RhBG and RhcG Proteins in Nonerythroid Tissues

描述(申请人提供)：我们以前已经证明了RhBG和RhCG是红细胞Rh抗原的新同系物。本项目围绕RhBG和RhCG的功能作用和作用模式展开，旨在阐明它们在哺乳动物非红系组织(肾、肝、皮肤、脑、睾丸和卵巢)中的底物特异性、差异表达和生理调节。这些研究是基于我们关于它们的主要序列特征、基因结构、组织特异性、上皮膜位置、蛋白质相互作用和从单细胞生物到人类的保守进化的研究结果。有了这些数据，我们现在可以研究Rh家族蛋白的功能，澄清它们是氨转运蛋白(AMT)、二氧化碳通道还是质膜重塑蛋白。我们假设：1)RhBG和RhCG在结构上与微生物Amts相连，但在功能上与原始生物的Rh蛋白同源。2)RhBG和RhCG可能不转运氨氮，但在介导CO2扩散和促进CO2通过功能结合蛋白转化为某些有机形式的通道型机制中起着相似的作用。3)RhBG和RhCG需要特定的蛋白质相互作用才能正常发挥作用，它们的差异表达是在基因转录和剪接水平上调节的。4)在与其伴侣的复合体中，RhBG和RhCG在特定的肾脏部位和靶器官执行相关但不同的生理作用。这些假说将以哺乳动物肾脏细胞和组织为主要模型进行验证。这四个目标是：1)确定RhBG和RhCG在不同肾段的分布，并确定它们是在顶膜还是在基底膜上呈现极化位置；2)通过解剖它们的启动子活性和剪接模式，确定RhBG和RhCG基因差异表达和调控的分子基础；3)鉴定与RhBG或RhCG基因物理相关的细胞成分，并通过细胞、生化或遗传学方法表征它们的相互作用；4)确定RhBG/RhCG及其相关蛋白的底物特异性，并阐明它们的作用模式，即Rh蛋白和伴侣(S)是平行还是协同发挥作用。这些结果有助于理解Rh家族的结构、功能和调控，揭示在人类和哺乳动物器官中发挥作用的一种潜在的重要的新的动态平衡机制。

DESCRIPTION (provided by applicant): We have shown previously that RhBG & RhCG are novel homologues of the red cell Rh antigens. This project centers on the functional role and mode of action of RhBG & RhCG, with the long-term goals to elucidate their substrate specificity, differential expression and physiological regulation in mammalian nonerythroid tissues (kidney, liver, skin, brain, testis and ovary). The proposed studies are based on our findings concerning their primary sequence features, gene structure, tissue specificity, epithelial membrane location, protein interaction and conserved evolution from unicellular organisms to man. With these data, we can now address the function of Rh family proteins, clarifying whether they are ammonia transporters (Amt), CO2 channels or plasma membrane-remodeling proteins. We postulate: 1) RhBG & RhCG are structurally connected to microbial Amts but functionally orthologous to the Rh proteins of primitive organisms. 2) RhBG & RhCG may not transport ammonium but act similarly in a channel-type mechanism mediating CO2 diffusion and facilitating CO2 conversion into certain organic forms via functionally associated proteins. 3) RhBG & RhCG require specific protein interactions to function properly, and their differential expression is regulated at the level of gene transcription and splicing. 4) In complex with their partners, RhBG & RhCG execute related but distinct physiologic roles in specific renal sites arid target organs. These hypotheses will be tested using mammalian kidney cells and tissues as a major model. The four specific aims are as follows: 1) resolve the distribution of RhBG & RhCG in the various renal segments, and establish whether they exhibit polarized location in the apical or basal membrane; 2) define the molecular basis for the differential expression and regulation of RhBG vs. RhCG gene by dissecting their promoter activity and splicing pattern; 3) identify the cellular components physically associated with RhBG or RhCG in renal tissues, and characterize their interactions by cell, biochemical or genetic approaches; 4) determine the substrate specificity of RhBG/RhCG and associated proteins, and elucidate the mode of their action as to whether the Rh proteins and the partner(s) function in parallel or cooperatively. The results obtained should contribute to understanding the structure, function and regulation of the Rh family, revealing a potentially important, new homeostatic mechanism operative in human and mammalian organs.