Multiple promoters drive tissue-specific expression of the human M muscarinic acetylcholine receptor gene.

Despite the wealth of information on the functional and pharmacological properties of the M2 muscarinic receptor, we know relatively little of structure and regulation of the M2 receptor gene. Here, we describe the organisation of the human M2 gene and its promoters. Four exons are present in the 5' untranslated region of the human M2 mRNA distributed over 146 kb on chromosome 7 which produce eight different splice variants in the IMR-32 neuroblastoma cell line. The unexpectedly large size of this gene indicates that transcription initiates much further upstream of the coding region than earlier studies had indicated. We present evidence that there are three distinct human M2 promoters. Analysis of endogenous transcripts revealed that promoter 2 is preferentially used in neuroblastoma cells, whereas promoter 1 in cardiac cells. All promoters are highly conserved across human, mouse, rat and pig. They contain multiple start sites and none possess a TATA-box. In addition, we describe another M2 promoter that is specific for rat. We show that GATA-4 transcription factor binds to two sites within the regulatory regions of the M2 gene using reporter gene assays, electromobility shift assays and mutational analysis.

Role of receptor protein and membrane lipids in xanomeline wash-resistant binding to muscarinic M1 receptors.

Xanomeline is a novel agonist functionally selective for muscarinic receptors of the M1 subtype. It binds to this receptor in two modes, reversible and quasi-irreversible (wash-resistant). We investigated the unknown mechanism of the wash-resistant binding in experiments with muscarinic M1 receptors expressed in transfected Chinese hamster ovary cells. Xanomeline's structure consists of two heterocycles and O-hexyl side chain. We compared the wash-resistant binding of xanomeline and its analogs with shorter O-alkyl side chains. For the wash-resistant binding to occur, the O-alkyl chain had to be at least O-butyl or longer. Accumulation of inositol phosphates was enhanced in washed cells that had been preexposed to xanomeline or its pentyl analog, whereas the agonistic effects of the methyl, propyl, and butyl analogs were abolished by washing. Only the reversible binding of xanomeline was detected purified soluble receptors, but both binding modes occurred purified receptors reconstituted into liposomes and exposed xanomeline only after reconstitution. The wash-resistant binding did not occur if the exposure of purified receptors or liposomes alone to xanomeline, followed by washing, reconstitution. Simultaneous presence of receptors and lipid environment is therefore essential for the binding to take place. We suggest that the binding of xanomeline involves interhelical penetration of M1 muscarinic receptor by xanomeline's O-alkyl chain and interaction with membrane lipids surrounding the receptor.

The effects of hydrocortisone on rat heart muscarinic and adrenergic alpha 1, beta 1 and beta 2 receptors, propranolol-resistant binding sites and on some subsequent steps in intracellular signalling.

Glucocorticoids affect the expression and density of neurotransmitter receptors in many tissues but data concerning the heart are contradictory and incomplete. We injected rats with hydrocortisone for 1-12 days and measured the densities of cardiac muscarinic receptors, alpha(1)-, beta(1)- and beta(2)-adrenoceptors and propranolol-resistant binding sites (formerly assumed to be the putative beta(4)-adrenoceptor). Some aspects of intracellular signalling were also evaluated: we measured adenylyl cyclase activity (basal, isoprenaline- and forskolin-stimulated and carbachol-inhibited), the coupling between muscarinic receptors and G proteins and basal and isoprenaline-stimulated heart rate. The density of cardiac muscarinic receptors increased (in both the atria and the ventricles). The density of beta(1)-adrenoceptors increased in the atria and was little changed in the ventricles. The density of beta(2)-adrenoceptors increased in both the atria and the ventricles. The number of alpha(1)-adrenoceptors decreased initially, followed by a transient increase in the atria and did not change in the ventricles. The density of propranolol-resistant binding sites first increased and then diminished in the atria and did not change in the ventricles. Although there were noticeable changes in receptor densities, the stimulatory and inhibitory effects on adenylyl cyclase, basal and isoprenaline-stimulated heart rate and the coupling between muscarinic receptors and G proteins were not significantly altered. This may indicate that changes in receptor densities might be one of the mechanisms maintaining stable functional output.

Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor.

Xanomeline is a potent agonist that is functionally selective for muscarinic M(1) receptors. We have shown previously that a significant fraction of xanomeline binding to membranes of Chinese hamster ovary (CHO) cells expressing the M(1) receptors occurs in a wash-resistant manner and speculated that this persistent binding likely does not take place at the primary binding site on the receptor. In the present work we investigated in depth the pharmacological characteristics of this unique mode of xanomeline binding and the effects of this binding on the interaction of classical competitive ligands with the receptor in CHO cells that express the M(1) muscarinic receptor. Onset of persistent binding of xanomeline to the M(1) muscarinic receptor was fast and was only slightly hindered by atropine. Its dissociation was extremely slow, with a half-life of over 30 h. Although persistently bound xanomeline strongly inhibited binding of the classical antagonist N-methylscopolamine (NMS) to the receptor, there are multiple indications that this is not the result of competition at the same binding domain. Namely, wash-resistant binding of xanomeline only slightly slowed the rate of NMS association, but enhanced the rate of NMS dissociation. Moreover, preincubation with xanomeline followed by extensive washing brought about an apparent decrease in the number of NMS binding sites. Our findings are best interpreted in terms of allosteric interactions between xanomeline-persistent binding to the M(1) muscarinic receptor and competitive ligands bound to the classical receptor binding site.

Quantitation of mRNAs for M(1) to M(5) subtypes of muscarinic receptors in rat heart and brain cortex.

It has been generally accepted that, of the five subtypes of muscarinic receptors (M(1)-M(5)), only the M(2) subtype is expressed in mammalian heart. This notion has recently been challenged by a series of reports indicating that mRNAs for some or all non-M(2) subtypes are also present in mammalian heart, in parallel with the M(2) mRNA. However, the quantities of relevant mRNAs reported to be present in the heart are not known, which makes it difficult to evaluate their likely significance. We measured the concentrations of the five muscarinic mRNAs by competitive reverse transcription-polymerase chain reaction and discovered that the M(2) mRNA represents more than 90% of total muscarinic mRNAs in rat atria and in either ventricle. The concentrations of total muscarinic mRNAs and of the M(2) mRNA were more than twice as high in the atria than in the ventricles. mRNAs for all non-M(2) muscarinic receptor subtypes were also detected but represented less than 1% (M(1) and M(4)), less than 3% (M(3)), and less than 5% (M(5)) of total muscarinic RNAs in the atria and ventricles. The findings support the concept of the prevalent role of the M(2) muscarinic receptors in the cholinergic control of the heart. When the same method of quantitation was applied to rat cerebral cortex, mRNAs for individual subtypes were found to represent 36% (M(1)), 21% (M(2)), 25% (M(3)), 11% (M(4)), and 7% (M(5)) of total muscarinic mRNAs.

Modelling the consequences of receptor-G-protein promiscuity.

Many G-protein-coupled receptors interact with more than one type of G protein, giving rise to extreme variability in the effects of receptor activation, depending on, for example, receptor density and desensitization, efficacy of agonists, and availability of specific G proteins. This leads to errors in interpretation of data. To facilitate understanding the consequences of receptor-G-protein promiscuity, we use two simplified models to simulate such consequences. Applied to the regulation of adenylyl cyclase and phosphoinositidase, the models predict seemingly paradoxical situations and explain some phenomena that, at first sight, might seem to require the induction of agonist-specific (G-protein-selective) receptor conformations.