Abstract
We have used a luciferase reporter gene under the transcriptional control of a cAMP response element as a sensitive monitor of the regulation by muscarinic acetylcholine receptors (mAChRs) of intracellular cAMP levels and cAMP-regulated gene expression. Treatment with the muscarinic agonist carbachol results in an increase in luciferase activity in JEG-3 cells transiently transfected with mouse m1 (8-10-fold) and chick m4 (3-5-fold) mAChRs. Control experiments indicate that these responses are not due to a calcium-mediated pathway and are dependent upon a functional protein kinase A. The m1 and m4 responses are not sensitive to pertussus toxin and the m4 response was potentiated by it. Thus, these responses do not result from direct stimulation of adenylate cyclase by beta gamma subunits released from pertussis toxin-sensitive G-proteins. Atropine treatment of cells transfected with high levels of m4 mAChR, but not m1, causes an elevation in basal levels of cAMP response element-mediated luciferase expression in the absence of agonist. This suggests that the m4 receptor is spontaneously active and can cause constitutive inhibition of adenylyl cyclase that is relieved by atropine treatment. Surprisingly, the m4 receptor exhibits little if any agonist-induced inhibition of luciferase expression at either low or high levels of receptor expression. JEG-3 cells express Gi alpha-1 and Gi alpha-3 but not Gi alpha-2. Cotransfection of Gi alpha subunits with m4 demonstrates that the m4 receptor requires Gi alpha-2 for optimal agonist-mediated inhibition. Even in the presence of Gi alpha-2, high levels of receptor increased luciferase expression at high concentrations of agonist. Thus, the m4 mAChR can undergo a switch in functional coupling from inhibition to stimulation of adenylyl cyclase. This switch is dependent on the level of receptor expression, the subtypes of G-proteins coexpressed with the receptor, and the concentration of agonist. Furthermore, we demonstrate that the Gi alpha-2 G-protein alpha subunit preferentially couples the m4 mAChR to inhibition of adenylyl cyclase in JEG-3 cells.
Highlights
We have used a luciferase reporter gene under the by seven putative transmembrane domains
Atropine treatment of cells transfected with high lev- system has a number of advantages which allow detection of els of m4 mAChR have been etylcholine receptors (mAChRs), but not ml, causes an elevation in subtlebut physiologically relevantchangesinintracellular basal levelsof CAMPresponse element-mediated lucifer-CAMP levelsas manifested in changes in CAMP-regulated gene ase expression in the absence of agonist
Addition of high concentrations of both forskolin that the m4 receptor is spontaneously active and can and PDE inhibitors are often used in direct biochemical detercause constitutive inhibition of adenylyl cyclasethat is minations of intracellular CAMP levels and can confound receprelieved by atropine treatment
Summary
Dansfections-Twelve wells were transfected with each concentration of mAChR DNA five wells for [3H~-methylscopolamine([3HlNMS). Levels of expression of m l and m4 in transient transfections were determined by measuring specific binding of the muscarinic antagonisct3H1NMSto intact cellass described by Nathanson et al (1992). These responses are transfection efficiency, we counted the numberof cells in 20 microscope fields fromwhich we determined an average numberof cells/field. 20 fieldswereexamined,and we counted thenumber of celldfield for the m4 receptor; m l mAChRs usually couple to a robust stained with X-gal. The mean number of X-gal-stained celldfield was increase in PI turnover, and m4 mAChRs usually couple t o divided by the mean numbeofr cells/field to determine what percentageinhibition of AC and sometimes to a lesser increase in PIturnof cells were transfected as indicatedby detectable levelsof P-galactosidaseexpression.Equivalent mAChR expressionlevelsweredetermined by dividing the transfection-specific levels of VHINMS binding sites by the transfectionefficiency percentage. We examined the DNA requirements for the m l and m4 responsesby transfecting JEG-3
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