Muscarinic cloning

CI-979 (29) is a balanced muscarinic agonist having equal affinities for cloned ml and m2 receptors (144). However, unlike prototypical muscarinic compounds such as (25), (29) increases central muscarinic tone, as indicated by behavioral and electroencephalogram (EEG) parameters, at doses lower than those requited to produce gastrointestinal effects (144). CI-979 is well tolerated in humans up to a dose of 1 mg. Dose-limiting side effects such as stomach pain and emesis were observed at a dose of 2 mg. 

The first and best-studied allosteric site on GPCRs is that on the muscarinic receptor [9,10,12,19,20]. For the five subtypes of these receptors that have been cloned and pharmacologically defined as Mi to M5, various agents have been identified that allosterically regulate selectively these... 

Jakubik J, Bacakova L, El-Fakahany EE, Tucek S. Subtype selectivity of the positive allosteric action of alcuronium at cloned M M5 muscarinic acetylcholine receptors. J Pharmacol Exp Ther 1995 274 1077-1083. 

Ellis J, Huyler J, Brann MR. Allosteric regulation of cloned mrm5 muscarinic receptor subtypes. Biochem Pharmacol 1991 42 1927-1932. 

Buckley, N. J. (1990). Molecular pharmacology of cloned muscarinic receptors. In Transmembrane Signalling, Intracellular Messengers and Implications for Drug Development, ed. S. R. Nahorski, pp. 11-30. Chichester John Wiley 8r Sons. 

At least five distinct muscarinic receptor genes have been cloned and sequenced. The genes are called Wj to m5. They correlate with the M to M5 receptors identified pharmacologically. The subtypes differ in their ability to couple to different G proteins and, hence, to elicit cellular signaling events. 

Thus, cholinergic receptor classification can be considered in terms of three stages of development. Initially, Dale [2] distinguished nicotinic and muscarinic receptor subtypes with crude alkaloids. Then, chemical synthesis and structure-activity relationships clearly revealed that nicotinic and muscarinic receptors were heterogeneous, but chemical selectivity could not come close to uncovering the true diversity of receptor subtypes. Lastly, analysis of subtypes came from molecular cloning, making possible the classification of receptors on the basis of primary structure (Fig. 11-2). 

The cDNAs for the muscarinic receptors encode apparent glycoproteins of 55-70 kDa, which contain seven predicted transmembrane-spanning regions, similar to what is seen for the [3-adrenergic receptor and other receptors that couple to G proteins (Fig. 11-11). There is only 38% amino acid identity between the proteins cloned from porcine brain and heart. The cDNA encoding the receptor initially cloned from the brain has been termed m whereas that cloned from the heart has been termed m2, in keeping with the names (Mj and M2) applied to the purified recombinant receptors. 

Hsieh, D.J. and Liao, C.F., Zebrafish M2 muscarinic acetylcholine receptor cloning, pharmacological characterization, expression patterns and roles in embryonic bradycardia, Br. J. Pharmacol, 137, 782-792, 2002. 

To date, five subtypes of these receptors have been cloned. However, initial studies relied on the pharmacological effects of the muscarinic antagonist pirenzepine which was shown to block the effect of several muscarinic agonists. These receptors were termed Mi receptors to distinguish them from those receptors for which pirenzepine had only a low affinity and therefore failed to block the pharmacological response. These were termed M2 receptors. More recently, M3, M4 and M5 receptors have been identified which, like the Mi and M2 receptors occur in the brain. Recent studies have shown that Mi and M3 are located posts)maptically in the brain whereas the M2 and M4 receptors occur pres)maptically where they act as inhibitory autoreceptors that inhibit the release of acetylcholine. The M2 and M4 receptors are coupled to the inhibitory Gi protein which reduces the formation of cyclic adenosine monophosphate (cyclic AMP) within the neuron. By contrast, the Mi, M3 and M5 receptors are coupled to the stimulatory Gs protein which stimulates the intracellular hydrolysis of the phosphoinositide messenger within the neuron (see Figure 2.8). 

Interaction of ACh with the postsynaptic ganglionic cell muscarinic receptor is responsible for slowly developing depolarization, the slow EPSP, which has a longer latency than the fast EPSP and a duration of 30 to 60 seconds. The slow EPSP is due to inhibition of a voltage-dependent K+ current called the M current, and inhibition of the M current involves activation of G proteins. At least five types of muscarinic receptors (Mj, M2, M3, M4 and M5) have been identified using functional studies and at least five subtypes (mj, m2, m3, rru, and m5) identified by molecular cloning techniques. The Mj receptor, which appears responsible for inhibiting the M current, can be blocked by atropine. 

As indicated in Chapter 6, muscarinic receptor subtypes have been characterized by binding studies and cloned. Several cellular events occur when muscarinic receptors are activated, one or more of which might serve as second messengers for muscarinic activation. All muscarinic receptors appear to be of the G protein-coupled type (see Chapter 2 and Table 7-1). Muscarinic agonist binding activates the inositol trisphosphate (IP3), diacylglycerol (DAG) cascade. Some evidence... 

Five subtypes of muscarinic receptors have been identified, primarily on the basis of data from ligand-binding and cDNA-cloning experiments (see Chapters 6 and 7). A standard terminology (M4 through M5) for these subtypes is now in common use, and evidence — based mostly on selective agonists and antagonists—indicates that functional differences exist between several of these subtypes. 

Kubo, T., Fukuda, K., Mikami, A., Maeda, A., Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A., Kangawa, K. Cloning, sequencing, and expression of complementary DNA encoding the muscarinic acetylcholine receptor, Nature 1986, 323, 411-416. 

All four receptor types have been cloned and belong to the large superfamily of receptors having seven membrane-spanning regions and intracellular association with G proteins. The structures of the Hi and H2 receptors differ significantly and appear to be more closely related to muscarinic and 5-HTi receptors, respectively, than to each other. The H4 receptor has about 40% homology with the H3 receptor but does not seem to be closely related to any other histamine receptor. 

Abstract Presynaptic metabotropic receptors for acetylcholine and adrenaline/ noradrenaline were first described more than three decades ago. Molecular cloning has resulted in the identification of five G protein-coupled muscarinic receptors (Mi — M5) which mediate the biological effects of acetylcholine. Nine adrenoceptors (ociabd,oc2abc,Pi23) adrenafine/noradrenaline signals between cells. 

Molecular cloning has led to the identification of a great complexity of receptor subtypes in the cholinergic and adrenergic systems. Five muscarinic GPCRs and altogether nine adrenoceptors mediate the biological effects of acetylcholine and... 

Olianas MC, Maullu C, Onali P. 1997. Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor. Br J Pharmacol 122 401-408. 

Olianas MC, Maullu C, Onali P. 1999. Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Neuropsychopharmacology 20 263-270. 

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