We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G(i/o)-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca2+-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP3 formation and intracellular Ca2+ mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca2+ ionophore and the activation of a typical G(q)-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results Suggest that EP3 elicits adenylyl cyclase superactivation via G(q)/phospholipase C activation and intracellular Ca2+ mobilization in a lipid raft microdomain-dependent manner. (C) 2009 Elsevier Inc. All rights reserved.