raised against the third cytoplasmic loop of the mouse M1 receptor was performed on equal amounts of membrane protein (55 µg) from M1-KO (-/-), M1-HET (+/-), and WT (+/+). H, heart; F, forebrain; and Cb, cerebellum. Membrane protein (55 µg each) from untransfected Y1 cells (U) and Y1 cells expressing M1 (T) were run as controls.

Decreased mAChR-mediated Activation of PLC in M1-deficient Cortical Cultures-- To measure the effects of inactivating the M1 receptor on mAChR-mediated activation of PLC, we measured the stimulation of PI hydrolysis in primary cortical cultures derived from WT and M1-KO mice in response to the muscarinic agonist carbachol. The mean maximum increase in inositol phosphates obtained with WT cultures was 250% above basal level (Fig. 2A). In M1-KO cultures this maximal level was reduced by a factor of 2.6 with a mean stimulation of 95% above basal.


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Fig. 2.   Muscarinic receptor-mediated phosphoinositide hydrolysis in cortical cultures from WT and M1-KO mice. Primary cortical cultures from WT and M1-KO mice were incubated with myo-[2-3H]inositol overnight and subjected to 15 min stimulations with the mAChR agonist, carbachol (A), or the metabotropic glutamate receptor agonist, ACPD (B) at the concentrations indicated. Results are expressed as percent stimulation above basal and represent the mean ± S.E. of four sets of cultures analyzed in quadruplicate at each concentration.

We previously showed by quantitative immunoprecipitation analyses that the level of the remaining mAChR subtypes was unaltered in the forebrain of M1-KO mice (19). In addition to M1, M3 couples via the Gq/11-family and is present in significant amounts in the forebrain. To ensure that levels of M3 were not altered in cortical cultures, we carried out quantitative immunoprecipitation analyses with an anti-M3-specific antibody. The levels of M3 were similar in cortical cultures prepared from WT (88 ± 17 fm/mg protein) and M1-KO (73 ± 18 fm/mg protein) mice (mean ± S.E.; n = 4).

Metabotropic glutamate receptors also activate PLC via Gq/11 proteins. ACPD, a selective agonist for the metabotropic glutamate receptor, was used as a positive control to ensure that no general defect in G protein or PLC function existed. Similar levels of maximal activation by ACPD were obtained from WT and M1-KO cortical cultures (Fig. 2B), indicating that the PLC signaling pathway downstream of the M1 mAChR is normal in M1-KO mice.

Decreased mAChR-mediated MAP Kinase Activation in M1-deficient Cortical Cultures-- Dual phosphorylation of the MAP kinases ERK1 and ERK2 on Thr-202 and Tyr-204 by MAPK kinase results in their activation; the detection of this dual phosphorylation using a dual-phospho-specific antibody is a widely used, sensitive method for the quantitation of the activation of MAPK in cultured cells and intact tissues (13, 15-17). We performed SDS-polyacrylamide gel electrophoresis and immunoblot analysis using antibodies specific to the activated (dually phosphorylated) forms of ERK to measure stimulation of MAPK in primary cortical cultures from WT and M1-KO mice. In WT cultures the time course of treatment with the agonist oxo showed maximal levels of phospho-ERK within 5-10 min of agonist stimulation with a return to basal level by 30 min (Fig. 3A, a). This blot was stripped and reprobed with an antibody to the non-phosphorylated forms of ERK to ensure that differences in band intensity observed with phospho-ERK antibodies were not due to differences in protein loading (Fig. 3A, b). Immunoblot analyses comparing stimulation of WT and M1-KO cultures showed a robust activation of MAPK by oxo in WT cortical neurons (Fig. 3B, a) and low to nonexistent activation in the M1-KO cultures (Fig. 3C, a). Quantitative analysis of the immunoblots showed that WT cultures treated with oxo exhibited a mean stimulation of >300% above basal compared with 31% for the M1-KO cultures (n = 5 for each genotype; Fig. 4). This activation is decreased by >75% when cultures are pretreated with the muscarinic-specific antagonist atropine, indicating that the effects of oxo are due to mAChR activation. PMA, a direct activator of PKC, induced a similar level of MAPK activation in WT (1050%) and M1-KO cultures (890%), respectively, indicating that the signaling pathway coupling stimulation of PKC to activation of the MAPK pathway is unaltered in M1-deficient neurons.


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Fig. 3.   Immunoblot analysis of mAChR-mediated MAPK activation in cortical cultures from WT and M1-KO mice. A, immunoblot analysis of the time-course of MAPK activation in WT cortical cultures. B and C, immunoblots of MAPK activation in WT and M1-KO cultures, respectively. Cultures were "turned down" for 2 h and treated with PBS (10 min), PMA (10 min), atropine (5 min) followed by oxo (5 min) (atr/oxo), or oxo alone for the times indicated. Membranes were probed with anti-phospho-p44/p42-MAPK antibody (a) and then stripped and probed with anti-MAPK antibody (b) to ensure similar levels of protein loading.


