Functions of Very Large G-protein Coupled Receptor-1

超大G蛋白偶联受体1的功能

• 批准号: 7112251
• 负责人: PERRIN C WHITE
• 金额: $ 29.4万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7112251
• 关键词: Usher syndrome; brain stem; cadherins; cell surface receptors; central nervous system; electron microscopy; electroretinography; eye fundus photography; gene mutation; gene targeting; genetically modified animals; immunocytochemistry; immunoprecipitation; in situ hybridization; laboratory mouse; labyrinth; light microscopy; otoacoustic emission; phenotype; protein protein interaction; protein structure function; retina; transfection

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.

描述（由申请人提供）：孤儿7-跨膜段受体，非常大的G蛋白偶联受体1（VLGR1，也称为MASSL），是最大的已知细胞外蛋白。 VLGR1中天然发生的突变会导致小鼠和人类癫痫，以及人类的usher综合征（感觉性耳聋和色素性视网膜炎）。该项目的总体目的是检验以下假设：VLGR1在视网膜和内耳具有重要的功能，导致小鼠和人类的usher综合征。使用原位杂交和免疫组织化学，我们将检验以下假设：VLGR1在中枢神经系统，视网膜和正常小鼠的内耳中表达与usher综合征的发病机理一致。我们将通过比较自然存在的V2250X突变的表型效应以及针对G蛋白蛋白解释性信号（GPS）和 /-Transmbrane semtem sement（7-tm）dellly prot（7-tm）dormain the contoment the contomain，我们将测试正常VLGR1功能需要细胞质和跨膜结构域的假设。使用基本摄影，电视图以及光和电子显微镜，我们将测试以下假设：VLGR1的突变导致突变小鼠的视觉系统异常。我们将检验以下假设：与VLGR1蛋白非功能形式的表达相关的外围听觉缺陷反映了异常的毛细胞转导。功能研究将包括听觉的脑干反应（ABR），失真产物耳声发射（DPOAE）和内核势。耳蜗形态将通过光和电子显微镜评估。我们将检验以下假设：VLGR1形成一个功能网络，具有一种或多种对听力和视觉至关重要的蛋白质，尤其是在其他形式的Usher综合征中受影响的蛋白质。为此，我们将与适当的VLGR1表达构建构建并构造编码其他候选蛋白的构造。我们将使用原位杂交和/或免疫组织化学来确定小鼠脑，眼睛和耳朵中的共表达，从而确定哪些相互作用在生物学上相关。我们将通过小鼠大脑和/或视网膜的共免疫沉淀在体内证实这些相互作用。为了测试VLGR1具有其他相互作用蛋白（尤其是其他Usher综合征基因产物）部分补充的功能的假设，我们将在VLGR1和携带最相关相互作用蛋白质中携带突变的小鼠之间产生双突变型线。我们特别对具有非常大的外胞菌蛋白蛋白（原粘蛋白-15和钙粘蛋白-23）的其他蛋白质感兴趣。这些研究应阐明控制视网膜和内耳发展的机制。