G Proteins and Signal Transduction in Neurospora crassa

超出所提供的空间。异三聚体G蛋白是真核生物对环境刺激的许多反应所必需的。丝状真菌粗神经孢子菌的基因组包含3个Gcc亚基（GNA-1、GNA-2、GNA-3）、1个GI3亚基（GNB-1）和1个GY NG-1，并预测cAMP、信息素和葡萄糖敏感G蛋白偶联受体（PCR’s）的存在。GNA-1是根尖生长、无性产孢和雌性生殖所必需的。GNA-3是有性孢子形成的主要调节因子。GNA-2功能与GNA-1和GNA-3功能是冗余的。GNA-1正调控gtp依赖性denylyl cyclase （CR-1）的活性，而GNA-3是正常水平的CR-1蛋白所必需的。然而，agna - 1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，性功能在很大程度上是cAMP独立的，而其他功能，如无性产孢，是通过cAMP依赖和独立的途径调节的。GNB-1通过转录后机制调节Gc_的数量，但GNB-1的某些表型不能用低G_蛋白水平来解释。G_在其他系统中调节丝裂原活化蛋白激酶（MAPK）通路。因此。我们假设G蛋白通过差异调控cAMP水平、MAPK 3通路和未知的效应物来调节油菜营养发育和性发育过程中的基因表达。具体目的是：1)突变6个藜草GPCR基因，并对表型和G_亚基偶联进行表征。f_表型分析将包括cAMP代谢缺陷。每个GPCR的定位和表达模式将使用抗血清来确定。gtpase缺陷Gc等位基因和双杂交试验将用于确定受体与G_的上位性关系和结合。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转录后调控Gc_水平的机制将通过脉冲追踪和体外翻译实验来确定。采用共免疫沉淀法和双杂交法检测GNB-1、GNG-1和3种G_蛋白之间的相互作用。在G蛋白突变背景下，利用gtpase缺陷G_等位基因来探索上位性关系。3)研究G(_和G_Y_’亚基对已知或疑似靶标的调控作用。纯化后的GNA-1将用于测试&gna-1制剂中AC活性的重建。分析cr-1和G_基因间的上位性，采用双杂交法和共免疫沉淀法检测G蛋白与gr -1的相关性。MAPK的活性将在GPCR和G蛋白亚基突变体中进行检测。4)鉴定未知的G蛋白信号成分。通过克隆Agna-1、Agna-3抑制因子和cr-2、cr-3、cr-4基因，通过转录谱实验鉴定未知组分。这些研究将阐明丝状真菌中G蛋白的信号通路，并对G蛋白的进化产生深入的了解。此外，由于许多真菌物种的毒力都需要N. crassa G_基因的同源物，这些研究也将为新出现的真菌病原体带来新的治疗方法。网站性能 ======================================== 节结束 ===========================================

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===========================================