M(1) 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 G(q) 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 G(q)/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 G(i)-linked M(2)mAChRs or in cells transfected with M(2)mAChRs. In contrast, in cells transfected with M(1)mAChRs, which effectively couple to G(q)/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as deter mined by immunofluorescence. Carbachol-mediated ANF gene expression was inhibited by the mAChR antagonist pirenzepine with a K-i value characteristic of an M(1)mAChR. Studies using chimeric M(1)- and M(2)mAChRs demonstrated that the N-terminal 21 amino acids of the third intracellular loop of the M(1)mAChR were required for receptor coupling to ANF gene expression. This region, previously shown to specify receptor coupling to G(q)/PLC, also conferred partial activity to a chimeric M(2) receptor. We further demonstrated that M(1)mAChR coupling to ANF gene expression was Ras-dependent since co-expression of dominant-interfering Ala-15 Ras inhibited M(1)mAChR-induced ANF expression by 60%. In contrast, ANF expression induced by the chimeric M(2) receptor was not blocked by dominant interfering Ras. We suggest that receptor coupling to G(q)/PLC is sufficient to induce ANF expression and that a Ras-dependent pathway contributes additional signals required for maximal M(1)mAChR-mediated ANF gene expression.