Ligand Recognition Among Mammalian Odorant Receptors

描述（由申请人提供）：哺乳动物可以区分数千种气味，而这一过程的第一步是气味配体和气味受体（OR）之间的相互作用。哺乳动物嗅觉受体是一个庞大的g蛋白偶联受体（gpcr）家族。序列分析将这些受体分为两大类，每一类又进一步分为许多亚科。包括我们的初步工作在内的几项研究表明，虽然OR亚家族的成员识别相似的化学结构，但它们可以区分这些配体。本项目旨在了解逆转录酶如何在分子水平上识别和区分配体。我们利用爪蟾卵母细胞表达系统和机器人电生理技术开发了一种ORs的功能测定方法。在目标1中，我们将从广泛的亚家族中筛选具有代表性的OR，以确定新的配体- OR配对。在目标2中，我们将克隆和功能表达特定OR亚家族的其他成员。将进行详细的药理学分析，以揭示亚家族成员之间配体特异性的差异。在我们的第三和第四个具体目标中，我们将结合计算同源性建模和配体对接与位点定向诱变和功能分析，以确定哺乳动物气味受体亚家族成员之间配体特异性差异的结构基础。在目标3中，取代半胱氨酸可及性方法将为我们计算导出的OR模型提供严格的测试。在Aim 4中，基于亚家族成员的序列分析进行的常规诱变和功能分析将确定赋予配体特异性差异的残基。本项目将在分子水平上对哺乳动物ORs的配体识别提供理解。除了ORs识别配体的具体信息外，这项工作将适用于一般的gpcr。这对人类健康很重要，因为许多当前和潜在的药物靶点都是gpcr。我们的研究将为这类重要受体识别配体提供基础信息。这些信息将有助于今后合理设计药物。

DESCRIPTION (provided by applicant): Mammals can distinguish among thousands of odorants and the first step in this process is the interaction between odorant ligand and odorant receptor (OR). The mammalian odorant receptors constitute an enormous family of G-protein coupled receptors (GPCRs). Sequence analysis has grouped these receptors into two broad classes, with each class further divided into many subfamilies. Several studies, including our preliminary work, indicate that while the members of an OR subfamily recognize similar chemical structures, they can distinguish among these ligands. This project is directed toward understanding how ORs recognize and distinguish among ligands at the molecular level. We have developed a functional assay for ORs using the Xenopus oocyte expression system and robotic electrophysiology. In Aim 1, we will screen representative ORs from a broad range of subfamilies to identify new ligand - OR pairings. In Aim 2, we will then clone and functionally express other members of the particular OR subfamilies. Detailed pharmacological analysis will be conducted to reveal differences in ligand specificity among subfamily members. In our third and fourth specific aims, we will combine computational homology modeling and ligand docking with site-directed mutagenesis and functional analysis to identify the structural basis for differences in ligand specificity among members of mammalian odorant receptor subfamilies. In Aim 3, the Substituted Cysteine Accessibility Method will provide a rigorous test of our computationally derived OR models. In Aim 4, conventional mutagenesis and functional analysis, conducted based on sequence analysis of the subfamily members, will identify residues that confer differences in ligand specificity. This project will provide an understanding of ligand recognition by mammalian ORs at the molecular level. In addition to specific information about ligand recognition by ORs, this work will be applicable to GPCRs in general. This is important to human health because many current and potential drug targets are GPCRs. Our studies will provide fundamental information about ligand recognition by this important class of receptors. This information will be useful to future efforts in rational drug design. PUBLIC HEALTH RELEVANCE: Humans and other mammals detect odors using an enormous family of receptors (odorant receptors), which are similar in structure to many receptors that are current and future drug targets. This project examines the molecular basis for the ability of odorant receptors to distinguish among the many thousands of odor molecules. Information derived from this work will be useful in understanding the interaction between therapeutic drugs and their target receptors, and may aid in the design of more effective therapeutic drugs.