A number of G protein-coupled receptors (GPCRs) have been shown to stimulate signal transducers and activators of transcription (STAT) activities while STAT3 activation by Galpha(0) can lead to neoplastic transformation in fibroblasts. In the present study we examined the ability of GPCRs to activate STAT3 via Galpha(16), a Galpha subunit which is primarily expressed in hematopoietic cells. In HEK 293 cells expressing a STAT3-driven luciferase reporter, the Galpha(16)-coupled ORL1 and fMLP receptors stimulated luciferase activity upon activation by their agonists. Agonist-induced STAT3 activity required coexpression of Galpha(16) and was resistant to PTX treatment. Upon activation of the ORL1 and fMLP receptors, phosphorylation of STAT3 at Tyr(705) was detected by immunoblot analysis. Additional experiments indicated that GPCR-mediated STAT3 activation was dependent on JAK and Raf1 signaling, but did not require phosphatidylinositol 3-kinase. This is the first study that demonstrates the stimulatory effect of ORL1 and fMLP receptors on STAT3 activity. (C) 2003 Elsevier Science (USA). All rights reserved.