Molecular, biochemical and physiological studies on bitter taste receptors

G蛋白偶联受体（GPCR）是最大和最多样化的一组膜蛋白。GPCR共享保守的七螺旋基序，并且响应于包括光、肽、激素和促味剂的多种细胞外刺激而激活同源G蛋白。味觉是负责基本食物评价的化学感觉系统，并且主要由GPCR介导。人类能够感知五种基本的味道，即甜、鲜、苦、酸和盐。甜味、鲜味和苦味的信号转导是通过GPCR进行的，而酸味和咸味则是通过离子通道进行的。在人类中，已知约有30种GPCR介导味觉感知，其中25种仅感知苦味。虽然甜味和鲜味味觉感知得到了很好的研究，但关于苦味感知知之甚少。到目前为止，大多数苦味受体（T2 R）的促味剂和阻断剂（拮抗剂或反向激动剂）的身份是未知的。实际上，即使对于具有已知配体的T2 R，也很少有结构-功能数据可用，并且这些受体的分子作用机制尚未被理解。我们的方法将是使用分子，生物化学和生理学技术的组合来解开T2 R的分布和作用机制的细节。申请人的研究计划可以分为两大领域，第一个涉及A类GPCR（前列腺素受体和β 2-肾上腺素能受体），第二个关注非A类和营养重要的GPCR，即人类味觉受体。该NSERC项目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T2 R比其他T2 R具有广泛的底物特异性？T2 R的分子和/或结构特征是什么，有助于这种广泛的特异性？此外，我们将试图阐明T2受体在口腔外组织中表达的生理作用。短期目标（未来5年）是：1）继续进行T2 R的结构-功能研究，表征其脱敏机制并鉴定翻译后修饰。2)发现可以降低这些受体活性的新型配体（苦味阻断剂）。3)表征口腔外组织（包括脑组织、脉管系统和气道）中表达的T2受体的生理作用。建议研究的意义味觉通常用于描述口腔产生的感觉。苦味已经演变为一种针对摄入潜在有毒物质的中央警告信号。苦味感知中的分子事件始于特定水溶性分子（促味剂或配体）与T2 R的结合，从而引发细胞内信号级联。本研究计划的重点是阐明T2 Rs信号转导的分子机制。关于T2 R如何与其配体相互作用的知识将使设计增强食品风味的人工促味剂成为可能。苦味阻断剂可用于抑制不愉快，从而增加促进健康的苦味食物如水果和蔬菜提取物的适口性。此外，表征口腔外组织中表达的T2 R的生理作用将刺激对GPCR结构和功能的额外基础科学研究。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学技术的专业知识。

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.