RUI: Production and Characterization of Membrane Steroid Receptors

类固醇激素在脊椎动物的生理和疾病过程中发挥着重要作用，如性行为、生殖和癌症。类固醇激素通常通过与经典的核受体相互作用来调节基因转录。类固醇还通过与一类未知的膜受体相互作用，在细胞表面启动转录无关的信号事件(非基因组)。由于缺乏膜类固醇受体的结构和序列信息，对这些非基因组类固醇作用的研究多年来一直受到阻碍。最近，一个新的膜类固醇受体家族被克隆并在鱼类和其他脊椎动物中鉴定，该家族与孕激素具有高亲和力，结构类似于G蛋白偶联受体。然而，在以前的研究中，由大肠杆菌产生的重组蛋白有几个缺点，例如它们不能结合其声称的天然配体，从而无法对受体进行功能和结构分析。本研究将利用昆虫和哺乳动物细胞系制备具有功能的斑马鱼膜受体蛋白，确定重组蛋白的类固醇结合特性，并研究这些蛋白在细胞表面信号转导通路中的作用。此外，本研究还将对已在斑马鱼中鉴定的膜类固醇受体亚型的功能进行表征和比较，从而为今后膜类固醇受体的研究和应用提供分子基础。这项拟议研究的长期目标是确定脊椎动物膜类固醇受体家族的功能。这项研究将在北卡罗来纳州农村地区的研究生和本科生的帮助下进行。这笔赠款将使这些学生能够在内分泌学和分子生物学领域接受最新知识和技术的培训。

Steroid hormones play important roles in vertebrate physiology and disease processes such as sexual behavior, reproduction, and cancer. Steroid hormones are generally known to modulate gene transcription through interactions with classical nuclear receptors. Steroids also initiate transcription-independent signaling events (nongenomic) at the cell surface through interactions with an unknown class of membrane receptors. Studies of these nongenomic steroid actions have been impaired for many years due to the lack of structure and sequence information for the membrane steroid receptors. Recently, a novel membrane steroid receptor family with high affinity to progestins and similar structure to G protein-coupled receptors has been cloned and characterized in fishes and other vertebrates. However, the recombinant proteins produced by the E. coli in previous studies have several disadvantages, such as their inability to bind its purported natural ligand and thus to allow functional and structural analyses of the receptors. The present research will produce functional zebrafish membrane receptor proteins using insect and mammalian cell lines, determine the steroid binding characteristics of the recombinant proteins, and investigate the involvement of the proteins in cell-surface signaling pathways. In addition, the proposed study will characterize and compare the function of the membrane steroid receptor subtypes that have been characterized in zebrafish, thus providing the molecular basis for future research and applications of the membrane steroid receptors. The long-term goal of the proposed study is to establish the functions of the membrane steroid receptor family in vertebrates. The study will be executed with the assistance from graduate and undergraduate students from rural areas of North Carolina. This grant will enable the training of these students in the latest knowledge and techniques in the fields of endocrinology and molecular biology.