IBA Publikation

Extern

Abstract:
To identify genes that are most responsive to a sustained activation of a Gs-protein coupled receptor, HEK293 cells were stably transfected with the β2-adrenergic receptor (HEK293-β2AR) and stimulated with agonist isoproterenol (1 μM). A microarray study indicated that the gene with the highest stimulation index (500-fold) encoded the common α-subunit of human glycoprotein hormones (GPHα). Induction of GPHα 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 timeresolved-immunofluorometric assay (TR-IFMA). Cloning and sequencing of the GPHα cDNA and mass spectrometric (MS/MS) analysis of HPLC-purified GPHα derived from serum-free HEK293-β2AR-stimulated cells verified the nature of the molecule. Enzymatic deglycosylation with subsequent western blots revealed that this was a large hyperglycosylated form of GPHα that had not been associated with a β-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 GPHα found at high concentrations in seminal plasma. As shown by a panel of endogenous or transfected G proteincoupled receptors in HEK293, the expression of large GPHα was controlled by Gs- and Gq- but not Gi-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 GPHα secretion under physiological and pathological conditions.