RhBG and RhcG Proteins in Nonerythroid Tissues

非红细胞组织中的 RhBG 和 RhcG 蛋白

• 批准号: 6914838
• 负责人: CHEN-HAN HUANG
• 金额: $ 25.32万
• 依托单位: NEW YORK BLOOD CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-20 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6914838
• 关键词: Chlamydomonas; DNA footprinting; DNA methylation; Dictyostelium; MDCK cell; ammonia; blood group antigens; blood proteins; carbon dioxide transport; clinical research; gel mobility shift assay; immunoelectron microscopy; kidney; membrane channels; membrane proteins; polymerase chain reaction; protein structure function; yeast two hybrid system

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.

描述（由申请人提供）：我们之前已经证明RhBG 和RhCG 是红细胞Rh 抗原的新型同源物。该项目以RhBG和RhCG的功能作用和作用方式为中心，长期目标是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。拟议的研究基于我们对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发现。有了这些数据，我们现在可以解决 Rh 家族蛋白的功能，阐明它们是否是氨转运蛋白 (Amt)、CO2 通道或质膜重塑蛋白。我们假设：1) RhBG 和 RhCG 在结构上与微生物 Amt 相关，但在功能上与原始生物体的 Rh 蛋白直系同源。 2) RhBG 和 RhCG 可能不运输铵，但在介导 CO2 扩散的通道型机制中发挥类似作用，并通过功能相关蛋白促进 CO2 转化为某些有机形式。 3）RhBG和RhCG需要特定的蛋白质相互作用才能正常发挥作用，并且它们的差异表达在基因转录和剪接水平上受到调节。 4) 与它们的伙伴一起，RhBG 和 RhCG 在特定的肾脏部位和靶器官中发挥相关但不同的生理作用。这些假设将使用哺乳动物肾细胞和组织作为主要模型进行测试。四个具体目标如下：1）解析RhBG和RhCG在肾各段的分布，并确定它们是否在顶膜或基底膜中表现出极化位置； 2) 通过剖析RhBG与RhCG基因的启动子活性和剪接模式，确定RhBG与RhCG基因差异表达和调控的分子基础； 3) 鉴定肾组织中与RhBG或RhCG物理相关的细胞成分，并通过细胞、生化或遗传方法表征它们的相互作用； 4) 确定RhBG/RhCG和相关蛋白的底物特异性，并阐明它们的作用模式，即Rh蛋白和伴侣是否并行或协同发挥作用。获得的结果应有助于理解 Rh 家族的结构、功能和调节，揭示在人类和哺乳动物器官中发挥作用的潜在重要的新稳态机制。