The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptormediated signaling in L␤T2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LH␤ genes in these cells. Signaling via the G i subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LH␤ induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters G␤␥-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of G␣ q (Q209L) and was blocked by a cell-permeable peptide that uncouples G␣ q from GPCRs. Furthermore, chronic activation of G␣ q signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples G␣ s from receptors was also able to inhibit ERK, c-Fos, and LH␤, indicating that both G q/11 and G s proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on G s and G q/11 and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of G␣ q (Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both G q and G s signaling to regulate gene expression in L␤T2 cells.The family of G protein-coupled receptors is the largest and most complex group of integral membrane proteins involved in signal transduction. These receptors can be activated by a diverse array of external stimuli, including growth factors, neurotransmitters, peptide, and protein hormones, chemokines, and other ligands. Agonist binding to a specific receptor on the cell surface causes a conformational change in the receptor that allows it to interact with its cognate G protein, stimulating guanine nucleotide exchange on the ␣-subunit of the G protein. The release of the GTP-bound ␣-subunit and ␤␥-subunits from the receptor-G protein complex initiates a broad range of intracellular signaling events, including the activation of classical effectors such as phospholipase C, adenylate cyclases, and ion channels, and regulation of the intracellular level of inositol phosphates, calcium, cyclic AMP, and other second messengers (for reviews see Refs. 1-8).Gonadotropin-releasing hormone (GnRH) 1 is a hypothalamic decapeptide, which serves as a key regulator of the reproductive system. In the pituitary, GnRH signals are transmitted via a specific cell surface receptor, which is a member of the G protein-coupled receptor superfamily. When GnRH binds to its receptor, it induces interaction of the receptor with heterot...