Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor.

The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.     

ADP-ribosylation factor-dependent phospholipase D activation by the M3 muscarinic receptor

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.     

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.     

Scanning mutagenesis of transmembrane domain 3 of the m1 muscarinic acetylcholine receptor

Scanning mutagenesis of transmembrane domain 3 of the M1 muscarinic acetylcholine receptor has revealed a highly-differentiated α-helical structure. Lipid-facing residues are distinguished from a patch of residues which selectively stabilise the ground state of the receptor, and from a band of amino acids extending the full length of the helix, which contribute to the active agonist-receptor-G protein complex. The most important residues are strongly conserved in the GPCR superfamily.     

Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. Analysis of the role of N-glycosylation in receptor expression and function

The cardiac m2 muscarinic acetylcholine receptor (mAChR) is a sialoglycosylated transmembrane protein which has three potential sites for N-glycosylation (namely, Asn2, Asn3, and Asn6). To investigate the role of N-linked oligosaccharide(s) in the expression and function of the receptor, we constructed glycosylation-defective mutant receptor genes in which the three asparagine codons were substituted by codons for either aspartate (Asp2,3,6), lysine (Lys2,3,6), or glutamine (Gln2,3,6). The glycosylation-defective and wild-type receptor genes were stably expressed in Chinese hamster ovary cells. Binding experiments with the membrane-permeable radioligand [3H]quinuclidinyl-benzilate and the membrane-impermeable radioligand [3H]N-methylscopolamine revealed that the Asp2,3,6, Gln2,3,6, and wild-type receptors were located exclusively on the cell surface and expressed in similar numbers. The Lys2,3,6 mutant receptor was expressed at a relatively low level and was therefore not included in subsequent experiments. Wheat germ agglutinin-Sepharose chromatography and sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis demonstrated that the wild-type receptor, but not the Asp2,3,6 and Gln2,3,6 mutant receptors were N-glycosylated. The Asp2,3,6 and Gln2,3,6 mutant receptors had the same affinities for mAChR ligands as wild-type receptors. The time courses for degradation of the Asp2,3,6, Gln2,3,6, and wild-type receptors were also similar. In vivo functional analysis of the ability of the glycosylation mutant receptors to inhibit forskolin-stimulated cAMP accumulation revealed that maximal inhibition of adenylate cyclase activity was similar in the mutant and wild-type receptors. The Asp2,3,6 mutant receptor had an unaltered IC50 value for carbachol while the IC50 value of the Gln2,3,6 mutant receptor was 2-fold higher than that of the wild-type receptor. These results indicate that N-glycosylation of the m2 mAChR is not required for cell surface localization or ligand binding and does not confer increased stability against receptor degradation. Furthermore, N-glycosylation of the m2 mAChR is not required for functional coupling of the m2 mAChR to inhibition of adenylate cyclase.     

Influence of phospholipase C on muscarinic acetylcholine receptor binding in rat brain

Treatment of neural membranes from rat cerebral cortex with phospholipase C (phosphatidylcholine cholinephosphohydrolase) inhibited the binding of radiolabelled antagonists to muscarinic acetylcholine receptors. This inhibition was incomplete, was not competitive, and did not appear to be related to the production of inhibitory products. The affinity of carbamylcholine for cortex muscarinic receptors was increased by phospholipase C action. The distribution of receptors between states of high and low affinity was not affected by phospholipase C; rather, the affinity for carbamylcholine of the lowest affinity receptors was selectively increased. This suggests that membrane lipids influence the interaction of the receptor binding subunit with other structures in the synaptic membrane.     

A role for phospholipase D3 in myotube formation

Phospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.     

The m1 muscarinic acetylcholine receptor transactivates the EGF receptor to modulate ion channel activity.

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.     

Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B

Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.     

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.     

Muscarinic acetylcholine receptor regulates phosphatidylcholine phospholipase D in canine brain

The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.     

Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.     

Phospholipase D activity and phosphatidylethanol formation in stimulated HeLa cells expressing the human m1 muscarinic acetylcholine receptor gene

The human m1 and m2 muscarinic acetylcholine receptor (AChR) genes were subcloned, permanently expressed in HeLa cells and analyzed for their pharmacological and biochemical profiles. Both subtypes displayed saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) which was displaced by muscarinic agonists and antagonists. Stimulation of intact HeLa cells expressing the human m1 AChR gene by the muscarinic agonist oxotremorine-M, in the presence of ethanol, resulted in the activation of phospholipase D (PLD) and the formation of phosphatidylethanol (PEt). In contrast, oxotremorine-M did not activate PLD in the HeLa cells expressing the human m2 AChR subtype. These data suggest that the human m1 AChR is linked to the signal transduction mechanism of PLD activation, whereas the human m2 AChR interacts with a different guanine nucleotide regulatory binding protein (G-protein) which does not cause the activation of PLD or the formation of PEt.     

