Schoderbek W E, Roberson M S, Maurer R A
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
J Biol Chem. 1993 Feb 25;268(6):3903-10.
Transient transfection studies using gonadotrope-derived, alpha T3-1 cells were used to determine the DNA sequences of the mouse glycoprotein hormone alpha-subunit gene that mediate the transcriptional response to gonadotropin releasing hormone (GnRH). The roles of phorbol esters and cyclic AMP in mediating the GnRH response were also investigated. The initial studies demonstrated that a construct containing approximately 500 base pairs of alpha-subunit flanking sequence was sufficient to mediate responses to a GnRH agonist (GnRHa), phorbol myristate acetate (PMA) and a cAMP analog. Responses to combinations of cAMP and GnRHa or cAMP and PMA were approximately additive, whereas the response to the combination of GnRHa and PMA was similar to that seen with either of the agents alone. Cotransfection studies with an expression vector for the heat-stable inhibitor of the cAMP-dependent protein kinase demonstrated that GnRHa and PMA responses are not dependent on the cAMP-dependent kinase. Deletion analysis indicated that sequences between -507 and -205 were involved in mediating responses to GnRHa and PMA. To determine if this region alone could support responses to these agents, the -507 to -205 region was linked to a minimal promoter and tested in transient transfections. The results demonstrated that this region supports responses to GnRHa, PMA, and cAMP. Clustered point mutations of this region were used to further characterize sequences involved in the GnRH response. Mutations in two regions, one at positions -406 to -399 and one at positions -337 to -330, resulted in decreased responses to GnRH and PMA. There is no obvious sequence similarity between the two regions that are required for the GnRH response. An enhancer test demonstrated that multimers of the -416 to -385 region were able to function as a GnRH-responsive element when linked to a minimal promoter, although a single copy of this region was not sufficient to permit a response to GnRH. In contrast, multimers of the -344 to -300 region did not permit a response to GnRH, but enhanced basal transcription. These findings are consistent with the identification of a two-component GnRH response unit, which probably involves the functional cooperation of two different transcription factors. The observation that GnRH responsiveness appears to co-localize with PMA responsiveness suggests that GnRH effects on the alpha-subunit transcription are likely mediated by the protein kinase C pathway.
利用源自促性腺激素细胞的αT3 - 1细胞进行瞬时转染研究，以确定小鼠糖蛋白激素α亚基基因的DNA序列，该序列介导对促性腺激素释放激素（GnRH）的转录反应。同时还研究了佛波酯和环磷酸腺苷（cAMP）在介导GnRH反应中的作用。初步研究表明，一个包含约500个碱基对α亚基侧翼序列的构建体足以介导对GnRH激动剂（GnRHa）、佛波醇肉豆蔻酸酯乙酸酯（PMA）和一种cAMP类似物的反应。对cAMP与GnRHa或cAMP与PMA组合的反应大致呈累加性，而对GnRHa与PMA组合的反应与单独使用这两种试剂中的任何一种时相似。用cAMP依赖性蛋白激酶的热稳定抑制剂的表达载体进行共转染研究表明，GnRHa和PMA反应不依赖于cAMP依赖性激酶。缺失分析表明， - 507至 - 205之间的序列参与介导对GnRHa和PMA的反应。为了确定仅该区域是否能支持对这些试剂的反应，将 - 507至 - 205区域与一个最小启动子连接，并在瞬时转染中进行测试。结果表明该区域支持对GnRHa、PMA和cAMP的反应。对该区域进行成簇点突变以进一步表征参与GnRH反应的序列。两个区域的突变，一个在 - 406至 - 399位，另一个在 - 337至 - 330位，导致对GnRH和PMA的反应降低。GnRH反应所需的这两个区域之间没有明显的序列相似性。增强子测试表明， - 416至 - 385区域的多聚体与最小启动子连接时能够作为GnRH反应元件发挥作用，尽管该区域的单拷贝不足以允许对GnRH产生反应。相比之下， - 344至 - 300区域的多聚体不允许对GnRH产生反应，但增强了基础转录。这些发现与鉴定出一个双组分GnRH反应单元一致，这可能涉及两种不同转录因子的功能协作。GnRH反应性似乎与PMA反应性共定位的观察结果表明，GnRH对α亚基转录的影响可能由蛋白激酶C途径介导。
