The C-terminal tail of the M3-muscarinic receptor possesses anti-apoptotic properties.

This study investigates the mechanisms by which the muscarinic receptor gene family can protect against apoptosis. Chinese hamster ovary cells transfected with human muscarinic receptor subtypes underwent apoptotic cell death following treatment with the DNA-damaging agent etoposide. Apoptosis was significantly reduced following muscarinic receptor stimulation of cells that were transfected with receptor subtypes that couple to the Gq/11/phospholipase C pathway, namely M1, M3, and M5. No protection was detected in cells transfected with the Gi-coupled M2 and M4 receptors. Further analysis of the Gq/11-coupled M3 receptor revealed that truncation of the carboxyl-tail (Delta 565-M3 mutant) removed the ability of the receptor to protect against etoposide-induced cell death. This mutation did not affect the ability of the receptor to signal through the phospholipase C pathway. Furthermore, activation of the Delta 565-M3 receptor resulted in robust activation of the extracellular-regulated kinase (ERK) and c-Jun kinase (JNK). The Delta 565-M3 receptor mutant also underwent agonist-driven phosphorylation in a similar manner to the wild-type receptor indicating that the anti-apoptotic effect of the M3 receptor is independent of receptor phosphorylation. Consistent with this was the fact that two M3-muscarinic receptor mutants deficient in agonist-induced receptor phosphorylation were capable of producing a full anti-apoptotic response. We conclude that the anti-apoptotic response of the muscarinic receptor family was confined to the Gq/11-coupled members of this family. The direct involvement of Gq/11/phospholipase C signaling and the ERK-1/2 and JNK pathways together with receptor phosphorylation in the anti-apoptotic response were eliminated. Mutation of a poly-basic region within the short C-terminal tail of the M3-muscarinic receptor inhibited the ability of the receptor to induce an anti-apoptotic response. We conclude that the conserved poly-basic region in the C-terminal tail of the M1, M3, and M5 receptors contributes to the ability of these receptors to mediate protection against apoptotic cell death.

Apoptosis or programmed cell death is the process by which cells initiate a series of biochemical reactions that ultimately results in the breakdown of the cell cytoskeleton, destruction of the integrity of the nucleus, and cleavage of cellular DNA, leading to cell death. Apoptosis is manifested during a number of physiological processes, including embryogenesis and development where specific populations of cells are targeted for elimination (1). However, it is also now widely appreciated that the process of programmed cell death can play a central role in the onset and development of various disease states, particularly in the mammalian CNS, and is characteristic of such neuronal disorders as Alzheimer's disease and ischemia (2).
The process of cellular degradation during apoptosis involves the activation of intracellular cysteine proteases (caspases) that cleave substrates after specific aspartate residues. Caspases exist in healthy cells as zymogens with low degradative activity but become fully activated via autocatalytic processing in response to an apoptotic signal (3). Apoptosis can be induced in mammalian cells by receptor-specific mechanisms (e.g. Fas ligand, tumor necrosis factor-related apoptosis-inducing ligand or TRAIL) or by the addition of cytotoxic agents (4). However, in the case of UV irradiation or the DNA-damaging agent etoposide, apoptosis in cells occurs via biochemical pathways that appear to involve mitochondrial depolarization, an increase in the mitochondrial permeability transition pore, and release of apoptogenic factors such as cytochrome c from the inner mitochondrial membrane to the cytosol. Cytosolic cytochrome c is able to interact with Apaf-1 and pro-caspase 9 to form the apoptosome (5,6). The formation of this large multimeric complex is the signal for the autocatalytic processing and activation of pro-caspase 9, which in turn lead to the sequential activation of downstream executioner caspases and degradation of cellular proteins, which ultimately leads to the destruction of the cell cytoskeleton, nuclear structure, and DNA (4).
It is now clear that a number of G-protein-coupled receptors (GPCRs) 1 have the ability to control apoptosis, either initiating a pro-or anti-apoptotic signal depending on the receptor subtype and cell type in which the receptor is expressed (7,8). Among those receptors initiating an anti-apoptotic signal is the muscarinic receptor family, which is composed of five distinct members, three of which (M 1 , M 3 , and M 5 ) are coupled to the G q/11 -phospholipase C pathway and two (M 2 and M 4 ) that in-hibit adenylate cyclase via coupling to G i/o (9). Muscarinic receptors (most likely the M 3 -subtype) expressed endogenously in cerebellar granule cells have been shown to protect against apoptotic-cell death induced by culturing in non-depolarizing conditions (10). Similarly, the G q/11 -coupled M 1 -muscarinic receptor expressed as a recombinant protein in PC-12 cells protected against apoptosis following serum deprivation (11). The mechanism by which muscarinic receptors are able to attenuate apoptosis is not clear with some studies demonstrating a role for the cell survival pathway mediated by phosphatidylinositol 3-kinase/Akt (8), and others reporting that this pathway in addition to the mitogen-activated protein kinase (MAPK) pathway and that downstream G-protein-mediated second messenger pathways (e.g. calcium, cAMP, and protein kinase C) are not important (11,12).