Choline produces cytoprotective effects against ischemic myocardial injuries: evidence for the role of cardiac m3 subtype muscarinic acetylcholine receptors.

BACKGROUND/AIMS: Accumulating evidence indicates the presence of functional M3 subtype of acetylcholine muscarinic receptors (M(3)-mAChR), in addition to the well-recognized M(2)-mAChR, in the heart of various species including man. However, the pathophysiological role of the cardiac M(3)-mAChR remain undefined. This study was designed to explore the possible role of M(3)-mAChR in cytoprotection of myocardial infarction and several related signaling pathways as potential mechanisms. METHODS: Studies were performed in a rat model of myocardial infarction and in isolated myocytes. RESULTS: We found that choline relieved myocardial injuries during ischemia or under oxidative stress, which was achieved by correcting hemodynamic impairment, diminishing ventricular arrhythmias and protecting cardiomyocytes from apoptotic death. The beneficial effects of choline were reversed by the M(3)-selective antagonists but not by the M(2)-selective antagonist. Choline/M(3)-mAChR activated several survival signaling molecules (antiapoptotic proteins Bcl-2 and ERKs), increased endogenous antioxidant reserve (SOD), and reduced apoptotic mediators (proapoptotic proteins Fas and p38 MAPK) and intracellular Ca2+ overload. CONCLUSION: Choline improves cardiac function and reduces ischemic myocardial injuries via stimulating the cardiac M(3)-mAChRs which in turn result in alterations of multiple signaling pathways leading to cytoprotection. The findings suggest M(3)-mAChR as a new target for drug development for improving cardiac function and preventing cardiac injuries during ischemia/reperfusion.     

The m3 muscarinic acetylcholine receptor differentially regulates calcium influx and release through modulation of monovalent cation channels.

Several types of transmembrane receptors regulate cellular responses through the activation of phospholipase C-mediated Ca2+ release from intracellular stores. In non-excitable cells, the initial Ca2+ release is typically followed by a prolonged Ca2+ influx phase that is important for the regulation of several Ca2+-sensitive responses. Here we describe an agonist concentration-dependent mechanism by which m3 muscarinic acetylcholine receptors (mAChRs) differentially regulate the magnitude of the release and influx components of a Ca2+ response. In transfected Chinese hamster ovary cells expressing m3 mAChRs, doses of the muscarinic agonist carbachol ranging from 100 nM to 1 mM evoked Ca2+ release responses of increasing magnitude; maximal Ca2+ release was elicited by the highest carbachol concentration. In contrast, Ca2+ influx was maximal when m3 mAChRs were activated by moderate doses (1-10 microM) of carbachol, but substantially reduced at higher agonist concentrations. Manipulation of the membrane potential revealed that the carbachol-induced Ca2+ influx phase was diminished at depolarized potentials. Importantly, carbachol doses above 10 microM were found to couple m3 mAChRs to the activation of an inward, monovalent cation current resulting in depolarization of the cell membrane and a selective decrease in the influx, but not release, component of the Ca2+ response. These studies demonstrate, in one experimental system, a mechanism by which a single subtype of G-protein-coupled receptor can utilize the information encoded in the concentration of an agonist to generate distinct intracellular Ca2+ signals.     

Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes

The human and rat genes for a fifth muscarinic receptor have been cloned and expressed in mammalian cells. The 532 amino acid human protein has 89% sequence identity to the 531 amino acid rat protein and is most closely related to the m3 receptor. Both proteins are encoded by single exons. The receptor has intermediate affinity for pirenzepine and low affinity for AF-DX 116, and it increases metabolism of phosphatidylinositol when stimulated with carbachol. Expression of mRNA has yet to be observed in brain or selected peripheral tissues, suggesting that either it is substantially less abundant than m1-m4 or its distribution is quite different.     

Identification and molecular characterization of m3 muscarinic receptor dimers.

Several studies suggest, but do not prove directly, that muscarinic receptors may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments using a modified version of the rat m3 muscarinic receptor (referred to as m3') as a model system. When membrane lysates prepared from m3' receptor-expressing COS-7 cells were subjected to Western blot analysis under non-reducing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers. However, under reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfide-linked m3 receptor complexes. Similar results were obtained when native m3 muscarinic receptors present in rat brain membranes were analyzed. Control experiments carried out in the presence of high concentrations of the SH group alkylating agent, N-ethylmaleimide, suggested that disulfide bond formation did not occur artifactually during the preparation of cell lysates. The formation of m3' receptor dimers/multimers was confirmed in coimmunoprecipitation studies using differentially epitope-tagged m3' receptor constructs. In addition, these studies showed that m3' receptors were also able to form non-covalently associated receptor dimers and that m3' receptor dimer formation was receptor subtype-specific. Immunological studies also demonstrated that m3' receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserved extracellular Cys residues (Cys-140 and Cys-220) play key roles in the formation of disulfide-linked m3' receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and highlight the specificity and molecular diversity of G protein-coupled receptor dimerization/oligomerization.