Regulation of the Hypothalamic GnRH Neuron

下丘脑 GnRH 神经元的调节

• 批准号: 7734672
• 负责人: Kevin J Catt
• 金额: $ 93万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734672
• 关键词: Adult; Agonist; Amino Acids; Angiotensin II; Animal Model; Anterior Pituitary Gland; Binding; Bioluminescence; COS-7 Cell; Cell Line; Cell physiology; Cells; Complementary DNA; Complex; Condition; Coupling; Cyclic AMP; Data; Dose; Energy Transfer; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Receptors; Estrus; Face; Family; Feedback; Female; Fire - disasters; G Protein-Coupled Receptor 54; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Gonadotropin Hormone Releasing Hormone; Gonadotropin Receptors; Gonadotropin-Releasing Hormone Receptor; Gonadotropins; Growth; Homo; Hormone Antagonists; Human; Hypothalamic structure; Individual; KISS1 gene; Klinefelter' s Syndrome; Label; Left; Ligand Binding; Ligands; Mediating; Membrane; Metabolic; Mus; Mutate; Neuraxis; Neurons; Neurosecretory Systems; Numbers; Ovary; Peptides; Peripheral; Pertussis Toxin; Phenylalanine; Pituitary Gland; Potassium; Production; Property; Protein Isoforms; Proteins; Rate; Rattus; Receptor Activation; Receptor Signaling; Regulation; Reproduction; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Serine; Side; Signal Pathway; Signal Transduction; Specificity; Structure; Study Section; System; Tissues; Transfection; Tyrosine; analog; asparaginyl-proline; autocrine; fetal; hypothalamic pituitary gonadal axis; immortalized cell; kisspeptin-10; male; mutant; neuroregulation; peptide hormone; prevent; programs; receptor; receptor function; reproductive function; response; seven-transmembrane G-protein-coupled receptor; vector

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. 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. 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. 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.

雌二醇（E2）充当卵巢和下丘脑GNRH神经元之间的有效反馈分子，并对GNRH合成和分泌产生阳性和负调节作用。 但是，这些在GNRH神经元中表达的雌激素受体介导的程度一直存在争议。在这项研究中，单细胞RT-PCR揭示了培养的胎儿和成年大鼠下丘脑GNRH神经元中雌激素受体α（Eralpha）和β（Erbeta）同工型的表达。 eralpha和Erbeta或单个ER均在94％的培养胎儿GNRH神经元中表达。 在成年雌性大鼠处于二肌的大鼠中，有68％的GnRH神经元表达了ER，其次是发情的54％，主雌感染了19％。在24％的成年雄性GNRH神经元中发现单个ER的表达。 Eralpha对GI介导的对自发性AP解散，营地产生和脉冲GNRH分泌的抑制作用标有明显的抑制作用，这表明其对GNRH神经元功能负调控的能力。 相比之下，E2浓度和ERBETA激动剂的增加提高了AP解火，GNRH分泌和cAMP生产的速度，与GnRH分泌的Erbeta依赖性阳性调节一致。 E2与Eralpha与百日咳毒素（PTX）敏感的GI/O蛋白的偶联还激活了G蛋白激活的G蛋白激活钾（Girk）通道（Girk）通道，膜状兴奋性降低，并减少hypothalamic gnrhneurons中自发性APS的触发性。 这些发现表明，E2对GNRH神经元兴奋性，营地产生和GNRH分泌的双重作用是由在下丘脑GNRH神经元中表达的Eralpha和Erbeta的剂量依赖性激活介导的。 七个跨膜，G蛋白偶联受体（GPCR）的ASN-Pro-任何氨基酸-TYR（NPX2-3Y）序列，其中X代表X代表脂肪族氨基酸，介导与细胞内效应子分子的相互作用。在GNRH受体中，通过分别在TM II和VII中存在的ASP和ASN残基来修改该序列为DPXXY，但保留了大多数GPCR的NPXXY序列基序中保守的TYR残基。在小鼠GNRHR中，Y324酪氨酸残基在Dpliy基序之外，代表面对受体细胞质侧的仅有的两个酪氨酸残基之一。在本研究中，将Tyr324突变为苯丙氨酸，以保留酪氨酸残基的原始芳香族成分。在表达内源性GNRHR和用小鼠GNRHR转染的内源性GNRHR和COS-7细胞的细胞中分析了结合特性和cAMP信号传导。在培养的下丘脑细胞和永生化的GNRH神经元（GT1-7细胞）中，内源性GNRHR的激活产生了双相cAMP反应。营地产生的刺激是百日咳毒素（PTX）不敏感，而对cAMP产生的抑制是通过先前用PTX治疗的。使用PGFP2-N载体在COS-7细胞中表达的野生类型的GnRHR的激动剂刺激导致营地产量单调剂量依赖性增加。与神经元细胞相反，GNRH在HEK-293 cAMP上表达GnRHR的WT的刺激作用是PTX敏感的，这表明GI/O激活中可能的细胞特异性。在表达Tyr324F突变受体的最大结合的COS-7细胞中，125i标记的GnRH激动剂类似物的最大结合仅减少了约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在中性杂种丝氨酸中，这些数据在125i标记的结合和cAMP信号传导中降低了70％。 Kisspeptins是由Kiss-1基因编码的肽家族，促进GNRH分泌，是GPCR的内源配体GPR54。 KISS-1和GPR54均在下丘脑中表达，这与观察到Kisspeptins和gpr54参与下丘脑 - 垂体 - 核轴轴的激活和调节。 破坏的GPR54信号传导导致啮齿动物和人类中的性腺功能不全。亲肽素的中央或外围给药可有效刺激下丘脑 - 垂体 - 轴轴，从而在许多动物模型中导致循环促性腺营养蛋白浓度增加。尽管这些作用似乎可能是由下丘脑促性腺营养蛋白释放激素系统介导的，但亲肽蛋白也可能对垂体前腺有直接作用。 GPR54在与生殖功能有关的许多组织中广泛表达，并且有越来越多的证据表明，亲肽素主要在中枢神经系统水平上起作用以调节GNRH分泌。 促性腺激素释放激素受体（GNRHR）和G蛋白偶联受体54（GPR54）及其相关的配体GNRH和Kisspeptin是下丘脑 - 坐核 - 核 - 核 - 基达轴的必不可少的调节剂。生物发光谐振能量转移（BRET）分析显示，在用GnRH-R-RLUC cDNA和GNRHR-GFP2 cDNA瞬时转染的HEK-293细胞中的组成型和GNRH激活的BRET2信号。 后一种构建体的浓度增加导致BRET2信号的双曲线增加，半最大响应为3.4 0.1 GFP2/RLUC比率。以1:12的GFP2/RLUC比达到了最大的BRET2信号传导，并通过GNRH-R激动剂类似物（D-Ala6Ag）的处理显着增加，并具有不变的半马日式响应。相反，当用GPR54激动剂Kisspeptin-10处理表达GNRH-R HOMO-OLIGOMES的HEK-293细胞时，BRET2信号没有显着变化。当用恒定量的GPR54-ruc cDNA瞬时转染HEK-293细胞并增加GPR54-GFP2 cDNA量时，还观察到GPR54同型异物体的形成。在本构相互作用中，这些条件给出了可饱和的Hyperbolic Bret2信号。在用Kisspeptin-10处理过程中，最大的BRET2信号显着降低，并没有变化的半大反应。同样，当用1 r d-ala6Ag处理表达GPR54同型植物的HEK-293细胞显着降低。这表明，抑制homo-Oligomer形成是由Kisspeptin-10直接激活GPR54或D-Ala6Ag对GPR54的共激活引起的。用固定量的GPR54-RLUC cDNA转染HEK-293细胞，GNRHR-GFP2的量增加导致GPR54/GNRH-R Hetero-Oligomers的形成。用D-Ala6Ag的GPR54激动剂或选择性GNRH-R激活的Kisspeptin-10处理这种复合物可显着增加BRET2信号。在表达GNRH-R HOMO-OLIGOMER的HEK-293细胞中观察到构成性和GNRH激活的BRET信号的增加。 通过用GnRH拮抗剂治疗GNRH诱导的BRET信号增加。 当用恒定量的GPR54-luc cDNA转染HEK-293细胞并增加GPR54-GFP2 cDNA时，观察到HEK-293细胞被转染时观察到GPR54同型异物体。在用Kisspeptin-10治疗期间，BRET2信号的强度显着降低。用固定量的GPR54-RLUC cDNA转染HEK-293细胞，GNRHR-GFP2的量增加导致GPR54/GNRH-R Hetero-Oligomers的形成。用GNRH的GPR54激动剂或选择性GNRH-R激活的Kisspeptin-10处理这种复合物可显着增加BRET2信号。这些结果表明，构成和激动剂介导的GnRH-R和GPR54均和异源 - 异物体的形成在下丘脑GNRH神经元中受体激活的调节中可能具有重要作用。

