Molecular, biochemical and physiological studies on bitter taste receptors

G蛋白偶联受体(GPCRs)是膜蛋白中最大、最多样的一类。GPCRs共有一个保守的七螺旋基序，并激活同源G蛋白，以响应不同的细胞外刺激，包括光、肽、激素和味觉。味觉是一种化学感觉系统，负责基本的食物评价，主要由GPCRs介导。人类能够辨别出五种基本的味道，即甜、鲜味、苦、酸和盐。甜味、鲜味和苦味的信号转导是通过GPCRs进行的，而酸味和盐味的信号转导是通过离子通道进行的。在人类中，大约有30个GPCR调节味觉，其中25个GPCR一项就感觉到了苦味。虽然人们对甜味和鲜味的知觉进行了很好的研究，但对苦味的知觉却知之甚少。到目前为止，大多数苦味受体(T2Rs)的味觉受体和阻滞剂(拮抗剂或反向激动剂)的身份尚不清楚，甚至对于已知配体的T2Rs，结构功能数据也很少，这些受体的分子作用机制尚不清楚。我们的方法将是使用分子、生化和生理技术的组合来揭开T2Rs的分布和作用机制的细节。 目标 申请者的研究计划可以分为两个大的领域，第一个领域涉及A类GPCRs(前列腺素受体和β2-肾上腺素能受体)，第二个领域专注于非A类和具有营养重要性的GPCRs，即人类的味觉受体。该NSERC计划的长期目标是阐明脊椎动物苦味信号转导的分子机制。我们将解决以下问题，为什么某些T2R比其他T2R具有广泛的底物特异性？导致这种广泛特异性的T2R的分子和/或结构特征是什么？此外，我们还将试图阐明在口腔外组织中表达的T2Rs的生理作用。 短期目标(未来5年)是： 1)开展T2Rs的结构-功能研究，研究其脱敏机制，并确定翻译后修饰。 2)寻找能够降低这些受体活性的新型配体(苦味阻滞剂)。 3)研究T2Rs在口腔外组织中表达的生理作用，包括脑组织、血管系统和呼吸道。 拟议研究的意义 味觉经常被用来描述口腔产生的感觉。苦味已经演变为防止摄入潜在有毒物质的中央警告信号。感受苦味的分子事件始于特定的水溶性分子(味精或配体)与T2Rs的结合，启动了细胞内的信号级联反应。本研究的重点是阐明T2Rs信号转导的分子机制。了解T2Rs如何与其配体相互作用，将有助于设计出增强食品风味的人工味料。苦味阻滞剂可以用来抑制不愉快，从而增加促进健康的苦味食物的可口性，如水果和蔬菜提取物。此外，对在口腔外组织中表达的T2Rs的生理作用的表征将促进对GPCRs结构和功能的进一步基础科学研究。这项拟议的研究将培养研究生，他们将获得广泛的现代分子和细胞生物技术方面的专业知识。

G protein-coupled receptors (GPCRs) constitute the largest and most diverse group of membrane proteins. GPCRs share a conserved seven-helix motif and activate cognate G-proteins in response to diverse extracellular stimuli including light, peptides, hormones and tastants. The sense of taste is a chemosensory system responsible for basic food appraisal and is mediated mostly by GPCRs. Humans are capable of detecting five basic tastes that are sweet, umami, bitter, sour and salt. While the signal transduction for sweet, umami and bitter tastes are through GPCRs, the sour and salt tastes are sensed by ion channels. In humans, around 30 GPCRs are known to mediate taste perception, with 25 of these sensing bitter taste alone. While sweet and umami taste perceptions are well studied, very little is known regarding the bitter taste perception. Thus far, the identities of the tastants and blockers (antagonists or inverse agonists) for a majority of the bitter taste receptors (T2Rs) are unknown, Indeed even for the T2Rs with known ligands, very little structure-function data is available and the molecular mechanisms of action of these receptors are not yet understood. Our approach will be to use a combination of molecular, biochemical and physiological techniques to unravel details of the distribution and mechanism of action of T2Rs. OBJECTIVES The applicant’s research program can be classified into two broad areas, the first dealing with the Class A GPCRs (Prostanoid receptors and Beta2-Adrenergic receptor) and the second focussed on non-Class A and nutritionally important GPCRs, the human taste receptors. The long-term objective of this NSERC program is to elucidate molecular mechanisms of vertebrate bitter taste signal transduction. We will address the following questions, why do certain T2Rs have broad substrate specificities than others? What are the molecular and/or structural features in the T2Rs that contribute to this broad specificity? In addition, we will attempt to elucidate the physiological roles of T2Rs expressed in extra-oral tissues. The short-term goals (next 5 years) are: 1) To pursue structure-function studies on T2Rs, characterize their desensitization mechanisms and identify post-translational modifications. 2) To discover novel ligands (bitter taste blockers) that can reduce the activity of these receptors. 3) To characterize the physiological role of T2Rs expressed in extra-oral tissues including the brain tissues, vasculature and airways. SIGNIFICANCE OF THE PROPOSED RESEARCH Taste is often used to describe sensations arising from the oral cavity. Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules (tastants or ligands) to T2Rs, initiating an intracellular signalling cascade. This research proposal focuses on elucidating the molecular mechanisms of signal transduction by T2Rs. Knowledge about how T2Rs interact with their ligand will enable design of artificial tastants that enhance food flavor. Bitter taste blockers can be used to suppress unpleasantness and thereby increase palatability of health-promoting bitter foods, such as fruit and vegetable extracts. Furthermore, characterization of the physiological roles of T2Rs expressed in extra-oral tissues will spur additional basic science research on GPCR structure and function. The proposed research would train graduate students, who will gain expertise in a broad range of modern molecular and cell biological techniques.