Cyclic GMP Phosphodiesterase in Signaling

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

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.