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

催乳素分泌的催乳素腺瘤(催乳素腺瘤)是人类最常见的脑下垂体肿瘤，会导致不孕症、骨质疏松、视力丧失和垂体腺功能丧失。催乳素基因的表达与催乳体特有的功能密切相关，受Pit-1调控，Pit-1是一种垂体特异的POU同源结构域转录因子，负责转导cAMP/PKA和RAS信号通路。Pit-1β亚型含有26个氨基酸的β结构域，其序列规定了其作为细胞型特定分子开关的作用。在GH4垂体细胞中，PIT-1β阻断基础催乳素启动子活性和RAS刺激的催乳素启动子活性，但在HeLa非垂体细胞中增强基础催乳素启动子和PKA模拟的催乳素启动子活性。这项建议的目标是确定Pit-1β结构域决定异构体特异性转录反应的精确分子机制(S)。为此，我计划使用分子、生化和结构方法，在我们为Pit-1和Pit-1β定义的不同生物反应的背景下，剖析Pit-1β结构域的机械结构-功能关系。我假设Pit-1异构体特异性反应是由β结构域插入到Pit-1βTAB上的特定氨基酸决定的，而由β结构域引起的一级结构变化引起Pit-1/Pit-1β的三维结构变化，这可能是导致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，是为了将我在研究生时学到的解决结构功能问题的分子方法应用到分子内分泌学的重要领域。我的工作导致了关于这一令人兴奋的主题的一份出版物、一篇评论、一份提交的手稿和一份在国际上发表的摘要。这一经历帮助我确定了一个我特别感兴趣的研究领域，具有未来独立发展的潜力。我将在古铁雷斯-哈特曼博士的实验室开展这项拟议中的工作，该实验室积极参与分子内分泌学研究，并拥有出色的生产力记录。这一奖项将使我能够推进我在Pit-1/Pit-1 Beta系统的工作，这样我将能够获得晋升到一个独立的终身教职职位，并赢得成功实现这一目标所需的有竞争力的资金。

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.