Mechanistic Investigations of Pheromone and Phytohormone G-Protein Coupled Receptors

G蛋白偶联受体（GPCR）形成细胞表面受体的超家族，其具有跨分类群鉴定的1000个代表性基因。它们构成了当今制药工业中绝大多数的当前药物靶标。Step2 p和Step 3 p是相反的交配型GPCR，负责酵母交配中基于信息素的信号传导。特别是在GPCR的结构-功能关系的基础研究中，Step2 p已经成为一个卓越的模型。以前，我们开发了一个高产量的重组表达系统，这些受体和特点的同源寡聚化的STE 2 P在体外。我们进一步证明了新的推定的作用，为STE 2 P在胞浆融合（胞质融合），包括介导细胞壁降解和可能的膜融合。 我们将这些功能性连接到受体的N-末端结构域和跨膜6的残基。现在，我们建议阐明与这些plasmogamy事件相关的Step2 p行动的机制。 与此同时，我们正在启动一个长期的机制调查，最近的特点植物气相色谱还原反应。 拟南芥GTG-1（GPCR Type G-Protein 1）是农业和环境相关植物激素脱落酸（阿坝）的受体，并且是迄今为止已知配体的唯一植物GPCR。我们已经开发了一种重组过表达/纯化系统，并证明了阿坝与重组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。 从长远来看，我们有兴趣尝试结晶这些受体，基于在过去5年中发表的6种哺乳动物GPCR结构的进展。总的来说，这项工作将增加我们对GPCR的结构和功能的了解，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.