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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.

环核苷酸的细胞内水平的调节是细胞信号传导中的主要范例，特别是， G蛋白偶联受体（GPCR）。最近，我们已经表明，两个成员的家族5的7-跨膜分段受体，包括卷曲蛋白，是关于几个下游信号通路。两个观察结果为本报告的具体目标提供了依据。建议：在小鼠F9畸胎瘤干细胞中抑制异源三聚体G蛋白亚基Go_t2/o 用环GMP PDE的抑制剂如IBMX、扎匹司特和双嘧达莫处理， GPCR大鼠Fz 2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，其三个细胞内环（loops 1-3）和C- Rfz 2末端胞质尾区已被剪接。这种新的嵌合体结合_肾上腺素能激动剂，拮抗剂，钙动员的信号传导和环GMP细胞内浓度的降低，但与野生型132-肾上腺素能受体一样，对G_s和腺苷酸环化酶无影响。我们已经验证了功能构建的能力，并提出了两个具体的目标，以阐明生物化学的新作用Go_t2/o 和环GMP PDE在信号传导中的作用：测试Fz 2和异源三聚体G蛋白之间的直接相互作用在Sf 9细胞中通过杆状病毒诱导的这些组分的表达和BRET的生物化学表达在F9细胞中;以及，通过以下方式确定负责该信号传导的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，以及互补的使用反义抑制/拯救以及蛋白质-蛋白质的BRET分析在F9细胞中的研究交互.我们将在这些细胞中采用一种新的药物诱导分泌系统来验证（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：以及阐明人类疾病的基础，其中信号通路的改变转化为异常的生物学