Non-Gonadotropin-Releasing Hormone-Mediated Transcription and Secretion of Large Human Glycoprotein Hormone α-Subunit in Human Embryonic Kidney-293 Cells

Abstract

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.

Neuropeptide S receptor 1 expression in the intestine and skin – putative role in peptide hormone secretion

“…Interestingly, SV2C , one of the three isoforms of the SV2 gene, was highly expressed after NPS stimulation in our microarray study and upregulated 22‐fold after 6 h NPS stimulation 24 . Moreover, the CGA‐encoded alpha subunit is also a product of the enteroendocrine cells, although its intestinal function is still unknown 29 . The more specific markers for the enteroendocrine cell neuropeptides that were upregulated by the NPS/NPSR1 pathway included the genes encoding tachykinin precursor (TAC1), neurotensin (NTS) and galanin (GAL) 24 .…”

Gonadotropin-releasing hormone modulates cholesterol synthesis and steroidogenesis in SH-SY5Y cells

“…Total RNA of SH-SY5Y and FNC (100 ng) was amplified with the primer set for LH␣ subunit [28] using the SuperScript TM One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions.…”

Decreased levels of genuine large free hCG alpha in men presenting with abnormal semen analysis

“…Herein, we demonstrate that hCGα purified from seminal plasma had the M r,app of large free hCGα, which is incapable of associating with β-subunits due to larger N-linked sugar chains [31]. Interestingly, the large free α subunit isolated from seminal fluid showed a different glycosylation pattern than large hCGα produced from HEK cells stably transfected with β 2 AR and stimulated with isoproterenol [28]. While the latter appeared to be more highly glycosylated at Asn 52 , seminal plasma-derived hCGα appeared to be more highly glycosylated at Asn 78 .…”

“…and, for comparative purposes, large free hCGα purified by HPLC from supernatant of HEK293 stably transfected with β 2 AR and specifically stimulated with 10 μM isoproterenol [28], as well as the frozen carrier-free concentrate (FC 862) of the WHO adopted 1 st International Reference Preparation for Immunoassay of hCGα (1 st IRP hCGα 99/720) were deglycosylated with peptide N-glycanase PNGase F (New England BioLabs). For digestion under non-reducing conditions to remove the glycan at Asn 52 [29,30], 15 μl samples equivalent to 150 ng hCGα were incubated with 1.5 μl 0.5 M sodium phosphate buffer (pH 7.5), 1.5 μl NP-40 and 1 μl Enzyme (PNGase F, 500 U) for 2 hrs at 37°C.…”