Study of the deafness gene Grxcr1 and a paralog, Grxcr2

耳聋基因 Grxcr1 和旁系同源基因 Grxcr2 的研究

• 批准号: 7015581
• 负责人: David C Kohrman
• 金额: $ 30.61万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7015581
• 关键词: actins; autosomal recessive trait; cell growth regulation; cell line; cellular pathology; deafness; disease /disorder model; ear hair cell; gene expression; gene mutation; genetic disorder; genetic mapping; genetically modified animals; immunocytochemistry; laboratory mouse; labyrinth disorder; microfilaments; oxidation reduction reaction; protein localization; protein protein interaction; protein structure function; sensorineural hearing loss; tissue /cell culture; transfection; transmission electron microscopy

DESCRIPTION (provided by applicant): Recessive mutations at the mouse pirouette (pi) locus cause sensorineural deafness and vestibular dysfunction due to defective maturation of sensory hair cells in the inner ear. We have recently identified the genetic basis of these pathologies as null mutations in a novel gene, Grxcr1. Pathology in pirouette sensory cells suggests that this gene is required for increasing the diameter of stereocilia during early postnatal maturation of sensory cells, potentially through regulation of actin filament distribution in the stereocilia core. Grxcr1 is expressed in sensory cells of the inner ear and encodes a 290 amino acid protein containing a central domain with significant similarity to glutaredoxin proteins, and a C-terminal cysteine-rich domain. Transfection of Grxcr1 constructs into cultured cells indicates that it localizes to actin filament-rich structures at the dorsal/apical surface. In addition, the actin filament content in dorsal projections in transfected fibroblasts often appears more prominent on cells expressing GRXCR1, suggesting a local, direct role for the protein in the induction and/or stabilization of the actin cytoskeleton of these structures. Grxcr1-related genes are present in a wide range of metazoan species, including a related paralogous gene (Grxcr1) also expressed selectively in the mouse inner ear. Using molecular genetic and cell biological approaches, we propose to investigate the biochemical, cellular, and physiological roles of GRXCR1 and GRXCR2. We will determine the sub cellular localization of these proteins in inner ear tissues (Aim 1), identify domains required for activities of the proteins in vitro, in cultured cells and inner ear tissue explants, and examine the interdependence of these activities (Aim 2), identify additional proteins that may interact directly with GRXCR1 (Aim 3), and generate a targeted mutation of Grxcr2 to characterize a potential novel model of inner ear dysfunction (Aim 4). Through these aims, we will investigate potential links between stereocilia development, actin dynamics, and processes influenced by reduction/oxidation pathways, and thereby provide insight into the molecular control of sensory cell development and function.

描述（由申请人提供）：小鼠旋转（pi）基因座的隐性突变会由于内耳感觉毛细胞的成熟缺陷而导致感音神经性耳聋和前庭功能障碍。我们最近发现这些病理的遗传基础是一个新基因 Grxcr1 的无效突变。旋转感觉细胞的病理学表明，在感觉细胞出生后早期成熟过程中，该基因是增加静纤毛直径所必需的，可能是通过调节静纤毛核心中肌动蛋白丝的分布。 Grxcr1 在内耳感觉细胞中表达，编码 290 个氨基酸的蛋白质，其中包含与谷氧还蛋白蛋白显着相似的中心结构域和 C 末端富含半胱氨酸的结构域。将 Grxcr1 构建体转染到培养细胞中表明它定位于背侧/顶端表面富含肌动蛋白丝的结构。此外，转染的成纤维细胞背侧投射中的肌动蛋白丝含量通常在表达 GRXCR1 的细胞上显得更为突出，表明该蛋白质在这些结构的肌动蛋白细胞骨架的诱导和/或稳定中具有局部、直接的作用。 Grxcr1 相关基因存在于多种后生动物物种中，包括也在小鼠内耳中选择性表达的相关旁系同源基因 (Grxcr1)。我们建议使用分子遗传学和细胞生物学方法来研究 GRXCR1 和 GRXCR2 的生化、细胞和生理作用。我们将确定这些蛋白质在内耳组织中的亚细胞定位（目标 1），在体外、培养细胞和内耳组织外植体中识别蛋白质活性所需的域，并检查这些活性的相互依赖性（目标 2），识别可能与 GRXCR1 直接相互作用的其他蛋白质（目标 3），并生成 Grxcr2 的靶向突变以表征潜在的内耳功能障碍新模型（目标 4）。通过这些目标，我们将研究静纤毛发育、肌动蛋白动力学和受还原/氧化途径影响的过程之间的潜在联系，从而深入了解感觉细胞发育和功能的分子控制。