促性腺激素释放激素配体诱导受体信号/功能选择性激活的分子机理

G protein-coupled receptors (GPCRs) modulate the majority of physiological and pathological processes and comprise the largest family of cell surface receptors. About 40% of current clinic drugs target GPCRs. Gonadotropin-releasing hormones (GnRHs) regulate the entire reproductive system by stimulating secretion of gonadotropins that in turn regulate steroidogenesis and gametogenesis, and also participates in the control of cellular energy expenditure and homeostasis that are important for reproductive competency. GnRH analogs can also directly inhibit cancer cell growth. Both GnRH peptide agonists and antagonists have been widely applied to treat infertility, hormone-dependent diseases and peripheral reproductive cancers such as breast and prostate cancers. There are two types of endogenous ligands (GnRH I and II), but only one functional type of receptor in humans.The type II receptor has been silenced by stop codons and frameshift deletions.The primary role of GnRH I is on the control of tonic pulsatile LH release, whereas GnRH II have roles in the generation of the preovulatory LH surge, sexual arousal and food intake. An outstanding question is how a single receptor mediates the effects of two distinct ligands. We proposed that binding of GnRH ligands to the receptor can induce different receptor conformations which preferentially activate differential signaling pathways, e.g.on trophoblast invasion, only GnRH II, but not GnRH I transactivates EGF receptor kinase. We termed this "ligand-induced selective signaling (LiSS)", which is an important concept for understanding the intricate functions of GPCRs and the future drug development. Our preliminary results have implied that the classic Gq/11-coupled GnRH receptor can activate other G proteins and non-G protein effectors, mediating differential functional responses. In this project, we will apply various techniques such as site-directed mutagenesis, signaling molecule silence/knock-down, pathway-specific inhibitors to identify novel GnRH receptor-interacting proteins such as G12/13, c- Src, SHP-2 and their interacting sites on the receptor. The roles of the two main G12/13 downstream signaling pathways: p115RhoGEF-Rho/Rock-SRE and E-cadherin-beta-catenin -T cell factors in the modulation of LH synthesis and exocytosis, as well as breast cancer cell growth.These functional studies will be extended into c-Src,SHP-2 and other newly identified effectors. The roles of c-Src and SHP-2 and their signaling to AMPK-mTOR/S6 kinase 1 that lead to the energy control and cell growth inhibition will be examined too. We will also investigate GnRH ligand-induced selective interactions of the receptor to the different effectors. The studies will both advance our current understanding of the molecular mechanisms of GnRH receptor signalling at the systems level, and also have great impact on the development of signal/function-selective drugs with improved therapeutic efficacy and reduced side-effects.

G蛋白偶联受体调节众多生理和病理过程，构成最重要的细胞表面药物靶蛋白超家族。人类存在二种促性腺激素释放激素(GnRH I和II)，但只有一种功能性受体。签于其生理及药理效应的多样性，本项目申请人及其同事提出了GnRH配体诱导受体信号/功能选择性理论, 但对其受体信号转导/偶联的多样性及其功能选择性的分子机理尚有待深入研究。本项目旨在探索经典Gq/11偶联GnRH受体与其它G蛋白如G12/13及非G效应蛋白如Src及SHP-2的相互作用及其细胞生理、药理效应；二种内源性配体的信号选择性及其结构基础。对GnRH配体诱导受体信号/功能选择性理论的纵深研究, 不仅对阐明GnRH在生殖、代谢/能量控制及其抑制肿瘤细胞如乳腺癌及前列腺癌生长的作用机制具有重要意义, 而且对揭示GPCR信号传递的系统生物学、信号选择性药物的全新设计、改善临床药物疗效、减少毒副作用等都具有深远的理论和现实意义。

人类促性腺激素释放激素(GnRH)受体为Gq/11偶联受体，存在两种内二种源性配体GnRH I和GnRH II，却只有一个功能性受体。本项目对GnRH受体信号转导/偶联的多样性及GnRH I 和GnRH II信号转导的选择性开展了深入的研究。发现，GnRH I 和GnRH II 均可激活G12/13信号转导途经，但两者对Gq/11和G12/13呈现差异性激活倾向，即一定程度的信号选择性。G12/13与Gq/11信号转导途经共同参与GnRH对黄体生成素合成与分泌的生理调节效应。此外，人类GnRH受体除了与G蛋白偶联之外，尚可直接通过Src激活SHP2磷酸化(激活)，参与调节ERK1/2的磷酸化程度。这一信号转导途经需要Gq/11的存在，但与其磷脂酶-磷酸肌醇信号转导途经无关。这一信号途径可以解释为什么众多Gq/11偶联受体促进肿瘤细胞生长，而GnRH受体却可抑制肿瘤细胞生长。这些新发现的信号途径与传统的Gq/11信号途径构成复杂的GnRH受体信号通信网络，调节GnRH的生理及药理效应。GnRH I 及GnRH II可差异性地影响不同的信号转导途经，从调节不同的生理效应。..Kisspeptins (KPs) 作为Kiss神经元分泌的神经肽参与GnRH的分泌调控，而外周KPs具有抑制肿瘤转移的重要功能。我们发现，KP受体可激活钙调蛋白依懒性激酶II，并催化KP受体磷酸化，反馈性抑制KP受体与Gq/11的偶联，从而调节胞内钙离子的波动强度与频率，参与KPs对GnRH/LH脉冲式释放的调节。此外，本项目还设计合成了二个KP 膦肽，其中一个(PKPR)可激活KP受体，并可选择性抑制基质金属蛋白酶2的活性，为进一步研发具有双重活性或代谢稳定性KP激动剂提供了一个先导化合物。也发现KP受体C末端半胱氨酸Cys340为棕榈酰化位点，参与受体的表达，内吞及细胞内的运输和受体信号转导过程。..上述研究对深入了解GnRH及KP受体在生殖及肿瘤生长转移的作用机制具有重要意义，对信号选择性药物的设计、改善临床药物疗效、减少毒副作用等也具有现实意义。..本项目也建立了RNA BSP克隆测序法(mC5)及MeRIP-Seq (m6A)测序法及其RNA甲基化测序结果的生物信息学分析技术。为今后研究疾病，药物及信号转导对RNA甲基化的影响奠定了基础。