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.
Casella
Ida
I
Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
Lindner
Herbert
H
Zenzmaier
Christoph
C
Riitano
Daniela
D
Berger
Peter
P
Costa
Tommaso
T
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008969:Molecular Sequence Data; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D011966:Receptors, LHRH / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D000318:Adrenergic beta-Agonists; D015292:Glycoprotein Hormones, alpha Subunit; D018343:Receptors, Adrenergic, beta-2; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D000242:Cyclic AMP; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol; D002118:Calcium