G Proteins and Signal Transduction in Neurospora crassa

超出提供的空间。三三聚体G蛋白在真核生物对环境刺激的许多反应中是必不可少的。粗毛脉孢霉基因组含有3个GCC亚基(GNA-1、GNA-2、GNA-3)、1个GI3(GnB-1)和1个GY NG-1亚基，并预测存在cAMP、信息素和葡萄糖敏感的G蛋白偶联受体。GNA-1是顶端生长、无性孢子形成和雌性生育所必需的。GNA-3是有性孢子形成的主要调节因子。GNA-2功能对GNA-1和GNA-3是冗余的。GNA-1正向调节GTP依赖的二酰环酶(CR-1)活性，而正常水平的CR-1蛋白则需要GNA-3。然而，AGNA-I和AGNA-3突变体的一些表型不能被外源cAMP拯救。这些和其他结果表明，有性繁殖在很大程度上是不依赖于cAMP的，而其他功能，如无性孢子形成，是通过cAMP依赖和独立的途径来调节的。GNB-1通过转录后机制调节GC量，但某些GNB-I表型不能用低G蛋白水平来解释。G_1在其他系统中调节丝裂原活化蛋白激酶(MAPK)途径。因此。我们推测G蛋白通过调节cAMP水平、MAPK途径和未知的效应物来调节粗毛裸藻营养和有性发育过程中的基因表达。具体目的是：1)突变6个粗面乳杆菌gpr基因，并对其表型和G_亚基偶联进行鉴定。F_表型分析将包括cAMP代谢缺陷。每个gpr的定位和表达模式将是：使用抗血清来确定。将利用GTPase缺失的GC(等位基因)和双杂交方法来确定受体与G_‘S之间的上位关系和结合。信息素、cAMP和其他分子将作为配体进行测试。2)确定G蛋白亚基之间的功能和物理关系。GNB-1对GC_2水平的转录后调控机制将通过脉冲追逐和体外翻译实验来确定。将使用免疫共沉淀和双杂交方法来检测GNB-1、GNG-1和三种G_蛋白之间的相互作用。上位性关系将利用G蛋白突变背景中缺乏GTP酶的G_等位基因来探索。3)研究G(_)和G_Y_‘亚基对已知或可疑靶标的调控。纯化的GNA-1将在&GNA-1制剂中进行AC活性重建测试。将分析cr-1和G_1基因之间的上位性，并将使用双杂交试验和免疫共沉淀来测试G蛋白和GR-I的相关性。将在GPCR和G蛋白亚单位突变体中测量MAPK活性。4)鉴定未知的G蛋白信号成分。未知成分将通过克隆AGNA-1、AGNA-3抑制子和cr-2、cr-3和cr-4基因以及通过转录图谱实验来鉴定。这些研究将阐明丝状真菌中的G蛋白信号通路，并对G蛋白的进化有深入的了解。此外，由于在许多真菌物种中毒力都需要粗毛奈瑟菌G基因的同源基因，这些研究也将为新出现的真菌病原体带来新的治疗方法。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. eterotrimeric (_) G proteins are essential for many responses to environmental stimuli in eukaryotes. The genome of e filamentous fungus Neurospora crassa contains three Gcc (GNA-1, GNA-2, GNA-3) one GI3 (GNB-1) and one GY NG-1) subunits, and also predicts the existence of cAMP, pheromone and glucose-sensing G protein coupled receptors PCR's). GNA-1 is required for apical growth, asexual sporulation and female fertility. GNA-3 is a major regulator of sexual sporulation. GNA-2 function is redundant to GNA-1 and GNA-3. GNA-1 positively-regulates GTP-dependent denylyl cyclase (CR-1) activity, while GNA-3 is required for normal levels of CR-1 protein. However, several phenotypes f Agna-I and Agna-3 mutants cannot be rescued by exogenous cAMP. These and other results indicate that sexual rtility is largely cAMP-independent, while other functions, such as asexual sporulation, are regulated using cAMP- ependent and independent pathways. GNB-1 modulates Gc_ amount via a post-transcriptional mechanism, but certain gnb-I phenotypes can not be explained by low G_ protein levels. G_ regulates Mitogen-Activated Protein Kinase ',MAPK) pathways in other systems. Therefore. we hypothesize that G proteins differentially regulate cAMP levels, MAPK 3athways and unknown effectors to modulate gene expression during vegetative and sexual development in N. crassa. the Specific Aims are: 1) Mutate six N. crassa GPCR genes and characterize phenotypes and G_ subunit coupling. F_henotypic analysis will include cAMP metabolism defects. Localization and expression patterns of each GPCR will be :letermined using antisera. GTPase-deficient Gc( alleles and the two-hybrid assay will be utilized to determine epistatic relationships and binding between receptors and G_'s. Pheromones, cAMP and other molecules will be tested as ligands. 2) Determine functional and physical relationships between G protein subunits. The mechanism of post- transcriptional regulation of Gc_ levels by GNB-1 will be determined using pulse-chase and in vitro translation experiments. Coimmunoprecipitation and two-hybrid assays will be used to test interactions between GNB-1, GNG-1, and the three G_ proteins. Epistatic relationships will be probed using GTPase-deficient G_ alleles in G protein mutant backgrounds. 3) Investigate regulation of known or suspected targets by G(_ and G_Y_'subunits. Purified GNA-1 will be tested for reconstitution of AC activity in &gna-1 preparations. Epistasis between cr-1 and G_ genes will be analyzed, and the two-hybrid assay and coimmunoprecipitation will be used to test for association of G proteins and GR-I. MAPK activity will be measured in GPCR and G protein subunit mutants. 4) Identify unknown G protein signaling components. Unknown components will be identified by cloning Agna-1 Agna-3 suppressors and the cr-2, cr-3 and cr-4 genes, and through transcriptional profiling experiments. These studies will elucidate G protein signaling pathways in filamentous fungi and yield insights into G protein evolution. Furthermore, since homologues of N. crassa G_ genes are required for virulence in numerous fungal species, these investigations will also lead to new therapies for emerging fungal pathogens. PERFORMANCE SITE ========================================Section End===========================================