Mechanistic Investigations of Pheromone and Phytohormone G-Protein Coupled Receptors

g蛋白偶联受体（gpcr）是细胞表面受体的一个超级家族，在整个分类群中有1000多个代表性基因。它们构成了当今制药工业中绝大多数的药物靶标。Ste2p和Ste3p是相反的交配型gpcr，在酵母的交配中负责信息素的信号传导。在gpcr结构-功能关系的基础研究中，step2尤其成为了一个卓越的模型。在此之前，我们开发了这些受体的高产重组表达系统，并在体外鉴定了Ste2p的同质寡聚化。我们进一步证明了Ste2p在细胞质融合中的新作用，包括介导细胞壁降解和可能的膜融合。我们将这些功能与受体的n端结构域和跨膜6的残基联系起来。我们现在建议阐明与这些等离子体事件相关的Ste2p作用机制。与此同时，我们正在启动一个长期的机制研究最近表征的植物GPCR。拟南芥GTG-1 （GPCR型g蛋白1）是与农业和环境相关的植物激素脱落酸（ABA）的受体，也是迄今为止唯一已知的植物GPCR配体。我们开发了一个重组过表达/纯化系统，并证明了ABA与重组GTG-1的结合。我们将继续开发工具来研究这种GPCR，包括基于酵母的生物测定，以进行筛选和诱变结构-功能分析。从更长远的角度来看，我们有兴趣尝试结晶这些受体，基于过去5年6种哺乳动物GPCR结构的研究进展。总的来说，这项工作将增加我们对gpcr结构和功能的了解，同时为未来的制药和农业生物技术发展带来新的前景。

G-protein coupled receptors (GPCRs) form a super-family of cell surface receptors with 1000's of representative genes identified across taxa. They comprise the vast majority of current drug targets in pharmaceutical industry today. Ste2p and Ste3p are the opposite mating type GPCRs responsible for pheromone based signalling in the mating of yeast. Ste2p in particular has served as a model par excellence in basic studies of structure-function relationships in GPCRs. Previously we developed a high-yield recombinant expression system for these receptors and characterized homo-oligomerization of Ste2p in vitro. We further demonstrated novel putative roles for Ste2p in plasmogamy (cytoplasmic fusion), including mediating cell wall degradation and possibly membrane fusion. We linked these functionalities to residues of the N-terminal domain and transmembrane 6 of the receptor. We now propose to elucidate the mechanisms of Ste2p action associated with these plasmogamy events. At the same time, we are initiating a long-term mechanistic investigation of a recently characterized plant GPCR. Arabipdopsis thaliana GTG-1 (GPCR Type G-Protein 1) is a receptor for the agriculturally and environmentally pertinent plant hormone abscisic acid (ABA) and is to date the only plant GPCR of known ligand. We have developed a recombinant overexpression/purification system and demonstrated binding of ABA to this reconstituted recombinant GTG-1. We will continue to develop tools to study this GPCR, including a yeast based bio-assay to enable screening and carry out mutagenic structure-function analyses. In the much longer term we are interested in attempting to crystallize these receptors, based on advances that have led to the publication of 6 mammalian GPCR structures in the last 5 years. Overall, this work will increase our knowledge of the structure and function of GPCRs while yielding novel prospects for pharmaceutical and agbiotech development in the future.