M1 muscarinic receptors heterologously expressed in cardiac myocytes mediate Ras-dependent changes in gene expression.
Stimulation of alpha 1-adrenergic receptors in neonatal ventricular cardiomyocytes induces hypertrophic changes including activation of the atrial natriuretic factor (ANF) gene. This receptor couples to Gq to activate phospholipase C (PLC) and protein kinase C, which have been implicated as mediators of the hypertrophic response. To directly determine whether receptor coupling to Gq/PLC is sufficient to induce ANF expression, we expressed wild-type and chimeric muscarinic cholinergic receptors (mAChRs) with altered G-protein coupling properties in cardiac myocytes and examined their ability to activate an ANF promoter/luciferase reporter gene. The cholinergic agonist carbachol failed to induce transcriptional activation of the ANF reporter gene through endogenous Gi-linked M2mAChRs or in cells transfected with M2mAChRs. In contrast, in cells transfected with M1mAChRs, which effectively couple to Gq/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as determined by immunofluorescence. Carbachol-mediated ANF gene expression was inhibited by the mAChR antagonist pirenzepine with a Ki value characteristic of an M1mAChR. Studies using chimeric M1- and M2mAChRs demonstrated that the N-terminal 21 amino acids of the third intracellular loop of the M1mAChR were required for receptor coupling to ANF gene expression. This region, previously shown to specify receptor coupling to Gq/PLC, also conferred partial activity to a chimeric M2 receptor. We further demonstrated that M1mAChR coupling to ANF gene expression was Ras-dependent since co-expression of dominant-interfering Ala-15 Ras inhibited M1mAChR-induced ANF expression by 60%. In contrast, ANF expression induced by the chimeric M2 receptor was not blocked by dominant-interfering Ras. We suggest that receptor coupling to Gq/PLC is sufficient to induce ANF expression and that a Ras-dependent pathway contributes additional signals required for maximal M1mAChR-mediated ANF gene expression.
Ramirez
M T
MT
Department of Pharmacology, University of California at San Diego, La Jolla 92093, USA.
Post
G R
GR
Sulakhe
P V
PV
Brown
J H
JH
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000235:genetics*; D002478:Cells, Cultured; D018076:DNA, Complementary; D005786:Gene Expression Regulation; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D018076:DNA, Complementary; D011976:Receptors, Muscarinic; D009320:Atrial Natriuretic Factor; D018631:ras Proteins