In the current study we use apoptosis induced by the DNAdamaging agent, etoposide, in Chinese hamster ovary (CHO) cells as our experimental model to investigate the properties of the anti-apoptotic response elicited by the muscarinic receptor family. We show that only the muscarinic receptor subtypes coupled to G q/11 -proteins (M 1 , M 3 , and M 5 ) elicit an anti-apoptotic response. Further analysis of the M 3 -muscarinic receptor revealed that the anti-apoptotic signal does not involve direct activation of the G q/11 /phospholipase C-signaling pathway, the MAPK pathway, or receptor phosphorylation but that a conserved poly-basic region within the C-terminal tail of the receptor contributes to the anti-apoptotic response.
The ⌬565-M 3 mutant was produced by introducing a stop codon at position 565 using the 5Ј primer, CCCGGATCCATGACCTTGCACAA-TAACAGTACA, and the 3Ј primer, CCCGAATTCCTAGTCACACTGG-CACAGCAGCAG. All mutant cDNAs were checked for the correct mutations by sequencing (AltaBioscience, Birmingham, UK). The K 3 A mutant was produced by mutating 565 KKKRRK 570 to alanine using the 5Ј primer, AAGATGCTGCTGCTGGTCCAGTGTGACGCCGCGGCGG-CGGCCGCGCAGCAGTACCAGCAGAGACAGTCGGTC, and the 3Ј primer, GACCGACTGTCTCTGCTGGTACTGCTGCGCGGCCGCCGC-CGCGGCGTCACACTGGCACAGCAGCAGCATCTT.
Tissue Culture-Chinese hamster ovary (CHO) cells were transfected with cDNA encoding for the wild-type human M 1 , M 2 , M 3 , M 4 , M 5 , mutant 6, T 3 A tail, or ⌬565-muscarinic receptor, and stably transfected clones were obtained by G418 sulfate selection. CHO cells stably expressing the muscarinic M 1 , M 2 , M 3 , M 4 , or M 5 receptors at 1.58, 0.75, 0.40, 1.08, and 0.85 pmol/mg of protein respectively, were chosen for further study. CHO cells stably expressing the ⌬565-M 3 , T 3 A tail, mutant 6, and K 3 A receptors at 0.35, 0.23, 0.36, and 0.31 pmol/mg of protein were chosen for further study. Stably transfected CHO cells were maintained in ␣-MEM media supplemented with 10% fetal calf serum, 100 IU/ml penicillin/streptomycin, 2.5 g/ml fungizone, and 500 g/ml G418 sulfate. In experiments investigating various M 3 -muscarinic receptor mutants, at least two clones were tested to eliminate clonal artifacts.
Annexin V-FITC and Propidium Iodide Staining-CHO-m3 cells were plated on 25-mm glass coverslips in six-well plates. Following overnight incubation with the appropriate drugs, cells were washed twice with binding buffer. Binding buffer (1 ml) was added together with 5 l each of annexin V-FITC and propidium iodide to each well. Plates were then incubated for 15 min in the dark at room temperature. Coverslips were washed 3ϫ with phosphate-buffered saline (PBS) and then coated with Citifluor to enhance fluorescence. Annexin V-FITCand propidium iodide-stained cells were visualized on an inverted fluorescence microscope using a dual filter set for FITC and rhodamine.
Measurement of Apoptotic DNA Laddering-CHO-m3 cells grown in 10-cm 2 plates were treated overnight with the appropriate drugs, and DNA laddering was measured using a commercially available apoptotic DNA laddering kit (Roche Applied Science, Lewes, UK). Floating and adhered cells were collected in PBS/0.5 mM EDTA. Cells were centrifuged at 1500 rpm for 3 min, and pellets were resuspended in 200 l of PBS. Cells were lysed with 200 l of lysis buffer (6 M guanidine-HCl, 10 mM urea, 10 mM Tris-HCl, 20% Triton x-100 (v/v), pH 4.4). Samples were then processed as described in the manufacturer's instructions.
