We have previously reported that: 1) inhibition of cyclooxygenase-2 and PGE2 production reduces hypertrophy following myocardial infarction in mice; and 2) PGE₂ acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVM) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP and protein kinase A (PKA). Our present studies were designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that the 90 kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVM with PGE₂ activated Rap and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant-negative mutant of Rap reduced PGE₂ activation of ERK1/2. PGE2 activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2 and p90RSK in PGE2 regulation of gene expression. PGE₂ stimulation of the BNP promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE₂ stimulation of c-Fos was dependent on EP4, PKA, ERK1/2 and p90RSK, whereas only the latter two kinases were involved in PGE₂ regulation of Egr-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVM in part involves PKA, Rap, ERK1/2, and p90RSK and results in increased expression of BNP and c-Fos.