G Proteins and Signal Transduction in Neurospora crassa

描述（由申请人提供）：异源三聚体（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。丝状真菌粗糙脉孢菌（Neurospora crassa）的基因组包含三个G-α（GNA-1、GNA-2、GNA-3）、一个G-β（GNB-1）和一个G-γ（GNG-1）亚基，并且还预测cAMP、信息素和葡萄糖敏感G蛋白偶联受体（GPCR）的存在。GNA-1是顶端生长、无性孢子形成和雌性生育力所必需的。GNA-3是无性孢子形成的主要调节因子。GNA-2功能相对于GNA-1和GNA-3是冗余的。GNA-1正调节GTP依赖性腺苷酸环化酶（CR-1）活性，而GNA-3是CR-1蛋白正常水平所必需的。然而，delta-gna-I和delta-gna-3突变体的几种表型不能被外源性cAMP拯救。这些和其他结果表明，性生育能力在很大程度上是cAMP独立的，而其他功能，如无性孢子形成，使用cAMP依赖性和独立的途径进行调节。GNB-1通过转录后机制调节G-α的量，但某些delta-gnb-I表型不能用低G-α蛋白水平来解释。G-β/γ调节其他系统中的促分裂原活化蛋白激酶（MAPK）途径。因此，我们假设G蛋白差异调节cAMP水平，MAPK途径和未知的效应器，以调节营养和性发育过程中的基因表达。粗鲁。具体目的是：1）突变6个N。crassa GPCR基因，并表征表型和G-α亚基偶联。 表型分析将包括cAMP代谢缺陷。将使用抗血清确定每个GPCR的定位和表达模式。GTP酶缺陷型G-α等位基因和双杂交试验将用于确定上位关系和受体与G-α之间的结合。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G-α水平的转录后调节机制将通过脉冲追踪和体外翻译实验来确定。将使用免疫共沉淀和双杂交测定来测试GNB-1、GNG-1和三种G-α蛋白之间的相互作用。将使用G蛋白突变体背景中GTP酶缺陷型G-α等位基因探索上位性关系。3)研究G-α和G-β/γ亚基对已知或疑似靶点的调节作用。将检测纯化的GNA-1在delta-gna-1制剂中的AC活性重建。将分析cr-1和G-α基因之间的上位性，并使用双杂交试验和免疫共沉淀法检测G蛋白和CR-1的相关性。将在GPCR和G蛋白亚基突变体中测量MAPK活性。4)识别未知的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，并通过转录谱实验来鉴定。这些研究将阐明丝状真菌中G蛋白的信号通路，并深入了解G蛋白的进化。此外，由于N. crassa G-α基因是许多真菌物种中毒力所必需的，这些研究也将导致新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.