Functions of Very Large G-protein Coupled Receptor-1

描述（由申请人提供）：孤儿7跨膜片段受体，超大G蛋白偶联受体-1（VLGR 1，也称为Mass 1），是已知最大的细胞外蛋白。VLGR 1中自然发生的突变导致小鼠和人类的癫痫，以及人类的Usher综合征（感觉神经性耳聋和色素性视网膜炎）。该项目的总体目的是测试VLGR 1在视网膜和内耳中具有引起小鼠和人类Usher综合征的基本功能的假设。使用原位杂交和免疫组化，我们将测试的假设，VLGR 1表达在中枢神经系统，视网膜和内耳的正常小鼠一致的发病机制的Usher综合征。我们将通过比较天然存在的V2250 X突变和靶向G蛋白水解信号（GPS）和/-跨膜区段（7-TM）结构域同时保持胞外域完整的工程化突变（VLGR/del 7 TM）的表型效应来检验正常VLGR 1功能需要胞质和跨膜结构域的假设。使用眼底照相术，视网膜电图，光学和电子显微镜，我们将测试VLGR 1突变导致突变小鼠视觉系统异常的假设。我们将测试这一假设，即与VLGR 1蛋白的非功能性形式的表达相关的外周听觉缺陷反映了异常的毛细胞转导。功能研究将包括听觉脑干反应（ABR）、畸变产物耳声发射（DPOAE）和耳蜗内电位。将通过光学显微镜和电子显微镜评估骨形态。我们将测试VLGR 1与一种或多种对听力和视力至关重要的蛋白质形成功能网络的假设，特别是在其他形式的Usher综合征中受影响的蛋白质。为此，我们将用适当的VLGR 1表达构建体和编码其他候选蛋白的构建体共转染哺乳动物细胞。我们将确定哪些相互作用最有可能是生物学相关的，通过确定在小鼠脑，眼和耳使用原位杂交和/或免疫组织化学共表达。我们将通过小鼠大脑和/或视网膜的共免疫沉淀来证实这些体内相互作用。为了检验VLGR 1具有由其他相互作用蛋白，特别是其他Usher综合征基因产物部分补充的功能的假设，我们将在VLGR 1和携带最生物相关的相互作用蛋白突变的小鼠之间产生双突变株系。我们特别感兴趣的其他蛋白质与非常大的胞外结构域，原钙粘蛋白-15和钙粘蛋白-23。这些研究将为控制视网膜和内耳发育的机制提供新的线索。

DESCRIPTION (provided by applicant): The orphan 7-transmembrane segment receptor, Very Large G-protein coupled Receptor-1 (VLGR1, also termed Massl), is the largest known extracellular protein. Naturally occurring mutations in VLGR1 cause epilepsy in mice and humans, and Usher syndrome (sensorineural deafness and retinitis pigmentosa) in humans. The overall Aim of this project is to test the hypothesis that VLGR1 has essential functions in the retina and inner ear that cause Usher syndrome in mice as well as humans. Using in situ hybridization and immunohistochemistry, we will test the hypothesis that VLGR1 is expressed in the CNS, the retina and the inner ear of normal mice consistent with the pathogenesis of Usher syndrome. We will test the hypothesis that normal VLGR1 function requires the cytoplasmic and transmembrane domains, by comparing phenotypic effects of the naturally-occurring V2250X mutation and an engineered mutation that targets the G-protein proteolytic signal (GPS) and /-transmembrane segment (7-TM) domains while leaving the ectodomain intact (VLGR/del7TM). Using fundal photography, electroretinography, and light and electron microscopy, we will test the hypothesis that mutations of VLGR1 lead to visual system abnormalities in mutant mice. We will test the hypothesis that peripheral auditory deficits associated with the expression of non-functional forms of the VLGR1 protein reflect abnormal hair cell transduction. Functional studies will include auditory brainstem responses (ABR), distortion product otoacoustic emissions (DPOAEs) and endocochlear potentials. Cochlear morphology will be assessed by light and electron microscopy. We will test the hypothesis that VLGR1 forms a functional network with one or more proteins that are critical for hearing and vision, particularly proteins affected in other forms of Usher syndrome. To do so, we will co-transfect mammalian cells with appropriate VLGR1 expression constructs and constructs encoding other candidate proteins. We will determine which interactions are most likely to be biologically relevant by determining co-expression in mouse brain, eye and ear using in situ hybridization and/or immunohistochemistry. We will confirm these interactions in vivo by coimmunoprecipitation from mouse brains and/or retinas. To test the hypothesis that VLGR1 has functions that are partially complemented by other interacting proteins, particularly other Usher syndrome gene products, we will generate double mutant lines between VLGR1 and mice carrying mutations in the most biologically relevant interacting proteins. We are specifically interested in the other proteins with very large ectodomains, protocadherin- 15 and cadherin-23. These studies should shed new light on mechanisms controlling development of the retina and inner ear.