Study of the deafness gene Grxcr1 and a paralog, Grxcr2

描述（由申请人提供）：小鼠pirouette （pi）位点的隐性突变导致内耳感觉毛细胞成熟缺陷导致感音神经性耳聋和前庭功能障碍。我们最近发现这些疾病的遗传基础是一个新基因Grxcr1的零突变。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，该基因是增加立体纤毛直径所必需的，可能通过调节立体纤毛核心的肌动蛋白丝分布。Grxcr1在内耳感觉细胞中表达，编码一个290个氨基酸的蛋白，包含一个与glutaredoxin蛋白非常相似的中心结构域和一个c端富含半胱氨酸的结构域。将Grxcr1构建体转染到培养细胞中，表明其定位于背/根尖表面富含肌动蛋白丝的结构。此外，在转染的成纤维细胞中，表达GRXCR1的细胞的背突肌动蛋白丝含量往往更突出，这表明GRXCR1在诱导和/或稳定这些结构的肌动蛋白细胞骨架中起着局部、直接的作用。Grxcr1相关基因广泛存在于后生动物物种中，包括一个相关的副同源基因（Grxcr1）也在小鼠内耳中选择性表达。利用分子遗传学和细胞生物学的方法，我们建议研究GRXCR1和GRXCR2在生化、细胞和生理方面的作用。我们将确定这些蛋白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鉴定这些蛋白活性所需的结构域，并检查这些活性的相互依赖性（目标2），鉴定可能直接与GRXCR1相互作用的其他蛋白（目标3），并产生Grxcr2的靶向突变，以表征内耳功能障碍的潜在新模型（目标4）。通过这些目标，我们将研究纤毛立体发育、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。

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.