GUANINE NUCLEOTIDE BINDING PROTEIN BETA-GAMMA DIMERS--STRUCTURE AND FUNCTION

鸟嘌呤核苷酸结合蛋白 β-γ 二聚体——结构和功能

• 批准号: 3776960
• 负责人: W SIMONDS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3776960
• 关键词: G protein; antibody; biological signal transduction; gene expression; gene mutation; protein structure function; site directed mutagenesis; tissue /cell culture

The quanine-nucleotide binding regulatory proteins (G-proteins) are alpha1beta1gamma1 heterotrimers which function as transmembrane signal transducers by coupling receptors for extracellular stimuli to intracellular effectors (enzymes, ion channels). G-proteins constitute a diverse family distinguished by specific receptor and effector interactions which in turn are determined by the structure of the three constituent subunits. The alpha subunit binds quanine nucleotides and has a well established role in effector modulation. The beta and gamma subunits are tightly associated as a beta gamma complex, comprising a single functional entity which, like the alpha subunit, is absolutely required for G-protein interaction with receptor. An effector modulatory role for the beta gamma complex is becoming increasingly apparent in several systems. The present research emphasizes the role of the beta gamma complex in G-protein-mediated signal transduction. We have used subunit specific peptide antibodies to probe regions of the beta gamma complex important for functional interaction with the alpha subunit and to monitor expression of recombinant subunits. Site directed mutagenesis has been used to study the assembly, processing and effector function of the beta gamma complex in both transient and stable transfected cell systems. These studies may elucidate the contribution of the beta gamma subunit complex to the receptor and effector selectivity characteristic of G-proteins and to the adaptive responses pursuant to agonist stimulation. GRANTS=Z01DK59005 Nephrogenic diabetes insipidus (NDI) is an inherited X-lined disorder in which affected subjects are resistant ot the actions of vasopressin (AVP) on renal medullary cells responsible for water concentration. Clinical manifestations include severe polydipsia and polyuria, and resultant severe dehydration can lead to cerebral swelling and death. Treatment with a potent AVP analog (DDAVP, useful in other forms of DI, is in-effective in NDI because of end-organ resistance to tahe hormone. The renal actions of AVP are mediated through a V2 type receptor linked via the Gs protein to stimulation of the 2nd messenger CAMP. In theory, the inherited gene defect could be located anywhere along the signal transduction path, but indirect evidence suggested a likely receptor defect. The recent cloning of a human V2 receptor permitted chromosomal localization studies which showed that the receptor is localized to Xq28, the site of the gene defect as determined by family linkage studies. This strongly suggested but did not prove that a receptor gene mutation is the underlying defect in NDI. We have obtained genomic DNA samples on multiple families with NDI, and in three families thus far have identified mutations predicted to disrupt formation of a normal V2 receptor. These findings have important implications for our under- standing of the pathogenesis of NDI and of normal V2 receptor structure and function, for identification of affected subjects and carriers, and eventually for gene therapy of the disease.

奎宁-核苷酸结合调节蛋白(G-蛋白)是 作为跨膜信号的α1β1伽马1异三聚体 通过将细胞外刺激受体偶联到 细胞内效应物(酶、离子通道)。G蛋白构成 以特定受体和效应器区分的不同家族 相互作用又由三者的结构决定 组成亚单位。α亚基与奎宁核苷酸结合， 在效应器调节中起到了很好的作用。贝塔和伽马 亚基作为β-伽马复合体紧密相连，包括一个 单个功能实体，就像阿尔法亚单位一样，绝对是 G蛋白与受体相互作用所必需的。效应器 贝塔-伽马复合体的调节作用越来越大 在几个系统中都很明显。本研究强调了这一角色 G蛋白介导的信号转导中的β-伽马复合体。 我们已经使用亚单位特异性多肽抗体来探测 β-伽马复合体对与阿尔法的功能相互作用很重要 并监测重组亚基的表达。立地 定向诱变技术已被用于研究DNA的组装、加工和 瞬变和稳定状态下β-伽马复合体的效应函数 转基因细胞系统。这些研究可能会阐明这一贡献 受体和效应器的β-γ亚单位复合体 G蛋白的选择性特征及其对适应性反应的影响 根据激动剂刺激。 赠款=Z01DK59005 肾源性尿崩症(NDI)是一种遗传性X线疾病 受影响的对象对加压素的作用有抵抗力 (AVP)在肾髓质细胞上与水分浓度有关。 临床表现包括严重的多饮多尿，以及 由此产生的严重脱水会导致脑肿胀和死亡。 用有效的AVP类似物(DDAVP，在其他形式的DI中有用， 在NDI中无效，因为终末器官对塔赫激素耐药。 AVP的肾脏作用是通过与V2型受体相连的途径实现的 通过Gs蛋白刺激第二信使cAMP。从理论上讲， 遗传基因缺陷可能位于信号的任何位置 转导途径，但间接证据表明一个可能的受体 叛逃。人类V2受体允许染色体的最新克隆 定位研究表明受体定位于 Xq28，由家系连锁决定的基因缺陷位置 学习。这强烈地表明，但并不能证明受体基因 突变是NDI的潜在缺陷。我们已经获得了基因组DNA 多个患有NDI的家庭的样本，到目前为止有三个家庭 已经确定了预测会扰乱正常V2形成的突变 受体。这些发现对我们的儿童-- NDI发病机制及正常V2受体结构的认识 和功能，用于识别受影响的对象和携带者，以及 最终用于疾病的基因治疗。