MTT Assay-CHO-M 3 cells grown in 10-cm 2 plates were treated overnight with the appropriate drugs, and MTT was added (0.5 mg/ml) to the plates. The plates were incubated at 37°C for 1 h, and then floating and adhered cells were collected in PBS/0.5 mM EDTA. An aliquot of cell suspension was used to measure protein concentration. The remainder of the CHO-M 3 cell suspension was then centrifuged at 1500 rpm for 3 min, and the resultant cell pellet was dissolved in 100% Me 2 SO to liberate and solubilize the insoluble formazen product. The level of formazen product was measured spectrophotometrically at 550 nm.
Caspase 3 Assay-Sub-confluent cells plated in 10-cm 2 plates were treated with the appropriate concentration of etoposide for the desired time. Floating and attached cells were harvested in PBS/0.5 mM EDTA, and cells were centrifuged at 1500 rpm in a bench-top centrifuge. The pellets were resuspended in lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM dithiothreitol, 0.1 mM EDTA), and the cells were kept on ice for 10 min. The cell lysates were pre-cleared by centrifugation at 10,000 ϫ g for 1 min. A Bradford assay was performed on the lysate, and 200 -400 g of cell lysate was diluted 1:2 in reaction buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 1 mM EDTA, 10% glycerol) in plastic 96-well plates. 1:10 v/v of 4 mM DEVD-pNA was added to the each well of the 96-well plate, and the plate was kept at 37°C for 2-4 h. Cleavage of the DEVD-pNA substrate was monitored colorimetrically at 405 nm in a microplate reader.
Receptor Phosphorylation-Stably transfected CHO cells were grown in six-well plates. Cells were incubated with 50 Ci/ml [ 32 P]orthophosphate for 1 h in phosphate-free Krebs/HEPES buffer (KHB: 10 mM HEPES, pH 7.4, 118 mM NaCl, 4.3 mM KCl, 1.17 mM MgSO⅐7H 2 O, 1.3 mM CaCl 2 , 25 mM NaHCO 3 , 11.7 mM glucose). CCH (1 mM) added for 5 min, and reactions were terminated by aspiration and the addition of 1 ml of radioimmune precipitation assay buffer (10 mM Tris, pH 7.4, 10 mM EDTA, 500 mM NaCl, 1% v/v Nonidet P-40, 0.5% w/v sodium deoxycholate). Receptor expression was determined by [ 3 H]N-methylscopolamine binding (see below) to ensure that an equal number of receptors was present in the lysates. Lysates were pre-cleared by centrifugation, and 3 l of anti-M 3 muscarinic receptor antisera was added. Immunocomplexes were precipitated on protein A-Sepharose. Samples were washed four times with TE buffer (10 mM Tris, pH 7.4, 2.5 mM EDTA) and resolved on 8% SDS-PAGE gels. Gels were stained with 0.2% Coomassie Blue to ensure that there was equal immunoprecipitation. The gels were then dried, and phosphoproteins were visualized by autoradiography.
Receptor Density Determination-Confluent CHO cells grown in sixwell plates were incubated with 0.14 Ci of [ 3 H]N-methylscopolamine for 1 h at 37°C. Cells were washed three times with ice-cold KHB and solubilized by the addition of 1 ml of ice-cold radioimmune precipitation assay buffer, and receptor number was determined by liquid scintillation counting. Nonspecific binding was determined by the inclusion of 10 M atropine sulfate.
ERK and JNK Assays-Stably transfected CHO cells were grown to confluence in six-well plates. CCH (1 mM) was added for the times indicated. For the ERK assay, phorbol-12,13-dibutyrate (1 M) was added for 5 min, and for the JNK assay, 0.3 M sorbitol was added for 30 min. Reactions were terminated by the addition of 0.5 ml of ice-cold lysis buffer (20 mM Tris, pH 7.6, 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , 1 mM dithiothreitol, 5 g/ml benzamidine), and cells were placed on ice for 5 min. Cells were then lysed by scraping, and lysates were pre-cleared by centrifugation. For ERK assays, 0.2 g of anti-ERK antisera (Santa Cruz Biotechnology) was added and immunoprecipitations were performed for 1 h at 4°C. For JNK assays, 5 g of GST-c-Jun was added and samples placed on a roller for 1 h at 4°C. For the ERK assay immunocomplexes were isolated on protein A-Sepharose beads, and for the JNK assay JNK⅐c-Jun complexes were isolated on glutathione-Sepharose beads. Samples were washed twice with lysis buffer and twice with assay buffer (20 mM HEPES, pH 7.2, 20 mM ␤-glycerophosphate, pH 7.2, 10 mM MgCl 2 , 1 mM dithiothreitol, 50 M Na 3 VO 4 ). ERK activity was measured by resuspending the immunocomplexes in assay buffer containing 2 Ci of [ 32 P]ATP, 20 M ATP, and 200 M epidermal growth factor receptor peptide (encompassing the region 661-681 of the epidermal growth factor receptor). For JNK assays, JNK⅐c-Jun complexes were resuspended in assay buffer containing 2 Ci/ml [ 32 P]ATP, 20 M ATP. For ERK assays, the reactions were terminated by the addition of 25% trichloroacetic acid, and samples were spotted on P81 phosphocellulose squares. P81 squares were washed four times with 0.05% orthophosphoric acid, and the incorporation of radioactivity was measured by scintillation counting. For JNK experiments, the assays were terminated by the addition of sample buffer. Samples were resolved on 12% SDS-PAGE gels, and phospho-c-Jun was visualized by autoradiography. Quantification of radioactivity incorporated into c-Jun was performed by excising the protein from the Coomassie Bluestained gel and measuring by scintillation counting.
