Non-gonadotropin-releasing hormone-mediated transcription and secretion of large human glycoprotein hormone alpha-subunit in human embryonic kidney-293 cells.

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 beta(2)-adrenergic receptor and stimulated with agonist isoproterenol (1 mum). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common alpha-subunit of human glycoprotein hormones (GPHalpha). Induction of GPHalpha 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. Cloning and sequencing of the GPHalpha cDNA and mass spectrometric analysis of HPLC-purified GPHalpha derived from serum-free HEK293-beta(2)-adrenergic receptor-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent Western blots revealed that this was a large hyperglycosylated form of GPHalpha that had not been associated with a beta-subunit previously. This uncombined variant is known to be either cosecreted 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 GPHalpha found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G protein-coupled receptors in HEK293 cells, the expression of large GPHalpha 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 G(s)-coupled receptors other than the classical GnRH receptor may play a role in the regulation of nonpituitary, nonplacental GPHalpha secretion under physiological and pathological conditions.

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