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IBA Publikation
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DATUM, UNTERSCHRIFT / DATE, SIGNATURE
BANK AUSTRIA CREDITANSTALT, WIEN (IBAN AT04 1100 0006 2280 0100, BIC BKAUATWW), DEUTSCHE BANK MÜNCHEN (IBAN DE16 7007 0024 0238 8270 00, BIC DEUTDEDBMUC)
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IBA Publikation, pp. 1144-1154, 2008/09/01
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.
Keywords: Large human glycoprotein hormone α-subunit, hCG, G-protein coupled receptors, HEK293, reporter gene, non-GnRH-mediated secretion