Gonadotropin-Releasing Hormone Couples to 3′,5′-Cyclic Adenosine-5′-Monophosphate Pathway through Novel Protein Kinase Cδ and -ε in LβT2 Gonadotrope Cells

Abstract

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.

Gonadotropin-Inhibitory Hormone Inhibits GnRH-Induced Gonadotropin Subunit Gene Transcriptions by Inhibiting AC/cAMP/PKA-Dependent ERK Pathway in LβT2 Cells

“…It has been reported that PKC␦ and PKC⑀ are the two endogenous isoforms mediating GnRH action on the activation of AC/cAMP pathway in L␤T2 cells (36). To date, AC2, AC5, and AC7 have been shown to be directly phosphorylated by PKC (54 -56), and two PKC-sensitive AC isoforms, AC5 and AC7, seem to be the potential targets for GnRH stimulation in L␤T2 cells (36).…”

“…To date, AC2, AC5, and AC7 have been shown to be directly phosphorylated by PKC (54 -56), and two PKC-sensitive AC isoforms, AC5 and AC7, seem to be the potential targets for GnRH stimulation in L␤T2 cells (36). These reports indicate that the AC/cAMP/PKA pathway is also activated via G␣ q/11 -mediated PKC pathway.…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…The former messenger diffuses through the cytoplasm, promoting the release of intracellular calcium and the release of both gonadotropins. More recently, coupling of the GnRHR to the cAMP pathway has been shown under conditions of sustained stimulation, which may be potentially important under intense GnRH release, such as the preovulatory GnRH surge (Larivière et al, 2007).…”

Multiple G proteins compete for binding with the human gonadotropin releasing hormone receptor

“…In LβT2 gonadotrope cells, GnRHR receptor stimulation was found to increase intracellular cAMP in a dose dependent fashion, and to increase a cAMP responsive reporter gene [23]. Also, in GH-secreting adenomas, GnRHR increased intracellular calcium levels, cAMP, and AC activity measured in membrane preparations [24].…”