The prostaglandin E(2) (PGE(2)) G protein-coupled receptor (GPCR), EP2, plays important roles in mouse skin tumor development (Chun, K. S., Lao, H. C., Trempus, C. S., Okada, M., and Langenbach, R. (2009) Carcinogenesis 30, 1620-1627). Because keratinocyte proliferation is essential for skin tumor development, EP2-mediated signaling pathways that contribute to keratinocyte proliferation were investigated. A single topical application of the EP2 agonist, butaprost, dose-dependently increased keratinocyte replication via activation of epidermal growth factor receptor (EGFR) and PKA signaling. Because GPCR-mediated activation of EGFR can involve the formation of a GPCR-β-arrestin-Src signaling complex, the possibility of a β-arrestin1-Src complex contributing to EP2-mediated signaling in keratinocytes was investigated. Butaprost induced β-arrestin1-Src complex formation and increased both Src and EGFR activation. A role for β-arrestin1 in EP2-mediated Src and EGFR activation was demonstrated by the observation that β-arrestin1 deficiency significantly reduced Src and EGFR activation. In agreement with a β-arrestin1-Src complex contributing to EGFR activation, Src and EGFR inhibition (PP2 and AG1478, respectively) indicated that Src was upstream of EGFR. Butaprost also induced the activation of Akt, ERK1/2, and STAT3, and both β-arrestin1 deficiency and EGFR inhibition (AG1478 or gefitinib) decreased their activation. In addition to β-arrestin1-dependent EGFR activation, butaprost increased PKA activation, as measured by phospho-GSK3β (p-GSK3β) and p-cAMP-response element-binding protein formation. PKA inhibition (H89 or R(P)-adenosine-3',5'-cyclic monophosphorothioate (R(P)-cAMPS)) decreased butaprost-induced cAMP-response element-binding protein and ERK activation but did not affect EGFR activation, whereas β-arrestin1 deficiency decreased EGFR activation but did not affect butaprost-induced PKA activation, thus indicating that they were independent EP2-mediated pathways. Therefore, the results indicate that EP2 contributed to mouse keratinocyte proliferation by G protein-independent, β-arrestin1-dependent activation of EGFR and G protein-dependent activation of PKA.