Measurement of [Ca 2ϩ ] i -CHO cells grown on 22-mm coverslips were loaded with 5 M fura-2-acetoxymethyl ester for 30 min at 37°C. Measurement of [Ca 2ϩ ] i was performed as previously described (13).

Anti-apoptotic Response of G q/11 -coupled Muscarinic Recep-
tor Subtypes-Apoptosis is mediated by the activation of caspases that was measured colorimetrically in this study by the cleavage of the peptide substrate DEVD-pNA (see "Materials and Methods"). Treatment of CHO cells stably expressing recombinant human M 1 -M 5 muscarinic receptors showed an increase in caspase activity following an overnight treatment with the DNA-damaging agent, etoposide (Fig. 1). This increase in caspase activity was significantly attenuated by muscarinic receptor stimulation in cells expressing the M 1 , M 3 , or M 5 muscarinic receptor subtypes ( Fig. 1: 59.0 Ϯ 1.0%, n ϭ 4; 51.6 Ϯ 1.2%, n ϭ 3; and 54.4 Ϯ 0.2%, n ϭ 4 for M 1 , M 3 , and M 5 , respectively). In contrast, muscarinic receptor stimulation of cells expressing the M 2 -or M 4 -muscarinic receptor subtypes had no significant affect on etoposide-mediated caspase activation ( Fig. 1: 95.0 Ϯ 11.0%, n ϭ 3; 93.1 Ϯ 1.0%, n ϭ 4 for the M 2and M 4 -muscarinic receptor, respectively).
Characterization of the Anti-apoptotic Response of the M 3muscarinic Receptor-Detailed characterization of the antiapoptotic response shown by the G q/11 -coupled muscarinic receptors was investigated in CHO cells expressing the recombinant human M 3 -muscarinic receptor (CHO-M 3 cells). The appearance of phosphatidylserine on the outer leaflet of the plasma membrane is a marker of apoptosis and can be detected by utilizing the extremely high affinity that annexin V exhibits for this phospholipid (14). Phase-contrast images of CHO-M 3 cells showed that an overnight treatment with etoposide resulted in a marked reduction in cell number with a large percentage of cells displaying phenotypic features characteristic of apoptosis such as cell rounding, membrane blebbing, and annexin V-FITC binding (Fig. 2). Significant inhibition of these markers of apoptosis was observed in CHO-M 3 cells treated with the muscarinic agonist carbachol (Fig. 2). In particular, the increase in annexin V-FITC staining observed following etoposide treatment was markedly reduced by muscarinic receptor stimulation (Fig. 2). As a control for necrotic cell death, CHO-M 3 cells treated with etoposide, etoposide and carbachol, or vehicle were stained with propidium iodide as a marker of necrosis. No significant difference in propidium iodide staining was observed with the different cell treatments, consistent with apoptotic rather than necrotic cell death (data not shown).
During the later stages of apoptosis caspase-dependent DNase activation results in the degradation of genomic DNA that is characterized by a 200-bp DNA ladder (15). Following an overnight treatment of CHO-M 3 cells with etoposide, the extracted genomic DNA shows a distinctive apoptotic DNA ladder that was attenuated by muscarinic receptor stimulation (Fig. 3). These data, combined with the measurement of caspase activity (Fig. 1) and the determination of annexin V staining (Fig. 2), are consistent with etoposide mediating apoptosis in CHO-M 3 cells, and stimulation of the M 3 -muscarinic receptor results in attenuation of this apoptotic response.
