Functions of Very Large G-protein Coupled Receptor-1

描述(申请人提供)：孤儿7-跨膜片段受体，非常大的G蛋白偶联受体-1(VLGR1，也称为MASL)，是已知的最大的胞外蛋白。自然发生的VLGR1突变会导致小鼠和人类的癫痫，以及人类的亚瑟综合征(感觉神经性耳聋和视网膜色素变性)。该项目的总体目标是测试VLGR1在视网膜和内耳具有基本功能的假设，这些功能在小鼠和人类中都会导致亚瑟综合征。利用原位杂交和免疫组织化学方法，我们将验证VLGR1在正常小鼠的中枢神经系统、视网膜和内耳表达与Usher综合征的发病机制相一致的假说。我们将通过比较自然发生的V2250X突变和针对G蛋白蛋白分解信号(GPS)和/-跨膜片段(7-TM)结构域的工程突变的表型效应，验证正常的VLGR1功能需要细胞质和跨膜结构域(VLGR/del7TM)的假设，同时保持胞外结构域的完整(VLGR/del7TM)。使用眼底照相、视网膜电描记术以及光学和电子显微镜，我们将检验VLGR1突变导致突变小鼠视觉系统异常的假设。我们将测试与非功能形式的VLGR1蛋白表达相关的外周听觉缺陷反映毛细胞转导异常的假设。功能研究将包括听性脑干反应(ABR)、失真产物耳声发射(DPOAEs)和耳蜗内电位。将通过光镜和电子显微镜评估耳蜗骨的形态。我们将测试这一假设，即VLGR1与一个或多个对听力和视觉至关重要的蛋白质形成一个功能网络，特别是在其他形式的亚瑟综合征中影响的蛋白质。为此，我们将用适当的VLGR1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。我们将通过原位杂交和/或免疫组织化学来确定哪些相互作用最有可能是生物相关的，方法是利用原位杂交和/或免疫组织化学来确定小鼠大脑、眼睛和耳朵中的共同表达。我们将通过小鼠大脑和/或视网膜的免疫共沉淀在体内证实这些相互作用。为了验证VLGR1具有其他相互作用蛋白，特别是其他亚瑟综合征基因产物部分互补的功能的假设，我们将在VLGR1和携带最具生物相关性的相互作用蛋白突变的小鼠之间产生双突变系。我们特别感兴趣的是其他具有非常大的胞外结构域的蛋白质，Protocadherin-15和cadherin-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.