STRUCTURE-FUNCTION ANALYSIS OF PIT-1/PIT-1BETA ISOFORM

泌乳素乳营养腺瘤（催乳素瘤）是人类最常见的垂体肿瘤，可导致不孕、骨质疏松、视力丧失和垂体功能丧失。催乳素基因表达与乳营养特异性功能密切相关，受垂体特异性POU同源结构域转录因子Pit-1调控，该因子可转导cAMP/PKA和Ras信号通路。pit -1 - β异构体包含26个氨基酸的β -结构域，其序列指定其作为细胞类型特异性分子开关的作用。pit -1 β阻断GH4垂体细胞中基础和ras刺激的催乳素启动子活性，但增强HeLa非垂体细胞中基础和pka模拟的催乳素启动子活性。这一建议的目标是确定精确的分子机制(s)，其中Pit-1 β结构域决定了亚型特异性转录反应。为此，我计划在我们定义的Pit-1与Pit-1 β的不同生物反应的背景下，使用分子、生化和结构方法来剖析Pit-1 β结构域的机制结构-功能关系。我假设，通过β结构域的插入，Pit-1异构体的特异性反应是由特定的氨基酸带入Pit-1 β TAB的，并且β结构域产生的初级结构变化诱导了Pit-1/Pit-1 β的三维结构变化，这可能是β结构域插入的功能后果的原因。我来到加州大学圣克鲁兹分校加入Gutierrez-Hartmann博士的实验室，目的是将我在研究生时期学到的分子方法应用于分子内分泌学这一重要领域，以解决结构功能问题。我的工作导致了一个出版物，一篇评论，一个提交的手稿和一个国际上展示的关于这个令人兴奋的主题的摘要。这段经历帮助我确定了一个我特别感兴趣的研究领域，并有可能在未来独立发展。Gutierrez-Hartmann博士的实验室，我将在那里进行拟议的工作，积极参与分子内分泌学研究，并有杰出的生产力记录。这个奖项将允许我推进我在Pit-1/Pit-1beta系统上的工作，这样我就能获得晋升到一个独立的终身职位，并为我成功实现这一目标赢得必要的竞争性资金。

Prolatin-secreting lactotroph adenomas (prolactinomas), the most common human pituitary tumors, cause infertility, osteoporosis, loss of vision and loss of pituitary functions. Prolactin gene expression, tightly linked to lactotroph-specific function, is governed by Pit-1, a pituitary-specific POU- homeodomain transcription factor, that transduces cAMP/PKA and Ras signaling pathways. The Pit-1beta isoform contains the 26 amino-acid beta- domain, whose sequence specifies its role as a cell-type specific molecular switch. Pit-1beta blocks basal and Ras-stimulated prolactin promoter activity in GH4 pituitary cells, yet enhances basal and PKA-simulated prolactin promoter activity in HeLa non-pituitary cells. The goal of this proposal is to determine the precise molecular mechanism(s) by which the Pit-1 beta-domain dictates isoform-specific transcriptional responses. To this end, I plan to use molecular, biochemical and structural approaches to dissect the mechanistic structure-function relationships of the Pit-1 beta-domain, in the context of the distinct biological responses we have defined for Pit-1 versus Pit-1beta. I hypothesize that Pit-1 isoform-specific responses are dictated by specific amino acids brought to the Pit-1beta TAB by the beta domain insertion, and that the primary-structural changed created by the beta-domain induce three- dimensional structural changes in Pit-1/Pit-1beta, which may be responsible for the functional consequences of the beta-domain insertion. I came to UCHSC to join Dr. Gutierrez-Hartmann's laboratory in order to apply the molecular approaches to structure-function questions that I learned as a graduate student to the important field of molecular endocrinology. My work has lead to one publication, a review, a submitted manuscript and an internationally presented abstract on this exciting subject. This experience has helped to define an area of research of particular interest to me with potential for future independent development. Dr. Gutierrez-Hartmann's laboratory, where I will be carrying out the proposed work, is actively involved in molecular endocrinology research, and has an outstanding record of productivity. This award will allow me to advance my work on the Pit-1/Pit-1beta system so that I will be able to gain promotion to an independent tenure-tract position and to win the competitive funding necessary for me to successfully pursue that goal.