Molecular analysis of GHRH receptor missense mutations

描述（由申请人提供）：孤立性生长激素缺乏症（IGHD）是家族性的，高达30%的病例。最常见的形式（IB型）具有常染色体隐性遗传。GHRH受体（GHRHR）基因的两个突变（产生截断受体）首次被描述为两大类群中IGHD IB的基础。通过鉴定10个额外的GHRHR突变，我们已经确定了这种疾病的分子异质性，证明GHRHR缺陷在家族性IGHD lB中是常见的。它们包括启动子缺陷、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：H137L、L144H、A176V、A222E、F242C、K329E。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后都不能产生正常的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受体结构域和特定氨基酸的重要信息，这些氨基酸控制着受体与GHRH和G蛋白的相互作用，以及受体脱敏。具体目标1：我们已经为每个含有GFP或FLAG标签的突变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，以确定其信号缺陷的基础。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：我们建议使用新的BRET技术来研究GHRHR是否以二聚体的形式存在，二聚体是否依赖于配体，以及它是否被六种突变中的任何一种阻止。总之，这些实验将告诉我们关于GHRHR信号在健康和疾病中的病理生理学的新信息。

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.