Finch Ann R, Caunt Christopher J, Armstrong Stephen P, McArdle Craig A
Laboratory for Integrative Neuroscience, Department of Clinical Sciences at South Bristol, Whitson Street, Bristol BS1 3NY, United Kingdom.
Mol Endocrinol. 2010 Feb;24(2):423-35. doi: 10.1210/me.2009-0343. Epub 2009 Dec 15.
Gonadotropin-releasing hormone acts via cell surface receptors but most human (h) GnRH receptors (GnRHRs) are intracellular. A membrane-permeant nonpeptide antagonist [(2S)-2-[5-[2-(2-axabicyclo[2.2.2]oct-2-yl)-1,1-dimethy-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine (IN3)] increases hGnRHR expression at the surface, apparently by facilitating its exit from the endoplasmic reticulum. Here we have quantified GnRHR by automated imaging in HeLa cells transduced with adenovirus expressing hemagglutinin-tagged GnRHR. Consistent with an intracellular site of action, IN3 increases cell surface hGnRHR, and this effect is not blocked or mimicked by membrane-impermeant peptide antagonists [Ac-D2Nal-D4Cpa-D3Pal-Ser-Tyr-d-Cit-Leu-Arg-Pro-d-Ala-NH(2) (cetrorelix) and antide]. However, when the C-terminal tail of a Xenopus (X) GnRHR was added (h.XGnRHR) to increase expression, both peptides further increased cell surface GnRHR. Cetrorelix also synergized with IN3 to increase expression of hGnRHR and a G-protein coupling-deficient mutant (A261K-hGnRHR). Cetrorelix also increased cell surface expression of hGnRHR, h.XGnRHR, and mouse GnRHR in gonadotrope-lineage LbetaT2 cells, and in HeLa cells it slowed h.XGnRHR internalization (measured by receptor-mediated antihemagglutinin uptake). Thus cetrorelix has effects other than GnRHR blockade; it acts as an inverse agonist in internalization assays, supporting the potential importance of ligand-biased efficacy at GnRHR. We also developed an imaging assay for GnRH function based on Ca(2+)-dependent nuclear translocation of a nuclear factor of activated T cells reporter. Using this in HeLa and LbetaT2 cells, IN3 and cetrorelix behaved as competitive antagonists when coincubated with GnRH, and long-term pretreatment (16 h) with IN3 reduced its effectiveness as an inhibitor whereas pretreatment with cetrorelix increased its inhibitory effect. This distinction between peptide and nonpeptide antagonists may prove important for therapeutic applications of GnRH antagonists.
促性腺激素释放激素通过细胞表面受体发挥作用，但大多数人（h）促性腺激素释放激素受体（GnRHRs）位于细胞内。一种可透过细胞膜的非肽拮抗剂[（2S）-2-[5-[2-（2-氮杂双环[2.2.2]辛-2-基）-1,1-二甲基-2-氧代乙基]-2-（3,5-二甲基苯基）-1H-吲哚-3-基]-N-（2-吡啶-4-基乙基）丙-1-胺（IN3）]可增加细胞表面hGnRHR的表达，显然是通过促进其从内质网中释放出来实现的。在此，我们通过自动成像对用表达血凝素标记的GnRHR的腺病毒转导的HeLa细胞中的GnRHR进行了定量。与细胞内作用位点一致，IN3增加了细胞表面的hGnRHR，而这种作用并未被不能透过细胞膜的肽拮抗剂[Ac-D2Nal-D4Cpa-D3Pal-Ser-Tyr-d-Cit-Leu-Arg-Pro-d-Ala-NH(2)（西曲瑞克）和抗肽]阻断或模拟。然而，当添加非洲爪蟾（X）GnRHR的C末端尾巴（h.XGnRHR）以增加表达时，这两种肽都进一步增加了细胞表面的GnRHR。西曲瑞克还与IN3协同作用，增加hGnRHR和一种G蛋白偶联缺陷突变体（A261K-hGnRHR）的表达。西曲瑞克还增加了促性腺激素细胞系LbetaT2细胞中hGnRHR、h.XGnRHR和小鼠GnRHR的细胞表面表达，并且在HeLa细胞中它减缓了h.XGnRHR的内化（通过受体介导的抗血凝素摄取来测量）。因此，西曲瑞克除了具有GnRHR阻断作用外还有其他作用；它在内化试验中作为反向激动剂起作用，支持了配体偏向性效能在GnRHR上的潜在重要性。我们还基于活化T细胞核因子报告基因的钙依赖性核转位开发了一种用于GnRH功能的成像试验。在HeLa细胞和LbetaT2细胞中使用该试验，当IN3和西曲瑞克与GnRH共同孵育时，它们表现为竞争性拮抗剂，并且用IN3进行长期预处理（16小时）会降低其作为抑制剂的有效性，而用西曲瑞克预处理则会增加其抑制作用。肽拮抗剂和非肽拮抗剂之间的这种区别可能对GnRH拮抗剂的治疗应用很重要。
