Cyclic GMP Phosphodiesterase in Signaling

信号转导中的环 GMP 磷酸二酯酶

• 批准号: 6986067
• 负责人: HSIEN-YU WANG
• 金额: $ 28.96万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6986067
• 关键词: G protein; biological signal transduction; cell differentiation; cell proliferation; cell surface receptors; cyclic GMP; fluorescence resonance energy transfer; green fluorescent proteins; ligands; pertussis toxin; phosphodiesterases; protein protein interaction

DESCRIPTION (provided by applicant): Regulation of intracellular levels of cyclic nucleotides is a major paradigm in cell signaling and, in particular, signaling by G-protein-coupled receptors (GPCRs). Recently, we have shown that two members of the family 5 of 7-transmembrane segmented receptors that includes Frizzleds are GPCRs with respect to several downstream signaling pathways. Two observations provide the basis for the specific aims in this proposal: suppression of the heterotrimeric G-protein subunits G(alpha)t2/o in mouse F9 teratocarcinoma stem cells and treatment with inhibitors of cyclic GMP PDE, such as IBMX, zaprinast, and dipyridamole, block the ability of the GPCR rat Fz2 to signal at the level of calcium transients and cyclic GMP. We have created a chimeric receptor composed of the transmembrane, ligand-binding domain and the exofacial segments of the well-known GPCR (beta)2-adrenergic receptor to which the three intracellular loops (iloops 1-3) and the C-terminal, cytoplasmic tail of Rfz2 have been spliced. This novel chimera binds beta-adrenergic agonists and antagonists, signaling to calcium mobilization and reduction in intracellular concentrations of cyclic GMP, but not to G(alpha)s and adenylylcyclase, like the wild-type (beta)2-adrenergic receptor. We have validated the functional capability of the construct and propose two specific aims to elucidate in a biochemical manner a new role for G(alpha)t2/o and cyclic GMP PDE in signaling: to test for the direct interaction between Fz2 and heterotrimeric G-proteins expressed in Sf9 cells biochemicaly by baculovirus-induced expression of these components and by BRET in F9 cells; and, to establish the molecular identity of the PDE(s) responsible for this signaling by expression, purification, and reconstitution of the triad of receptor/G-protein/PDE in vitro and complementary studies in F9 cells using antisense suppression/rescue as well as BRET analysis of protein-protein interactions. We shall employ a novel drug-induced secretion system in these cells to validate (with native ligand and receptor) the observations exploited by use of the chimera. These studies are highly relevant to signaling as well as to elucidation of basis for human diseases in which alterations in signaling pathways translate into aberrant biology.

描述（由申请人提供）： 细胞内环核苷酸水平的调节是细胞信号转导，特别是 G 蛋白偶联受体 (GPCR) 信号转导的主要范例。最近，我们发现，包括卷曲蛋白在内的 7 次跨膜分段受体家族 5 的两个成员是与多个下游信号通路相关的 GPCR。两项观察为该提案中的具体目标提供了基础：抑制小鼠 F9 畸胎癌干细胞中的异三聚体 G 蛋白亚基 G(α)t2/o 以及使用环 GMP PDE 抑制剂（例如 IBMX、扎普司特和双嘧达莫，阻断 GPCR 大鼠 Fz2 在钙瞬变和环 GMP 水平上发出信号的能力。我们创建了一种嵌合受体，由众所周知的 GPCR (β)2-肾上腺素能受体的跨膜、配体结合结构域和外表面片段组成，三个细胞内环 (iloops 1-3) 和 C 末端， Rfz2 的细胞质尾已被剪接。这种新型嵌合体结合β-肾上腺素能激动剂和拮抗剂，向钙动员和细胞内环鸟苷酸浓度降低发出信号，但不与野生型(β)2-肾上腺素能受体一样与G(α)和腺苷酸环化酶结合。我们验证了该构建体的功能能力，并提出了两个具体目标，以生化方式阐明 G(α)t2/o 和环 GMP PDE 在信号传导中的新作用：测试 Fz2 和异源三聚体 G- 之间的直接相互作用通过杆状病毒诱导的这些成分的表达以及通过F9细胞中的BRET在Sf9细胞中生化表达的蛋白质；并且，通过受体/G蛋白/PDE三联体的体外表达、纯化和重建以及使用反义抑制/救援在F9细胞中的补充研究来确定负责该信号传导的PDE的分子身份作为蛋白质-蛋白质相互作用的 BRET 分析。我们将在这些细胞中采用一种新型药物诱导的分泌系统来验证（使用天然配体和受体）使用嵌合体所利用的观察结果。这些研究与信号传导以及阐明人类疾病的基础高度相关，其中信号传导途径的改变转化为异常的生物学。