The reduction of MTT to an insoluble formazen product is catalyzed by mitochondrial succinate dehydrogenase and is used as measure of cellular viability (16). An overnight treatment of CHO-M 3 cells with etoposide resulted in an inhibition in the level of MTT reduction compared with CHO-M 3 cells treated with vehicle only, confirming that the apoptotic agent reduces the viability of the cell population (Fig. 4). Stimulation of the M 3 -muscarinic receptor prevented this reduction in cellular viability induced by etoposide treatment (Fig. 4).
Characterization of the M 3 -muscarinic Receptor Anti-apoptotic Response-We explored the kinetics of the muscarinicinduced protection of CHO-M 3 cells by treating cells with carbachol for various time periods. M 3 -muscarinic receptor signaling was terminated by the addition of atropine (0.5 M) followed by three washes with ␣-MEM. Apoptosis was then induced by an overnight treatment of cells with etoposide. Surprisingly, muscarinic receptor-mediated protection was induced extremely rapidly, because a brief 0.5-min exposure of CHO-M 3 cells to carbachol resulted in a maximal anti-apoptotic response to a subsequent overnight exposure to etoposide (Fig.  5A). It should be noted that the continued presence of muscarinic receptor stimulation was not necessary to provide protection from etoposide-induced cell death. The suppression of cell death by the M 3 -muscarinic receptor was also dose-dependent with an EC 50 of 4.9 M (Fig. 5, B and C).
We have utilized chemical inhibitors of ERK, p38, and phosphatidylinositol 3-kinase (PI3K) to determine whether these signaling components play any role in mediating the antiapoptotic response of the M 3 -muscarinic receptor. All the chemical inhibitors were tested for their expected activity in cell signaling experiments in CHO-M 3 cells (data not shown). Inhibition of ERK and p38 with PD98059 (50 M) and SB202190 (10 M), respectively, had little affect on the M 3 -muscarinic receptor protective response (data not shown). Importantly, these inhibitors did not mediate an apoptotic response in their own right. Likewise, inhibition of PI3K with wortmannin (100 nM) alone did not induce cell death. However, incubation of CHO-M 3 cells with wortmannin (100 nM) had no significant affect on the ability of the M 3 -muscarinic receptor to induce protection against etoposide-mediated cell death (data not shown).
Functional Role of the C-terminal Tail of the M 3 -muscarinic Receptor in the Anti-apoptotic Mechanism-Sequence align-ment of the five members of the muscarinic receptor family revealed that the C-terminal tail of the G q/11 -coupled receptors all share a degree of homology in the membrane distal portion that is not present in the G i/o -coupled members (Fig. 6A). To address the functional role of the C-terminal tail in mediating the anti-apoptotic effects of the G q/11 -coupled muscarinic receptors, we truncated the M 3 -muscarinic receptor C-terminal tail at position 565 (⌬565-M 3 , Fig. 6B). These receptors were stably expressed in CHO cells at the plasma membrane as determined by ligand binding using the hydrophilic muscarinic receptor antagonist [ 3 H]N-methylscopolamine (see "Materials and Methods"). Furthermore, this mutant receptor was able to signal through phospholipase C/calcium and the MAPK kinase pathways in a similar manner to the wild-type receptor (see below). Care was taken to select mutant receptor cell lines that expressed similar levels of receptor to wild-type receptor cell lines for these studies (see "Materials and Methods").
Overnight treatment of CHO cells stably expressing the ⌬565-M 3 receptor with etoposide lead to a significant increase in caspase activity. However, in contrast to the wild-type receptor, activation of the ⌬565-M 3 muscarinic receptor was unable to prevent etoposide-mediated caspase activation (Fig.  7A). These data, together with the sequence alignment of the C-terminal tails of muscarinic receptor, suggest that the Cterminal tail region of the M 3 -muscarinic receptor and particularly the poly-basic region in the membrane proximal region (Fig. 6A) may be central in allowing the receptor to inhibit etoposide-mediated cell death in CHO cells.
To test the importance of the poly-basic region of the Cterminal tail in mediating the anti-apoptotic response of the M 3 -muscarinic receptor, we produced a receptor mutant where the basic residues in the region 565 KKKRRK 570 were mutated to alanine (K 3 A) (Fig. 6B). These receptors were stably expressed in CHO cells at the plasma membrane as determined by ligand binding using the hydrophilic muscarinic receptor antagonist [ 3 H]N-methylscopolamine (see "Materials and Methods"). As observed with CHO cells expressing the ⌬565-M 3 receptor, activation of the K 3 A receptor significantly attenuated the ability of etoposide to induce activation of caspase 3 (Fig. 7B). These data strongly suggest that the conserved polybasic region in the C-terminal tail of the muscarinic M 3 receptor plays an important role in mediating the anti-apoptotic effects of the receptor.
