Desensitization and internalization of human and Xenopus gonadotropin-releasing hormone receptors expressed in αT4 pituitary cells using recombinant adenovirus
Desensitization and internalization of human and Xenopus gonadotropin-releasing hormone receptors expressed in αT4 pituitary cells using recombinant adenovirus
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DOI：
10.1210/en.141.12.4564
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发表时间：
2000-12-01
期刊：
影响因子：
4.8
通讯作者：
McArdle, CA
中科院分区：
文献类型：
--
作者：
Hislop, JN;Madziva, MT;McArdle, CA
非哺乳类脊椎动物表达至少两种形式的GnRH，并且不同形式的GnRH受体（GnRH-R）与其配体共同进化。哺乳动物和非哺乳动物：GnRH-R具有关键的结构差异（特别是哺乳动物GnRH-R缺乏C-末端尾），比较研究开始揭示其功能相关性。然而，细胞环境和受体密度的影响G蛋白偶联受体的功能，并可能在这样的工作中使用异源表达系统的重要变量。在这里，我们报告了一项比较研究，使用α T4细胞（缺乏内源性GnRH-R的促性腺细胞祖细胞）转染哺乳动物（人类）或非哺乳动物（非洲爪蟾I型）GnRH-R。由于传统的转染策略证明效率低下，重组腺病毒表达这些受体的构建，使控制和有效的GnRH-R的表达。当在α T4细胞中以生理密度表达时，这些GnRH-R保留了其内源性对应物的药理学（根据放射性配体结合和磷酸肌醇蓄积试验中的配体特异性判断），但不激活腺苷酸环化酶，并且不具有组成性活性。此外，非洲爪蟾GnRH-R在这些细胞中迅速脱敏和内化，而人GnRH-R则不然，并且内化率不依赖于受体数量。这些数据扩展了在COS、HEK和GH（3）细胞中的研究，表明其他具有C-末端尾的GnRH-R可迅速脱敏和内化，而无尾哺乳动物GnRH-R则不然。在促性腺激素谱系细胞的生理受体密度保留这些区别，支持的论点，即非脱敏哺乳动物GnRH-Rs的进化是功能相关的，并与哺乳动物生殖策略的发展。
Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian:GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alpha T4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alpha T4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH(3) cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.