Functions of Very Large G-protein Coupled Receptor-1

描述（由申请人提供）：孤儿7跨膜段受体，甚大g蛋白偶联受体-1 (VLGR1，也称为Massl)，是已知最大的细胞外蛋白。自然发生的VLGR1突变导致小鼠和人类癫痫，以及人类Usher综合征（感音神经性耳聋和视网膜色素变性）。该项目的总体目标是验证这样一个假设，即VLGR1在视网膜和内耳中具有导致小鼠和人类Usher综合征的基本功能。我们将采用原位杂交和免疫组织化学方法，验证VLGR1在正常小鼠中枢神经系统、视网膜和内耳中表达的假设，与Usher综合征的发病机制相一致。我们将通过比较自然发生的V2250X突变和针对g蛋白蛋白水解信号（GPS）和/-跨膜段（7-TM）结构域而保持外结构域完整（VLGR/del7TM）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的假设。通过基础摄影、视网膜电图、光学和电子显微镜，我们将验证VLGR1突变导致突变小鼠视觉系统异常的假设。我们将验证与VLGR1蛋白非功能形式表达相关的外周听觉缺陷反映异常毛细胞转导的假设。功能研究将包括听觉脑干反应（ABR）、畸变产物耳声发射（dpoae）和耳蜗电位。耳蜗形态将通过光镜和电子显微镜进行评估。我们将测试这一假设，即VLGR1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，特别是在其他形式的Usher综合征中受到影响的蛋白质。为此，我们将用合适的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/或免疫组织化学测定小鼠脑、眼和耳中的共表达来确定哪些相互作用最有可能具有生物学相关性。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。为了验证VLGR1的功能部分被其他相互作用蛋白（特别是其他Usher综合征基因产物）补充的假设，我们将在VLGR1和携带最具生物学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外结构域的蛋白质，原钙粘蛋白- 15和钙粘蛋白-23。这些研究将为视网膜和内耳发育的控制机制提供新的线索。

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.