Cyclic GMP Phosphodiesterase in Signaling

信号转导中的环 GMP 磷酸二酯酶

基本信息

批准号：
7333975
负责人：
HSIEN-YU WANG
金额：
$ 10.7万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-01-01 至 2010-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7333975
关键词：
Adrenergic Agents Adrenergic Agonists Adrenergic Receptor Affinity Agonist Baculoviruses Binding Biological Assay Biology Bioluminescence C-terminal Calcium Cells Chimera organism Coupled Coupling Cyclic GMP Cyclic Nucleotides Cytoplasmic Tail Development Dipyridamole Elements Embryonal Carcinoma Energy Transfer Enzyme Inhibitor Drugs Enzyme Inhibitors FZD2 gene Family member G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Goals Guanosine Triphosphate Phosphohydrolases Heterotrimeric G Protein Subunit Heterotrimeric GTP-Binding Proteins High Pressure Liquid Chromatography Immunoblotting In Vitro Ligand Binding Domain Ligands Mass Spectrum Analysis Measures Mediating Molecular Mus Pathway interactions Pharmaceutical Preparations Phototransduction Play Proteomics RNA Splicing Rattus Regulation Resistance Role Signal Pathway Signal Transduction Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Stem cells System Technology Teratocarcinoma Testing Translating Triad Acrylic Resin adrenergic base human FZD2 protein human disease inhibitor/antagonist liquid chromatography mass spectrometry loss of function mature animal member novel phosphoric diester hydrolase protein protein interaction receptor reconstitution release of sequestered calcium ion into cytoplasm research study response zaprinast

项目摘要

Regulation of intracellular levels of cyclic nucleotides is a major paradigm in cell signaling and, in particular, signaling by G-protein-coupled receptors (GPCRs). Recenlty, 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 Go_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 132-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 _-adrenergic agonists and antagonists, signaling to calcium mobilization and reduction in intracellular concentrations of cyclic GMP, but not t0 G_.s and adenylylcyclase, like the wild-type 132-adrenergic receptor. We have validated the functional capability of the construct and propose two specific aims to elucidate biochemicaHy a new role for Go_t2/o and cyclic GMP PDE in signaling: to test for the direct interaction between Fz2 and heterotrimeric G-proteins expressed in Sf9 cells biochemically 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.

细胞内环核苷酸水平的调节是细胞信号传导的主要范例，特别是， G 蛋白偶联受体 (GPCR) 的信号传导。最近，我们已经证明了两名成员包括卷曲蛋白在内的 7 次跨膜分段受体家族 5 是 GPCR 多个下游信号通路。两项观察为本报告的具体目标提供了基础提案：抑制小鼠 F9 畸胎瘤干细胞中的异源三聚体 G 蛋白亚基 Go_t2/o 细胞和使用环 GMP PDE 抑制剂（例如 IBMX、扎普司特和双嘧达莫）治疗可阻断 GPCR 大鼠 Fz2 在钙瞬变和环 GMP 水平上发出信号的能力。我们创建了一个嵌合受体由跨膜、配体结合结构域和外表面片段组成众所周知的 GPCR 132-肾上腺素能受体，三个细胞内环（iloops 1-3）和 C- Rfz2 的末端、细胞质尾已被剪接。这种新颖的嵌合体结合了_-肾上腺素能激动剂和拮抗剂，向钙动员发出信号并降低细胞内环 GMP 浓度，但是不是 t0 G_.s 和腺苷酸环化酶，如野生型 132-肾上腺素能受体。我们已经验证了功能结构的能力并提出两个具体目标来阐明 Go_t2/o 的生化新作用信号传导中的环 GMP PDE：测试 Fz2 和异源三聚体 G 蛋白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和 BRET 在 Sf9 细胞中进行生物化学表达在F9细胞中；并且，通过以下方式确定负责此信号传导的 PDE 的分子身份：受体/G蛋白/PDE三联体的体外表达、纯化和重建以及互补使用反义抑制/拯救以及蛋白质-蛋白质的 BRET 分析对 F9 细胞进行研究互动。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（使用天然的配体和受体）通过使用嵌合体进行观察。这些研究与以下方面高度相关信号通路的改变以及阐明人类疾病的基础转化为异常的生物学。