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Molecular analysis of GHRH receptor missense mutations

GHRH 受体错义突变的分子分析

基本信息

批准号：
6718364
负责人：
Roberto Salvatori
金额：
$ 8.18万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-04-01 至 2005-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6718364
关键词：
G protein biological signal transduction fluorescence resonance energy transfer gene deletion mutation growth hormone releasing hormone hormone receptor ligands pathologic process point mutation polymerase chain reaction protein protein interaction protein structure function receptor binding receptor expression tissue /cell culture

项目摘要

DESCRIPTION (provided by applicant): Isolated GH deficiency (IGHD) is familial in up to 30% of the cases. The most common form (type IB) has autosomal recessive transmission. Two mutations (generating truncated receptor) of the GHRH receptor (GHRHR) gene were first described as the basis for IGHD IB in two large kindreds. We have established the molecular heterogeneity of this disease through identification of ten additional mutations of the GHRHR, proving that defects in the GHRHR are common in familial IGHD lB. They consist of a promoter defect, one splice mutation, one nonsense mutation, one deletion, and six missense mutations that replace conservative amino acids: H137L, L144H, A176V, A222E, F242C, K329E. Preliminary characterization of the missense mutant receptors expressed in CHO cells shows that all these mutant receptors fail to produce a normal a cAMP response after exposure to GHRH, confirming that they are mutations and not innocent polymorphisms. Further characterization will reveal important information about receptor domains and specific amino acids that control receptor interaction with GHRH and with G proteins, and receptor desensitization. Specific aim 1: We have created cDNA's encoding for each of the mutant GHRHR's containing GFP or FLAG tag. We will express these fusion receptors in mammalian cells to determine the basis for their defective signaling. If proper cell surface expression is confirmed, binding studies will determine if the mutant receptors are able to bind their ligand. If the mutations cause abnormal cell surface expression, further experiments will determine the mechanism of such lack of expression. The wild type and mutant receptors will also allow us to study if and how specific amino acid substitutions interfere with receptor interaction with Gs alpha and other downstream proteins. Specific aim 2: We propose to study if the GHRHR exists as dimer using the novel BRET technique, if dimerization is ligand-dependent, and if it is prevented by any of the six the mutations. Altogether, these experiments will teach us new information about the pathophysiology of GHRHR signaling in health and disease.

描述（由申请人提供）：高达30%的病例中，孤立性生长激素缺乏症（IGHD）是家族性的。最常见的形式（IB型）具有常染色体隐性传播。GHRH受体（GHRHR）基因的两个突变（产生截短的受体）首次被描述为两个大激酶中IGHD IB的基础。我们已经通过鉴定GHRHR的另外10个突变建立了这种疾病的分子异质性，证明GHRHR的缺陷在家族性IGHD 1B中是常见的。它们由一个启动子缺陷、一个剪接突变、一个无义突变、一个缺失和六个替换保守氨基酸的错义突变组成：H137 L、L144 H、A176 V、A222 E、F242 C、K329 E。对CHO细胞中表达的错义突变受体的初步表征表明，所有这些突变受体在暴露于GHRH后均未能产生正常的cAMP反应，证实它们是突变而不是无辜的多态性。进一步的表征将揭示有关受体结构域和控制受体与GHRH和G蛋白相互作用的特定氨基酸以及受体脱敏的重要信息。具体目标1：我们已经创建了编码每个含有GFP或FLAG标签的突变体GHRHR的cDNA。我们将在哺乳动物细胞中表达这些融合受体，以确定其缺陷信号的基础。如果证实了适当的细胞表面表达，结合研究将确定突变受体是否能够结合其配体。如果突变导致异常的细胞表面表达，进一步的实验将确定这种缺乏表达的机制。野生型和突变型受体也将使我们能够研究特定的氨基酸取代是否以及如何干扰受体与Gs α和其他下游蛋白质的相互作用。具体目标2：我们建议研究GHRHR是否使用新的BRET技术作为二聚体存在，二聚化是否是配体依赖性的，以及是否被六个突变中的任何一个阻止。总之，这些实验将教会我们关于健康和疾病中GHRHR信号传导的病理生理学的新信息。