The M 3 -muscarinic Receptor-mediated Anti-apoptotic Response Is Independent of Receptor Phosphorylation-We have previously shown that M 3 -muscarinic receptors are subject to rapid agonist-driven phosphorylation (17,18). Here we exam- ined the role of receptor phosphorylation in the M 3 -muscarinic receptor anti-apoptotic response by generating two receptor mutants that were deficient in their ability to undergo agonistmediated receptor phosphorylation. The T 3 A tail mutant was produced by site-directed mutagenesis of three threonine resi-dues (threonines 551, 553, and 554) to alanine in the membrane proximal region of the C-terminal tail of the M 3 -muscarinic receptor (Fig. 6B). This mutant receptor showed a 54.3 Ϯ 8.6% decrease in agonist-mediated receptor phosphorylation compared with wild-type receptor (Fig. 8, A and D). A second mutant, designated mutant-6, was generated by site-directed mutagenesis of the serine phospho-acceptor sites in the third intracellular loop of the M 3 -muscarinic receptor (Fig. 6C). Mutant-6 also showed a significant reduction in agonist-mediated receptor phosphorylation (Fig. 8, B and D, 62.9 Ϯ 1.9% reduction compared with wild-type receptor). It should be noted that both mutant-6 and the T 3 A tail mutants were expressed at the cell surface and were able to couple to downstream signaling pathways such as calcium/phospholipase C and the MAPK pathway (data not shown). Importantly, mutant-6, which contains point changes in the region to which the M 3 -muscarinic receptor antibody was raised (i.e. Ser 345 -Lys 463 in the third intracellular loop of the human M 3 -muscarinic receptor), was recognized by the anti-M 3 -muscarinic receptor antibody as determined by immunoprecipitation of biotinylated receptor with an efficiency equivalent to the wild-type receptor (data not shown).
Despite the fact that the mutant-6 and the T 3 A tail mutant receptors showed reduced levels of agonist-mediated phospho- rylation, both were able to mediate an anti-apoptotic response similar to that observed for the wild-type receptor ( Fig. 9: 56.2 Ϯ 2.4%, 46.8 Ϯ 6.4%, and 65.0 Ϯ 3.9% attenuation of the etoposide activation of caspase by wild type, T 3 A tail mutant, and mutant-6, respectively; n ϭ 4). In addition, the ⌬565-M 3 truncation mutant, which was unable to mediate an antiapoptotic response (Fig. 7A), did undergo agonist-mediated phosphorylation in a manner similar to the wild-type receptor (Fig. 8, C and D). These data strongly suggest that phosphorylation of the M 3 -muscarinic receptor is not involved in the anti-apoptotic response.
Signaling Properties of the ⌬565-M 3 Mutant-GPCR activation of the MAPK signaling pathways has been shown to be central in the regulation of cell death in a variety of cell lines (19 -22). To test this in the current study, the ability of the truncation mutant ⌬565-M 3 to stimulate the ERK and JNK pathways was investigated. Stimulation of the ⌬565-M 3 receptor induced a robust increase in ERK activity in a manner similar to the wild-type M 3 -muscarinic receptor (Fig. 10A). Similarly, stimulation of the ⌬565-M 3 receptor also resulted in activation of the JNK pathway that was similar to that seen following wild-type M 3 -muscarinic receptor activation (Fig. 10, B and C: 134.2 Ϯ 26.0 and 118.7 Ϯ 33.1 fmol of phosphate incorporated/mg/min following a 60-min stimulation with 1 mM carbachol for the wild-type M 3 -muscarinic receptor and ⌬565-M 3 , respectively; n ϭ 3). These data, coupled with the inhibitor studies above, strongly imply that the M 3 -muscarinic receptor anti-apoptotic affects are not mediated via stimulation of the MAPK family.
