Abstract
Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LβT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved.
Highlights
Reproductive function is under the control of the hypothalamic neurohormone gonadotropinreleasing hormone (GnRH), which activates an heptahelical transmembrane receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR)
We observed that GnRHa dose-dependently decreased SET protein expression with an EC50 of 7.3 ± 0.9 nM and a maximal decrease of 36 ± 3% at 10−7 M of GnRHa, that was maintained at 10−6 M of GnRHa (Fig 1A)
To elucidate the mechanisms involved in SET protein down-regulation upon GnRHa stimulation, we first determined the kinetics of GnRHa action on SET protein expression in αT3-1 cells
Summary
Reproductive function is under the control of the hypothalamic neurohormone gonadotropinreleasing hormone (GnRH), which activates an heptahelical transmembrane receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). Activation of GnRHR leads to synthesis and secretion of the two gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), both regulating gametogenesis and steroidogenesis in gonads. GnRHR activates a complex signaling network involving notably calcium and cyclic AMP (cAMP) pathways. GnRHR is mainly coupled to phospholipase Cβ via Gαq/11, leading to a rapid increase of intracellular calcium concentrations and activation of several PKC isoforms [1]. Recruitment of the cAMP/PKA pathway by GnRH contributes to the regulation of a limited set of gene expression including Gnrhr [2,3]
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