G Proteins and Signal Transduction in Neurospora crassa

粗糙脉孢菌中的 G 蛋白和信号转导

• 批准号: 6827820
• 负责人: KATHERINE A BORKOVICH
• 金额: $ 26.95万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6827820
• 关键词: Proteins; Signal; Transduction; Neurospora; crassa; Proteins; Signal; Transduction; Neurospora; crassa

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

超过提供的空间。在真核生物中对许多对环境刺激的反应至关重要。 E丝真菌神经孢子的基因组包含三个GCC（GNA-1，GNA-2，GNA-3）一个GI3（GNB-1）和一个GY NG-1）亚基，并且还可以预测CAMP，ETHOMONE和GEL糖素和葡萄糖感应G蛋白耦合受体PCR的存在。 GNA-1是顶端生长，无性孢子形成和女性生育能力所必需的。 GNA-3是性孢子形成的主要调节剂。 GNA-2功能对GNA-1和GNA-3是冗余的。 GNA-1呈阳性调节GTP依赖性拒绝环酶（CR-1）活性，而对于正常水平的Cr-1蛋白需要GNA-3。但是，几种表型F AGNA-I和AGNA-3突变体不能通过外源营地挽救。这些结果和其他结果表明，性rt型在很大程度上是与营地无关的，而其他功能（例如无性孢子形成）是使用营地和独立途径来调节的。 GNB-1通过转录后机制调节GC_量，但是某些GNB-I表型无法用低的G_蛋白水平来解释。 G_调节其他系统中的有丝分裂原激活的蛋白激酶'，MAPK）途径。所以。我们假设G蛋白会差异地调节cAMP水平，MAPK 3径和未知效应子，以调节N. Crassa的营养和性发育期间的基因表达。具体目的是：1）突变六乳杆菌GPCR基因并表征表型和G_亚基耦合。 F_HENOTYPIC分析将包括营地代谢缺陷。每个GPCR的定位和表达模式将是：使用抗血清进行letermer。 GTPASE缺陷型GC（等位基因和两杂化测定法将用于确定受体和G_之间的上皮关系和结合。信息素，cAMP和其他分子将被测试为配体。2）确定G蛋白亚基之间的功能和物理关系。 GNB-1对GC_水平的转录后调节机制将使用脉冲练习和体外翻译实验确定。共免疫沉淀和两种杂交分析将用于测试GNB-1，GNG-1和三种G_蛋白之间的相互作用。在G蛋白突变体背景中使用GTPase缺陷型G_等位基因探测上皮关系。 3）调查通过G（_和g_y_'subunits。将测试纯化的GNA-1。将测试纯化的GNA-1在＆GNA-1制备中重构AC活性。将分析CR-1和G_基因之间的静脉症，并将使用两种杂交分析和coMmunopricition tockipition和GR-gropceck和GR-grop- gropck and grop grop grop grop groun和grp。蛋白质亚基突变体。将通过克隆AGNA-1 AGNA-3抑制剂以及CR-2，CR-3和CR-4基因以及通过转录分析实验来鉴定未知成分。这些研究将阐明丝状真菌中的G蛋白信号传导途径，并对G蛋白演化产生见解。此外，由于在许多真菌物种中需要毒力的N. crassa g_基因的同源物，因此这些研究还将导致新兴的真菌病原体的新疗法。 表演站点=============================================================================================