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Fig. 4.   Quantitative analysis of mAChR-mediated MAPK activation in WT and M1-KO primary cortical cultures. Data represent the mean ± S.E. of five sets of cultures for each genotype, which were treated with PBS (10 min), oxo (5, 10 min), and atropine (5 min) followed by oxo (5 min), (atr/oxo), or PMA (10 min). Intensities of p42 phospho-MAPK bands on immunoblots were corrected for variations in protein loading using the normalized p42 band intensities from the anti-MAPK immunoblots and expressed as percent stimulation above basal.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A growing number of studies show that functionally significant intermolecular interactions between muscarinic receptors can occur and that the third cytoplasmic loop is vital for these potential heteromolecular interactions. In the original construct designed to ablate the M1 gene product by homologous recombination, nucleotides coding for the N terminus and first transmembrane domain were deleted (19). However, the nucleotide sequence for >85% of the coding sequence, including the third cytoplasmic loop, is still present in these mice. To ensure that this portion of the receptor was not expressed, we performed immunoblot analyses using an M1-specific antibody raised against the third cytoplasmic loop of M1 (amino acids 233-332). Our results confirm that this key region of the receptor is significantly reduced in the forebrain of the M1-HET mice and is not detected in the M1-KO mice.

Studies using recombinant M1 mAChR show that it couples via the Gq/11 protein family to the activation of PLC (24, 30). The M1 receptor constitutes approximately half of the total mAChR in adult mouse forebrain (19). In primary cortical cultures from newborn M1-KO mice, agonist-stimulated PI hydrolysis was reduced by >60% compared with WT cultures. Thus, the M1 receptor is the predominant mAChR subtype responsible for PLC activation in mouse cortical neurons. Despite the loss of M1, there was still significant carbachol-stimulated hydrolysis of PI. This is not unexpected because M3 also couples to PLC and is expressed to similar extents in WT and KO cortical cultures. The similar levels of PI hydrolysis obtained with the metabotropic glutamate receptor agonist ACPD in both WT and M1-KO cultures demonstrate that the G protein/PLC interactions are unaltered in the M1-KO mice.

Although activation of mAChR in the brain has recently been shown to activate MAPK in rat cortical cultures (16), most work demonstrating the relationship between mAChR and MAPK has been performed in established cell lines. In human neuroblastoma SH-SY5Y cells, carbachol was found to activate MAPK via M3 and a PKC-dependent pathway (31). When M1 is overexpressed in PC-12 cells, agonist-induced stimulation leads to MAPK activation (14). Although the predominant mAChR subtype in native PC-12 cells is M4 (~95%), carbachol-induced MAPK activation was completely abolished with a highly selective M1 toxin (32). However, pretreatment with pertussis toxin, which uncouples members of the Gi/o family from their receptors, inhibited 70% of this agonist-induced activation, suggesting that either ectopic coupling of M1 to Gi/o proteins or signal transduction downstream of M4 also contributes to the muscarinic agonist activation in this neuronal cell line.

MAPK activation in our WT primary cortical cultures ranged from 180 to 540% over basal values, similar to the values reported in rat cortical cultures (Fig. 3A, Ref. 16)). The maximal MAPK activation occurred in our WT mouse cultures within 5-10 min of agonist stimulation and rapidly returned to baseline, in contrast to the more persistent (30-60 min) activation reported for rat cultures (16). This may be due to differences in species or tissue culture conditions. In M1-KO cultures the range of MAPK stimulation by agonist was reduced to -70 to 120%. The average magnitude of stimulation decreased from 320% over basal in cultures from WT mice to 31% in the cultures from M1-KO mice. Thus M1 is the mAChR responsible for coupling to ERK activation in our primary cortical neuron culture system. The M3 receptor is present in similar amounts in our WT and KO cultures and most likely accounts for the agonist-induced PLC activation remaining in the M1-KO cultures. The much greater impairment in MAPK activation compared with PLC activation observed in the M1-KO cultures suggests that the M3 receptor, although still able to evoke significant activation of PLC, is relatively ineffective in mediating activation of MAPK in these neuronal cultures. PMA-induced activation of MAPK was similar in WT and M1-KO cultures indicating that signaling downstream of PKC is unaltered in mice lacking M1 mAChR.

In summary, these results show that there are significant defects in mAChR-mediated activation of PLC and MAPK in the forebrains of mice lacking the M1 receptor. Both the M1 and M3 receptors couple to members of the Gq-family of G proteins, although it is not known if there are subtle differences in the specificity of the two receptors for either alpha  or beta gamma subunits; the results here suggest that the signaling pathways activated by the two receptors in cortical neurons appear to be different. The M1-KO mice will be useful in determining the PKC-dependent and PKC-independent pathways by which mAChR mediate secretion of soluble amyloid precursor protein. In addition, these mice should be useful in determining the relationship between M1 mAChR, MAPK activation, long-term potentiation, and learning and memory. Indeed, preliminary data indicate that mice lacking the M1 mAChR exhibit defects in the consolidation of hippocampus-dependent learning and show a deficit in long term potentiation (33).

    ACKNOWLEDGEMENTS

We thank Dr. Guy C.-K. Chan for his generous advice regarding primary cortical culture techniques. We also thank Drs. Robin M. Gibson, Laurie S. Nadler, and Michael L. Schlador for helpful comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant NS26920.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: Dept. of Pharmacology, P. O. Box 357750, Univ. of Washington School of Medicine, Seattle, WA 98195-7750. Tel.: 206-543-9457; Fax: 206-616-4230; E-mail: nathanso@u.washington.edu.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M011563200

    ABBREVIATIONS

The abbreviations used are: mAChR, muscarinic acetylcholine receptor; PLC, phospholipase C; PI, phosphoinositide; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; MAP, mitogen-activated protein; ERK(s), extracellular signal-regulated kinase(s); PMA, phorbol 12-myristate 13-actetate; oxo, oxotremorine methiodide; APV, (+)-2-amino-5-phosphonopentanoic acid; ACPD, trans-(1S, 3R)-1-amino-1,3-cyclopentanedicarboxylic; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; PBS, phosphate-buffered saline; KO, knock-out; WT, wild type; HET, heterozygote.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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