Regulation of the Hypothalamic GnRH Neuron

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Estradiol (E2) acts as a potent feedback molecule between the ovary and hypothalamic GnRH neurons, and exerts both positive and negative regulatory actions on GnRH synthesis and secretion. However, the extent to which these are mediated by estrogen receptors expressed in GnRH neurons has been controversial. In this study, Single-cell RT-PCR revealed the expression of both estrogen receptor alpha (ERalpha) and beta (ERbeta) isoforms in cultured fetal and adult rat hypothalamic GnRH neurons. Both ERalpha and ERbeta or individual ERs were expressed in 94% of cultured fetal GnRH neurons. In adult female rats at diestrous, 68% of GnRH neurons expressed ERs, followed by 54% in estrous and 19% in proestrous. Expression of individual ERs was found in 24% of adult male GnRH neurons. ERalpha exerts marked Gi-mediated inhibitory effects on spontaneous AP firing, cAMP production, and pulsatile GnRH secretion, indicating its capacity for negative regulation of GnRH neuronal function. In contrast, increased E2 concentrations and ERbeta agonists increase the rate of AP firing, GnRH secretion, and cAMP production, consistent with ERbeta-dependent positive regulation of GnRH secretion. Consonant with the coupling of ERalpha to pertussis toxin (PTX)-sensitive Gi/o proteins, E2 also activates G protein-activated inwardly rectifying potassium (GIRK) channels, decreasing membrane excitability and slowing the firing of spontaneous APs in hypothalamic GnRH neurons. These findings demonstrate that the dual actions of E2 on GnRH neuronal membrane excitability, cAMP production, and GnRH secretion are mediated by the dose-dependent activation of ERalpha and ERbeta expressed in hypothalamic GnRH neurons. [unreadable] [unreadable] The Asn-Pro-any amino acid-Tyr (NPX2-3Y) sequence of the seven transmembrane, G protein-coupled receptors (GPCRs) where X represents an aliphatic amino acid, mediates interactions with intracellular effectors molecules. In the GnRH receptor, this sequence is modified to DPXXY by exchange of the Asp and Asn residues normally present in TM II and VII, respectively, but retains the Tyr residue that is conserved in the NPXXY sequence motif of most GPCRs. In the mouse GnRHR, the Y324 tyrosine residue is outside the DPLIY motif, and represents one of only two tyrosine residues that face the cytoplasmic side of the receptor. In the present studies Tyr324 was mutated to phenylalanine to preserve the original aromatic component of the tyrosine residue. The binding properties and cAMP signaling were analyzed in cells expressing endogenous GnRHR and COS-7 cells transfected with mouse GnRHR. In cultured hypothalamic cells and immortalized GnRH neurons (GT1-7 cells) activation of endogenous GnRHR gives a biphasic cAMP response. Stimulation of cAMP production was pertussis toxin (PTX) insensitive, while inhibition of cAMP production was reversed by prior treatment with PTX. Agonist stimulation of the wild type of GnRHR expressed in COS-7 cells using a pGFP2-N vector caused a monotonic dose-dependent increase in cAMP production. In contrast to neuronal cells, the stimulatory action of GnRH on cAMP in HEK-293 expressing WT of GnRHR was PTX-sensitive, suggesting possible cell specificity in Gi/o activation. In COS-7 cells expressing Tyr324F mutant receptor maximal binding of 125I-labeled GnRH agonist analog was reduced by only about 10%. cAMP production remained monotonic, dose-dependent, and ED50 of the Tyr324F mutant receptor was shifted to left (63.2 nM, WT vs 0.3 nM, mutant. In contrast to the WT GnRH-R, pretreatment with PTX of COS-7 cells expressing the Tyr324F mutant receptor had no effect on cAMP production. Substitution of aromatic-hydrophobic Tyr234 of GnRHR with neutral-hydrophilic serine caused a 70% decrease in 125I-labeled binding and loss of cAMP signaling. In summary, these data present evidence that Tyr234 has an important role in ligand binding, cAMP signaling, and GnRHR-Gi/o interaction.