Study of the deafness gene Grxcr1 and a paralog, Grxcr2

描述（由申请人提供）：由于内耳中感觉毛细胞的成熟缺陷，小鼠旋转（pi）位点的隐性突变导致感觉神经性耳聋和前庭功能障碍。我们最近确定了这些病理的遗传基础为一种新基因Grxcr 1的无效突变。在pirouette感觉细胞的病理学表明，该基因是必要的增加直径的静纤毛在出生后早期成熟的感觉细胞，可能通过调节肌动蛋白丝分布在静纤毛核心。Grxcr 1在内耳感觉细胞中表达，编码一个290个氨基酸的蛋白质，含有一个与谷氧还蛋白具有显著相似性的中心结构域和一个C-末端富含半胱氨酸的结构域。Grxcr 1结构转染到培养的细胞中表明，它定位于肌动蛋白亲和丰富的结构在背侧/顶面。此外，在转染的成纤维细胞的背侧突起中的肌动蛋白丝含量通常在表达GRXCR 1的细胞上显得更突出，这表明该蛋白在这些结构的肌动蛋白细胞骨架的诱导和/或稳定中的局部、直接作用。Grxcr 1相关基因存在于多种后生动物物种中，包括一个相关的旁系同源基因（Grxcr 1）也选择性地在小鼠内耳中表达。使用分子遗传学和细胞生物学方法，我们建议调查的生化，细胞和生理作用的GRXCR 1和GRXCR 2。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），鉴定体外、培养细胞和内耳组织外植体中蛋白质活性所需的结构域，并检查这些活性的相互依赖性（目标2），鉴定可能与GRXCR 1直接相互作用的其他蛋白质（目标3），并产生Grxcr 2的靶向突变以表征内耳功能障碍的潜在新模型（目的4）。通过这些目标，我们将调查静纤毛的发展，肌动蛋白动力学和还原/氧化途径的影响过程之间的潜在联系，从而提供深入了解感觉细胞的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.