项目成果

Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons.

永生化促性腺激素释放激素神经元中 Gi 偶联质膜雌激素受体对环腺苷 3,5- 单磷酸信号传导和脉动神经分泌的调节。

• 发表时间: 2003
• 期刊: Molecular endocrinology (Baltimore, Md.)
• 作者: Navarro,CarlosE;Saeed,SheikhAbdul;Murdock,Cynthia;Martinez-Fuentes,AntonioJ;Arora,KrishanK;Krsmanovic,LazarZ;Catt,KevinJ
• 通讯作者: Catt,KevinJ

Estrogen stimulation of COX-2-derived PGI2 confers atheroprotection.

雌激素刺激 COX-2 衍生的 PGI2 具有动脉粥样硬化保护作用。

• DOI: 10.1016/j.tem.2005.05.008
• 发表时间: 2005
• 期刊: Trends in endocrinology and metabolism: TEM
• 作者: Shah,BukhtiarH

Control of action potential-driven calcium influx in GT1 neurons by the activation status of sodium and calcium channels.

通过钠通道和钙通道的激活状态控制 GT1 神经元中动作电位驱动的钙流入。

• DOI: 10.1210/mend.13.4.0261
• 发表时间: 1999
• 期刊: Molecular endocrinology (Baltimore, Md.)
• 作者: VanGoor,F;Krsmanovic,LZ;Catt,KJ;Stojilkovic,SS
• 通讯作者: Stojilkovic,SS

Expression and function of the gonadotropin-releasing hormone receptor are dependent on a conserved apolar amino acid in the third intracellular loop.

促性腺激素释放激素受体的表达和功能依赖于第三细胞内环中的保守非极性氨基酸。

• DOI: 10.1074/jbc.274.50.35756
• 发表时间: 1999
• 期刊: The Journal of biological chemistry
• 作者: Chung,HO;Yang,Q;Catt,KJ;Arora,KK
• 通讯作者: Arora,KK

Autocrine regulation of gonadotropin-releasing hormone secretion in cultured hypothalamic neurons.

培养的下丘脑神经元中促性腺激素释放激素分泌的自分泌调节。

• DOI: 10.1210/endo.140.3.6588
• 发表时间: 1999
• 期刊: Endocrinology
• 影响因子: 4.8
• 作者: Krsmanovic,LZ;Martinez-Fuentes,AJ;Arora,KK;Mores,N;Navarro,CE;Chen,HC;Stojilkovic,SS;Catt,KJ
• 通讯作者: Catt,KJ