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: 7467899
• 负责人: PERRIN C WHITE
• 金额: $ 28.69万
• 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/7467899
• 关键词: Affect; Anatomy; Auditory; Auditory Brainstem Responses; Autosomal Dominant Partial Epilepsy with Auditory Features; Body Size; Brain; Brain Stem; Breeding; Chickens; Co-Immunoprecipitations; Complement; Development; Ear; Electron Microscopy; Electroretinography; Engineering; Epilepsy; Extracellular Protein; Eye; Fertility; G-Protein-Coupled Receptors; GTP-Binding Proteins; Growth; Hair Cells; Hearing; Histologic; Human; Immune Sera; Immunization; Immunohistochemistry; In Situ Hybridization; Knock-in Mouse; Labyrinth; Lead; Left; Light; Longevity; Mammalian Cell; Morphology; Mus; Mutant Strains Mice; Mutate; Mutation; Nonsense Mutation; Orphan; Pathogenesis; Peripheral; Phenotype; Photography; Predisposition; Proteins; Rate; Research Personnel; Retina; Retinal; Retinitis Pigmentosa; Scanning Transmission Electron Microscopy Procedures; Sensorineural Hearing Loss; Signal Transduction; Techniques; Testing; Transmembrane Domain; Usher Syndrome; Vision; Visual system structure; audiogenic seizure; cellular transduction; deafness; human CDH23 protein; in vivo; interest; light microscopy; mutant; otoacoustic emission; programs; receptor; response

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次跨膜片段受体，超大G蛋白偶联受体-1（VLGR1，也称为Mass1），是已知的最大的细胞外蛋白。 VLGR1 自然发生的突变会导致小鼠和人类癫痫，以及人类 Usher 综合征（感音神经性耳聋和色素性视网膜炎）。该项目的总体目标是检验以下假设：VLGR1 在视网膜和内耳中具有导致小鼠和人类亚瑟综合症的重要功能。利用原位杂交和免疫组织化学，我们将检验VLGR1在正常小鼠的中枢神经系统、视网膜和内耳中表达的假设，这与Usher综合征的发病机制一致。我们将通过比较自然发生的 V2250X 突变和针对 G 蛋白水解信号 (GPS) 和 /-跨膜片段 (7- TM) 结构域，同时保持胞外域完整 (VLGR/del7TM)。使用眼底摄影、视网膜电图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。我们将检验以下假设：与 VLGR1 蛋白非功能形式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）和耳蜗电位。将通过光学和电子显微镜评估耳蜗形态。我们将验证以下假设：VLGR1 与一种或多种对听力和视力至关重要的蛋白质（特别是受其他形式的亚瑟综合症影响的蛋白质）形成功能网络。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。我们将通过使用原位杂交和/或免疫组织化学确定小鼠大脑、眼睛和耳朵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/或视网膜的免疫共沉淀来确认这些体内相互作用。为了检验 VLGR1 具有由其他相互作用蛋白（特别是其他亚瑟综合症基因产物）部分补充的功能的假设，我们将在 VLGR1 和在生物学上最相关的相互作用蛋白中携带突变的小鼠之间产生双突变系。我们对具有非常大的胞外域的其他蛋白质特别感兴趣，即原钙粘蛋白-15 和钙粘蛋白-23。这些研究应该为控制视网膜和内耳发育的机制提供新的线索。

项目成果

A novel Usher protein network at the periciliary reloading point between molecular transport machineries in vertebrate photoreceptor cells.

脊椎动物感光细胞分子运输机器之间纤毛周围重新加载点的新型亚瑟蛋白网络。

• 发表时间: 2008-01-01
• 影响因子: 3.5
• 作者: Maerker, Tina;van Wijk, Erwin;Overlack, Nora;Kersten, Ferry F J;McGee, Joann;Goldmann, Tobias;Sehn, Elisabeth;Roepman, Ronald;Walsh, Edward J;Kremer, Hannie;Wolfrum, Uwe
• 通讯作者: Wolfrum, Uwe