[unreadable] [unreadable] Kisspeptins, a family of peptides encoded by the KiSS-1 gene, promote GnRH secretion and are endogenous ligands for the GPCR, GPR54. Both KiSS-1 and GPR54 are expressed in the hypothalamus, consistent with observations that kisspeptins and GPR54 are involved in activation and regulation of the hypothalamic-pituitary-gonadal axis. Disrupted GPR54 signaling causes hypogonadotrophic hypogonadism in rodents and humans. Central or peripheral administration of kisspeptin potently stimulates the hypothalamic-pituitary-gonadal axis, causing increased circulating gonadotrophin concentrations in a number of animal models. Although these effects appear likely to be mediated by the hypothalamic gonadotrophin-releasing hormone system, kisspeptins may also have direct effects on the anterior pituitary gland. GPR54 is widely expressed in many tissues related to reproductive function, and there is increasing evidence that kisspeptin acts predominantly at the level of the central nervous system to regulate GnRH secretion. [unreadable] [unreadable] The gonadotropin-releasing hormone receptor (GnRHR) and G protein-coupled receptor 54 (GPR54) and their related ligands, GnRH and kisspeptin, are essential regulators of the hypothalamic-pituitary-gonadal axis. Bioluminescence resonance energy transfer (BRET) analysis revealed constitutive and GnRH-activated BRET2 signals in HEK-293 cells transiently transfected with of GnRH-R-Rluc cDNA and GnRHR-GFP2 cDNA. Increasing concentrations of the latter construct caused a hyperbolic rise in the BRET2 signal with half-maximal response at 3.4 0.1 GFP2/Rluc ratios. Maximal BRET2 signaling was reached at a 1:12 GFP2/Rluc ratio and was significantly increased by treatment with a GnRH-R agonist analog (D-Ala6Ag), with unchanged half-maximal response. In contrast, when HEK-293 cells expressing GnRH-R homo-oligomers were treated with kisspeptin-10, a GPR54 agonist, there was no significant change in the BRET2 signal. Formation of GPR54 homo-oligomers was also observed when HEK-293 cells were transiently transfected with a constant amount of GPR54-Rluc cDNA and increasing amounts of GPR54-GFP2 cDNA. In constitutive interaction these conditions give a saturable-hyperbolic BRET2 signal. The maximal BRET2 signal decreased significantly during treatment with kisspeptin-10 with, unchanged half-maximal response. Similarly, when HEK-293 cells expressing GPR54 homo-oligomers were treated with 1 M D-Ala6Ag the BRET2 was significantly reduced. This suggests that inhibition of homo-oligomer formation is caused by direct activation of GPR54 by kisspeptin-10, or co-activation of GPR54 by D-Ala6Ag. Transfection of HEK-293 cells with a fixed amount of GPR54-Rluc cDNA and increasing amounts of GnRHR-GFP2 caused formation of GPR54/GnRH-R hetero-oligomers. Treatment of such complex with kisspeptin-10, a GPR54 agonist, or selective GnRH-R activation by D-Ala6Ag, significantly increased the BRET2 signal. Constitutive and GnRH-activated increase in BRET signals was observed in HEK-293 cells expressing GnRH-R homo-oligomers. The GnRH-induced increase in BRET signal was prevented by prior treatment with a GnRH antagonist. GPR54 homo-oligomers were observed when HEK-293 cells were transfected with a constant amount of GPR54-Rluc cDNA and increasing amounts of GPR54-GFP2 cDNA. The intensity of BRET2 signal decreased significantly during treatment with kisspeptin-10. Transfection of HEK-293 cells with a fixed amount of GPR54-Rluc cDNA and increasing amounts of GnRHR-GFP2 caused formation of GPR54/GnRH-R hetero-oligomers. Treatment of such complex with kisspeptin-10, a GPR54 agonist, or selective GnRH-R activation by GnRH, significantly increased the BRET2 signal. These results suggest that constitutive and agonist-mediated formation of GnRH-R and GPR54 homo- and hetero-oligomers may have a significant role in the regulation of receptor activation in hypothalamic GnRH neurons.