It has been widely recognized that cytosolic increases in intracellular calcium [Ca 2ϩ ] i can either protect or induce apoptosis under certain experimental conditions (23). In this study we have demonstrated that the G q/11 /phospholipase C-coupled muscarinic receptors are able to protect CHO cells against etoposide-mediated cell death, whereas the G i/o -coupled muscarinic receptor family members are not (Fig. 1). To determine whether activation of the phospholipase C pathway is essential in mediating the anti-apoptotic effects of the G q/11 -coupled members of the muscarinic receptor family, we examined whether the ⌬565-M 3 receptor, which is unable to protect against etoposide-mediated cell death, was able to produce a [Ca 2ϩ ] i transient following receptor activation. Fig. 11A shows that activation of the ⌬565-M 3 receptor with a maximal dose of carbachol initiates a [Ca 2ϩ ] i transient that was similar to that observed following activation of wild-type M 3 -muscarinic receptors. Similarly, submaximal doses of carbachol gave a comparable response in the mutant and wild-type expressing cell lines (Fig. 11B). As an internal control, endogenous purinergic receptors expressed in CHO cells were stimulated with 100 M ATP, which resulted in a robust [Ca 2ϩ ] i transient (Fig. 11A). The conclusion from these data is that the anti-apoptotic properties of the G q/11 -coupled muscarinic receptors is not due to their ability to activate the phospholipase C pathway but is due to a conserved poly-basic region found within the membrane distal portion of the C-terminal tail of these receptors that is not shared by the G i/o -coupled members of the receptor family. DISCUSSION We report here that the G q/11 -coupled members of the muscarinic receptor family expressed in CHO cells protect against apoptotic cell death. Detailed analysis of the M 3 -muscarinic receptor demonstrated that the distal portion of the C-terminal tail provided a motif that was essential in this anti-apoptotic response.
One of most intriguing characteristics of the M 3 -muscarinic receptor cell survival response was the rapid time course. A short 0.5-min pulse of muscarinic receptor agonist was sufficient to protect against apoptosis induced by an overnight treatment with etoposide. This suggested that the mechanism of the M 3 -muscarinic receptor response was rapid in its onset and was then maintained for an extended period after agonist withdrawal. A strong candidate for mediating such a mechanism was rapid changes in intracellular calcium, which in the case of a number of GPCRs has been shown to encode for longer adaptive responses such as changes in gene transcription (24,25). However, analysis of a truncated mutant of the M 3 -muscarinic receptor (⌬565-M 3 ), which was unable to mediate an anti-apoptotic response, revealed that this receptor was still coupled to the calcium/PLC pathway in a manner similar to the wild-type receptor. These data would appear to eliminate a role for calcium/PLC signaling in the M 3 -muscarinic receptor cell survival response. In this regard our work is consistent with that of Lindenboim and colleagues (11) who demonstrated that the M 1 -muscarinc receptor in PC-12 cells protected against serum deprivation-induced cell death via a Ca 2ϩ -independent mechanism.
Detailed analysis of the truncation mutant ⌬565-M 3 provided a unique opportunity to probe other signaling pathways for their involvement in the M 3 -muscarinic receptor anti-apoptotic response. In particular, focus was placed on the MAPK pathway that has previously been shown to be involved in the anti-apoptotic response of a number of GPCRs, including the neurokinin-1, lysophosphatidic acid, and endothelin-1 receptors (20,21,26). In the current study a robust activation of both ERK and JNK was clearly detectable following activation of ⌬565-M 3 receptor, indicating that neither of these MAPK pathways are involved in the M 3 -muscarinic receptor-induced protection from cell death. We have also tested chemical inhibitors of ERK and p38 MAPK and found that the receptor-induced anti-apoptotic response was not inhibited in the presence of either inhibitor. These data would indicate that MAPK pathways are not involved in the anti-apoptotic response of the M 3 -muscarinic receptor observed here.
Previous studies in COS-7 cells have demonstrated that M 1and M 2 -muscarinic receptors can protect against cell death, in part, through the pro-survival PKB/Akt pathway (8). Activation of PKB/Akt is downstream of the phosphoinositide lipid products of the PI3Ks (27). By using wortmannin, an inhibitor of PI3K, previous studies have demonstrated that M 3 -muscarinic receptors can activate PKB/Akt via PI3K (28). However, the involvement of PKB/Akt in the M 3 -muscarinic receptor anti-apoptotic response in the current study appears unlikely, because treatment with wortmannin at a concentration reported to inhibit PI3K had no significant effect on the antiapoptotic response.
There appear to be discrepancies in the muscarinic antiapoptotic responses reported here in CHO cells compared with those previously reported in COS-7 cells (8). The anti-apoptotic response in CHO cells is confined to the G q/11 -coupled subtypes, whereas in COS-7 cells both G q/11 -and G i/o -coupled receptor subtypes provide protection. These discrepancies may be due to cell and receptor-specific differences, but it is also possible that the mechanisms adopted by GPCRs to modulate cell death might be influenced by the method of inducing cell death (e.g. UV irradiation in COS-7 cells and etoposide in the current study). We are currently testing in CHO cells whether muscarinic receptors can protect against cell death induced by apoptotic stimuli other than etoposide.
