G Proteins and Signal Transduction in Neurospora crassa

描述(申请人提供)：异三聚体(α/β/伽马)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。粗丝状脉孢霉基因组含有3个G-α(GNA-1、GNA-2、GNA-3)、1个G-β(GNB-1)和1个G-伽马(GNG-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(GPCRs)。GNA-1是顶端生长、无性孢子形成和雌性生育所必需的。GNA-3是无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正向调节依赖GTP的腺苷环化酶(CR-1)活性，而GNA-3是正常水平的CR-1蛋白所必需的。然而，外源cAMP不能挽救Delta-gna-I和Delta-gna-3突变体的几个表型。这些和其他结果表明，有性生殖在很大程度上是不依赖于cAMP的，而其他功能，如无性产孢子，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G-α的数量，但某些Delta-GNB-I表型不能用低G-α蛋白水平解释。G-β/γ在其他系统中调节丝裂原活化蛋白激酶(MAPK)通路。因此，我们推测粗毛裸藻营养发育和有性发育过程中，G蛋白通过差异调节cAMP水平、MAPK通路和未知的效应因子来调节基因表达。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-α亚基偶联进行研究。表型分析将包括cAMP代谢缺陷。每个gpr的定位和表达模式将使用抗血清来确定。GTPase缺失的G-α等位基因和双杂交试验将被用来确定受体和G-α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转录后调节G-α水平的机制将通过脉冲追逐和体外翻译实验来确定。免疫共沉淀和双杂交试验将用于测试GNB-1、GNG-1和三种G-α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GTPase的G-α等位基因进行探索。3)研究G-α和G-β/γ亚基对已知或可疑靶点的调节作用。纯化的GNA-1将在Delta-GNA-1制剂中用于AC活性的重建测试。将分析CR-1和G-α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CR-I的关联性。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Delta-GNA-1、Delta-GNA-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糙奈瑟氏菌G-α基因的同源基因，这些研究也将导致对新出现的真菌病原体的新疗法。

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.