Abstract
Receptors coupled to heterotrimeric G proteins are linked to activation of mitogen-activated protein kinases (MAPKs) via receptor- and cell-specific mechanisms. We have demonstrated recently that gonadotropin-releasing hormone (GnRH) receptor occupancy results in activation of extracellular signal-regulated kinase (ERK) through a mechanism requiring calcium influx through L-type calcium channels in alphaT3-1 cells and primary rat gonadotropes. Further studies were undertaken to explore the signaling mechanisms by which the GnRH receptor is coupled to activation of another member of the MAPK family, c-Jun N-terminal kinase (JNK). GnRH induces activation of the JNK cascade in a dose-, time-, and receptor-dependent manner in clonal alphaT3-1 cells and primary rat pituitary gonadotrophs. Coexpression of dominant negative Cdc42 and kinase-defective p21-activated kinase 1 and MAPK kinase 7 with JNK and ERK indicated that specific activation of JNK by GnRH appears to involve these signaling molecules. Unlike ERK activation, GnRH-stimulated JNK activity does not require activation of protein kinase C and is not blocked after chelation of extracellular calcium with EGTA. GnRH-induced JNK activity was reduced after treatment with the intracellular calcium chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), whereas activation of ERK was not affected. Chelation of intracellular calcium also reduced GnRH-induced activation of JNK in rat pituitary cells in primary culture. GnRH-induced induction and activation of the JNK target c-Jun was inhibited after chelation of intracellular calcium, whereas induction of c-Fos, a known target of ERK, was unaffected. Therefore, although activation of ERK by GnRH requires a specific influx of calcium through L-type calcium channels, JNK activation is independent of extracellular calcium but sensitive to chelation of intracellular calcium. Our results provide novel evidence that GnRH activates two MAPK superfamily members via strikingly divergent signaling pathways with differential sensitivity to activation of protein kinase C and mobilization of discrete pools of calcium.
Highlights
G protein-coupled receptors activate mitogen-activated protein kinases (MAPKs) signaling pathways through mechanisms that vary with specific ligand-receptor interactions, heterotrimeric G protein subtypes, and cellular phenotype [1]
We have demonstrated recently that calcium influx through VGCCs is absolutely required for activation of extracellular signal-regulated kinase (ERK) by the gonadotropin-releasing hormone (GnRH) receptor agonist buserelin in both ␣T3-1 cells and pituitary cells maintained in primary culture [10]
To provide evidence that GnRH receptor occupancy was required for Jun N-terminal kinase (JNK) activation, the cells were pretreated with the specific GnRH receptor antagonist, antide
Summary
G protein-coupled receptors activate MAPK signaling pathways through mechanisms that vary with specific ligand-receptor interactions, heterotrimeric G protein subtypes, and cellular phenotype [1]. Activation of the Gq/11-coupled receptor for GnRH in the clonal gonadotrope ␣T3-1 cell line or in pituitary cells in primary culture results in stimulation of a complex signaling cascade that includes activation of phospholipase C, production of IP3 and diacylglycerol, and subsequent activation of PKC [4, 5]. In contrast to the signaling pathway for activation of ERK, GnRH-induced activation of JNK occurs independently of both PKC activation and extracellular calcium. Inhibition of GnRH-induced JNK activity by chelation of intracellular calcium was observed in studies using rat pituitary cells in primary culture. Our results support the conclusion that there is divergence in the signaling pathways coupling the GnRH receptor to multiple MAPKs with selective requirements for classical downstream signaling molecules as well as PKC and pharmacologically distinct calcium signals. Defining specific elements that are critical for activating MAPKs is fundamental to the understanding of GnRHmediated regulation of immediate early and late response genes that control gonadotrope function
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