To identify genes that are most responsive to a sustained activation of a G_s-protein coupled receptor, HEK293 cells were stably transfected with the ₂-adrenergic receptor (HEK293-₂AR) and stimulated with agonist isoproterenol (1 µM). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common -subunit of human glycoprotein hormones (GPH). Induction of GPH transcription in response to cAMP elevations resulted in a dramatic increase (600-fold) of protein secretion as shown by RT-PCR and a highly specific time-resolved-immunofluorometric assay (TR-IFMA). Cloning and sequencing of the GPH cDNA and mass spectrometric (MS/MS) analysis of HPLC-purified GPH derived from serum-free HEK293-₂AR-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent western blots revealed that this was a large hyperglycosylated form of GPH that had not been associated with a -subunit previously. This uncombined variant is known to be either co-secreted with GPHs from the pituitary, the placenta and a variety of tumors or secreted without GPHs from APUD cells and rare tumors. Moreover, it is similar to GPH found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G protein-coupled receptors in HEK293, the expression of large GPH was controlled by G_s- and G_q- but not G_i-dependent receptors and mediated via cAMP and Ca⁺⁺ release. This suggests that Gq or Gs-coupled receptors other than the classical GnRH-receptor may play a role in the regulation of non-pituitary, non-placental GPH secretion under physiological and pathological conditions.