We addressed the requirement of M 3 -muscarinic receptor phosphorylation in the induction of the anti-apoptotic response. This is important given the growing body of evidence that suggests the formation of phosphorylated GPCR⅐arrestin complexes appears to be essential in allowing for GPCR modulation of the cell death pathway (20,29). Indeed, the formation of rhodopsin⅐arrestin complexes is essential in inducing retinal degeneration in Drosophila (30). Light-induced photoreceptor apoptosis in Drosophila appears to involve the formation of membrane complexes of phosphorylated and activated rhodopsin and arrestin and, subsequently, clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment (31). It has been proposed that similar phosphorylated GPCR⅐arrestin complexes may be required to allow GPCR regulation of apoptosis in mammalian cells (29). Indeed prevention of stable neurokinin-1 receptor⅐␤-arrestin complexes by mutating the C-terminal tail of the receptor is sufficient to prevent substance P-mediated anti-apoptosis (20). Intriguingly, in the current study phosphorylation of the M 3 -muscarinic receptor does not appear to be central in allowing for the anti-apoptotic effects of the receptor, because the ⌬565-M 3 receptor, which lacks the anti-apoptotic properties of the wildtype receptor, is phosphorylated normally in response to agonist addition. Furthermore, we have also shown that two M 3muscarinic receptor mutants that exhibit significantly reduced agonist-induced phosphorylation, are able to protect CHO cells against etoposide-induced cell death in an identical manner to wild-type M 3 -muscarinic receptors. Therefore, it appears that the ability of G q/11 -coupled muscarinic receptors to inhibit apoptotic cell death proceeds via a mechanism that does not involve receptor phosphorylation.
The inability of the C-terminal tail truncation mutant, ⌬565-M 3 , to mediate an anti-apoptotic response indicates the existence of structural determinants within the distal portion of the C-terminal tail that are essential for mediating the pro-survival response of the receptor. Unlike many other type I GPCRs (e.g. the adrenergic receptor family) the muscarinic receptor family have relatively short C-terminal tails (23-39 amino acids). There is a large degree of conservation between the five muscarinic receptor subtypes in the membrane proximal region of the C-terminal tail up to the putative cysteine palmitoylation site (see Fig. 5A). This conservation across the family is, however, lost downstream of the cysteine palmitoylation site. In the case of the G q/11 -coupled receptors (M 1 , M 3 , and M 5 ) there is a poly-basic motif that is not present in the G i/o -coupled members (M 2 and M 4 ). We assessed whether this poly-basic region was important in mediating the anti-apoptotic effects of the G q/11coupled muscarinic receptors by mutating the basic residues in this region of the M 3 -muscarinic receptor to alanine (K 3 A). Indeed activation of the K 3 A mutant significantly reduced the ability of etoposide to induce activation of caspase 3 in CHO cells. Because it is only the G q/11 -coupled members of the muscarinic receptor family that are able to protect against cell death, these data strongly imply that this poly-basic region is the common structural element that is essential for the antiapoptotic response of the M 1 , M 3 , and M 5 -muscarinic receptors.
The conclusions from the current study are that the M 3muscarinic receptor (and most probably the M 1 and M 5 receptors) are able to protect against etoposide-mediated cell death in CHO cells by a mechanism that is rapid in its onset, is independent of calcium/PLC signaling, receptor phosphorylation, and the MAPK and PI3K pathways, and is dependent on a conserved poly-basic region within the distal region of the C-terminal tail. The physiological importance of these findings has yet to be clearly defined. However, an anti-apoptotic response mediated by M 3 -muscarinic receptors in native tissues has been demonstrated. For example, muscarinic activation in cerebellar granule cells has been shown to protect from apoptosis induced by culturing in non-depolarizing conditions suggesting that this process may be involved in regulating neuronal apoptosis in the developing central nervous system (10). Furthermore, the recent findings that acetylcholine can be released from cells of hematopoietic lineage (32) and that both T-and B-lymphocytes express functional M 3 -muscarinic receptors (33), lead to the intriguing possibility that the immune function of circulating T-and B-lymphocytes may be controlled in an autocrine/paracrine manner by circulating acetylcholine via M 3 -muscarinic receptors. Because the function, growth, and differentiation of lymphocytes are highly dependent on apoptosis, this may provide a novel, physiological setting whereby M 3 -muscarinic receptor modulation of programmed cell death may be important in mediating immune responses.