Molecular analysis of GHRH receptor missense mutations

GHRH 受体错义突变的分子分析

• 批准号: 6612163
• 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
• 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/6612163
• 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

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% 的孤立性 GH 缺乏症 (IGHD) 具有家族性。最常见的形式（IB 型）为常染色体隐性遗传。 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家族中 IGHD IB 的基础。我们通过鉴定 GHRHR 的十个额外突变，确定了该疾病的分子异质性，证明 GHRHR 缺陷在家族性 IGHD IB 中很常见。它们由一个启动子缺陷、一个剪接突变、一个无义突变、一个缺失和六个取代保守氨基酸的错义突变组成：H137L、L144H、A176V、A222E、F242C、K329E。 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后均无法产生正常的a cAMP反应，证实它们是突变而不是无害的多态性。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：我们为每个包含 GFP 或 FLAG 标签的突变体 GHRHR 创建了 cDNA 编码。我们将在哺乳动物细胞中表达这些融合受体，以确定其信号传导缺陷的基础。如果确认细胞表面表达正确，结合研究将确定突变受体是否能够结合其配体。如果突变导致细胞表面表达异常，进一步的实验将确定这种表达缺失的机制。野生型和突变型受体还将使我们能够研究特定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：我们建议使用新型 BRET 技术研究 GHRHR 是否以二聚体形式存在，二聚化是否是配体依赖性的，以及是否被六种突变中的任何一种所阻止。总而言之，这些实验将告诉我们有关健康和疾病中 GHRHR 信号传导的病理生理学的新信息。