The Role of Phosphorylation in the Regulation of NHE3

描述（由申请人提供）： 该研究职业奖的主要研究者的目标是发展成为肾脏病理生理学的独立基础科学研究者。申请人是耶鲁大学医学院内科和儿科的儿科肾病学家。这两个部门都对青年科学家的发展作出了坚定的承诺。通过这些部门，申请人将有一个教学课程的重点计划，参加研究研讨会，并保护研究时间。这里详细介绍的研究项目旨在提供一个优秀的培训工具，以促进申请人向独立调查员的过渡。 该项目概述的具体目标为申请人提供了发展多种实验室技术专业知识的机会，完善实验设计和数据解释技能的能力，以及她的导师Peter Aronson博士的持续监督。拟议的项目集中在NHE 3，在近端小管的主要顶端膜Na/H交换的调节磷酸化的作用。该交换器负责肾脏重吸收大部分碳酸氢钠，因此在酸碱和容量稳态中起着至关重要的作用。 申请人已经成功地产生了针对NHE 3的C末端上的两个不同推定磷酸化位点丝氨酸552和605的磷酸化特异性抗体。她建议研究NHE 3磷酸化在这些网站在响应各种生理和药理演习。然后，这些抗体将用于确定磷酸化NHE 3在基线和响应于已知改变NHE 3活性的各种刺激的亚细胞定位。最后，将寻找与磷酸化或非磷酸化NHE 3特异性相互作用的相关NHE 3蛋白，并进一步表征关于NHE 3功能和分布的任何鉴定的相互作用。 本提案中的实验将加深我们对NHE 3磷酸化作为调节机制的理解，从而有助于理解以异常体积和酸碱平衡为特征的疾病，如高血压和骨质疏松症。

DESCRIPTION (provided by applicant): The objective for the Principal Investigator of this Research Career Award is to evolve into an independent basic science investigator in renal pathophysiology. The applicant is a pediatric nephrologist working in the Departments of Medicine and Pediatrics at the Yale University School of Medicine. Both departments have a strong commitment to the development of young scientists. Through these departments the applicant will have a focused program of didactic courses, participation in research seminars, and protected research time. The research project detailed here has been designed to provide an excellent training vehicle for furthering the applicant's transition to an independent investigator. The specific aims outlined for this project provide the applicant with the opportunity to develop expertise in multiple laboratory techniques, the ability to refine experimental design and data interpretation skills, and continued supervision by her mentor, Dr. Peter Aronson. The proposed project concentrates on the role of phosphorylation in the regulation of NHE3, a principal apical membrane Na/H exchanger in the proximal tubule. This exchanger is responsible for the majority of sodium bicarbonate reabsorption by the kidney and therefore plays a vital role in acid-base and volume homeostasis. The applicant has successfully generated phosphospecific antibodies to two different putative phosphorylation sites on the C-terminus of NHE3, serines 552 and 605. She proposes to study NHE3 phosphorylation at each of these sites in response to various physiologic and pharmacologic maneuvers. These antibodies will then be used to determine the subcellular localization of phosphorylated NHE3 at baseline and in response to various stimuli known to alter NHE3 activity. Finally, associated NHE3 proteins interacting specifically with either phosphorylated or non-phosphorylated NHE3 will be sought, and any identified interactions further characterized with regard to NHE3 function and distribution. The experiments in this proposal will deepen our understanding of NHE3 phosphorylation as a regulatory mechanism, and thereby contribute to understanding disorders characterized by abnormal volume and acid-base balance, such as hypertension and osteoporosis.