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 c ardiomyocytes induces hypertrophic changes including activation of the atrial natriuretic factor (ANF) gene. This receptor couples to G(q) t o activate phospholipase C (PLC) and protein kinase C, which have been implicated as mediators of the hypertrophic response. To directly det ermine whether receptor coupling to G(q)/PLC is sufficient to induce A NF expression, we expressed wild-type and chimeric muscarinic choliner gic receptors (mAChRs) with altered G-protein coupling properties in c ardiac 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 en dogenous G(i)-linked M(2)mAChRs or in cells transfected with M(2)mAChR s. In contrast, in cells transfected with M(1)mAChRs, which effectivel y couple to G(q)/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as deter mined by immunofluor escence. Carbachol-mediated ANF gene expression was inhibited by the m AChR antagonist pirenzepine with a K-i value characteristic of an M(1) mAChR. Studies using chimeric M(1)- and M(2)mAChRs demonstrated that t he N-terminal 21 amino acids of the third intracellular loop of the M( 1)mAChR were required for receptor coupling to ANF gene expression. Th is region, previously shown to specify receptor coupling to G(q)/PLC, also conferred partial activity to a chimeric M(2) receptor. We furthe r demonstrated that M(1)mAChR coupling to ANF gene expression was Ras- dependent since co-expression of dominant-interfering Ala-15 Ras inhib ited M(1)mAChR-induced ANF expression by 60%. In contrast, ANF express ion induced by the chimeric M(2) receptor was not blocked by dominant interfering Ras. We suggest that receptor coupling to G(q)/PLC is suff icient to induce ANF expression and that a Ras-dependent pathway contr ibutes additional signals required for maximal M(1)mAChR-mediated ANF gene expression.