G Proteins and Signal Transduction in Neurospora crassa

描述（由申请人提供）：异三聚体（α / β / γ） 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G- α （GNA-1、GNA-2、GNA-3）、1个G- β (GNB-1)和1个G- γ (GNG-1)亚基，并预测了cAMP、信息素和葡萄糖传感G蛋白偶联受体（GPCR’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是无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依赖性腺苷酸环化酶（CR-1）活性，而GNA-3是正常水平CR-1蛋白所必需的。然而，外源cAMP无法挽救delta-gna- 1和delta-gna-3突变体的几种表型。这些和其他结果表明，性生殖在很大程度上是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g - α的量，但某些δ - GNB-1表型不能用低g - α蛋白水平来解释。g - β / γ调节其他系统中的丝裂原活化蛋白激酶（MAPK）途径。因此，我们假设G蛋白通过差异调控cAMP水平、MAPK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 - α亚基偶联进行表征。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确定。gtpase缺陷g - α等位基因和双杂交试验将用于确定受体和g - α之间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g - α水平的机制将通过脉冲追踪和体外翻译实验来确定。共免疫沉淀法和双杂交法将用于测试GNB-1、GNG-1和三种g - α蛋白之间的相互作用。在G蛋白突变背景下，利用gtpase缺陷G- α等位基因来探索上位性关系。3)研究g - α和g - β / γ亚基对已知或可疑靶标的调节作用。纯化后的GNA-1将用于测试δ GNA-1制剂中AC活性的重建。将分析cr-1和G- α基因之间的上位性，并采用双杂交试验和共免疫沉淀法检测G蛋白与cr-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。未知成分将通过克隆δ -gna-1 δ -gna-3抑制因子和cr-2、cr-3和cr-4基因，并通过转录谱实验进行鉴定。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 - α基因的同源物，这些研究也将为新兴真菌病原体带来新的治疗方法。

DESCRIPTION (provided by applicant): Heterotrimeric (alpha/beta/gamma) G proteins are essential for many responses to environmental stimuli in eukaryotes. The genome of the filamentous fungus Neurospora crassa contains three G-alpha (GNA-1, GNA-2, GNA-3) one G-beta (GNB-1) and one G-gamma (GNG-1) subunits, and also predicts the existence of cAMP, pheromone and glucose-sensing G protein coupled receptors (GPCR's). GNA-1 is required for apical growth, asexual sporulation and female fertility. GNA-3 is a major regulator of asexual sporulation. GNA-2 function is redundant to GNA-1 and GNA-3. GNA-1 positively-regulates GTP-dependent adenylyl cyclase (CR-1) activity, while GNA-3 is required for normal levels of CR-1 protein. However, several phenotypes of delta-gna-I and delta-gna-3 mutants cannot be rescued by exogenous cAMP. These and other results indicate that sexual fertility is largely cAMP-independent, while other functions, such as asexual sporulation, are regulated using camp-dependent and independent pathways. GNB-1 modulates G-alpha amount via a post-transcriptional mechanism, but certain delta-gnb-I phenotypes can not be explained by low G-alpha protein levels. G-beta/gamma regulates Mitogen-Activated Protein Kinase (MAPK) pathways in other systems. Therefore, we hypothesize that G proteins differentially regulate cAMP levels, MAPK pathways 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-alpha subunit coupling. Phenotypic analysis will include cAMP metabolism defects. Localization and expression patterns of each GPCR will be determined using antisera. GTPase-deficient G-alpha alleles and the two-hybrid assay will be utilized to determine epistatic relationships and binding between receptors and G-alpha's. Pheromones, cAMP and other molecules will be tested as ligands. 2) Determine functional and physical relationships between G protein subunits. The mechanism of posttranscriptional regulation of G-alpha 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-alpha proteins. Epistatic relationships will be probed using GTPase-deficient G-alpha alleles in G protein mutant backgrounds. 3) Investigate regulation of known or suspected targets by G-alpha and G-beta/gamma subunits. Purified GNA-1 will be tested for reconstitution of AC activity in delta-gna-1 preparations. Epistasis between cr-1 and G-alpha genes will be analyzed, and the two-hybrid assay and coimmunoprecipitation will be used to test for association of G proteins and CR-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 delta-gna-1 delta-gna-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-alpha genes are required for virulence in numerous fungal species, these investigations will also lead to new therapies for emerging fungal pathogens.