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

催乳素腺瘤是最常见的垂体瘤，可导致不孕、骨质疏松、视力丧失和垂体功能丧失。催乳素基因表达与催乳素特异性功能紧密相关，受Pit-1（一种垂体特异性POU同源域转录因子）控制，Pit-1可转导cAMP/PKA和Ras信号通路。Pit-1 β同种型含有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刺激的催乳素启动子活性。该提议的目标是确定Pit-1 β结构域决定同种型特异性转录反应的精确分子机制。为此，我计划使用分子，生物化学和结构的方法来剖析的Pit-1 β结构域的机械结构功能的关系，在不同的生物反应，我们已经定义的Pit-1与Pit-1 β的背景下。我假设Pit-1亚型特异性反应是由β结构域插入Pit-1 β TAB所带来的特定氨基酸决定的，并且β结构域产生的一级结构变化诱导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UCHSC加入Gutierrez-Hartmann博士的实验室，以便将我作为研究生学习的结构-功能问题的分子方法应用于分子内分泌学的重要领域。我的工作已经导致了一个出版物，一个评论，一个提交的手稿和一个国际上提出的关于这个令人兴奋的主题的摘要。这段经历有助于确定一个我特别感兴趣的研究领域，并具有未来独立发展的潜力。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，并有出色的生产力记录。这个奖项将使我能够推进我在Pit-1/Pit-1beta系统上的工作，这样我就能够晋升到一个独立的终身职位，并赢得成功实现这一目标所需的竞争性资金。

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.