Chemosensory receptors and the basis of specificity

描述（由申请人提供）：哺乳动物使用几种化学感觉系统来检测和编码它们的化学环境。这些系统如何区分相关的化学线索是一个尚未解决的主要问题。我们假设不同群体的化学感觉细胞在刺激选择性上的差异在很大程度上反映了其中表达的化学感觉受体（CRs）的配体选择性和敏感性的差异。难以获得大量适合生化或结构分析的受体蛋白，以及已知配体的cr数量较少，阻碍了表征配体特异性基础的努力。其中一组CR，即T1R味觉受体，提供了独特的优势，将允许首次系统分析CR结构/功能关系如何影响化学感觉细胞群体检测和区分生理相关配体的能力。我们将利用已证实的T1Rs对甜味配体的敏感性，以及可用于生化纯化和结构表征的细胞外n端配体结合域，来确定不同T1Rs在甜味刺激检测中的作用。目的1：利用圆二色分光光度法和T1R n端结构域的x射线晶体学相结合的方法，研究在配体存在和不存在的情况下，T1R配体结合袋的结构。目的2：为了确定配体结合对味觉功能的具体贡献，将通过体外和小鼠基因靶向两种方法在T1R n端结构域的配体结合口袋中引入靶向突变。配体结合动力学的变化将使用等温滴定量热法来测量，而T1R缺失或突变对味觉功能的影响将通过短暂访问行为任务来分析，其中将确定目标小鼠对甜味刺激的敏感性。总之，这些研究将首次对化学感觉受体及其配体之间的相互作用进行深入的结构和定量分析，并将为个体味觉受体如何参与对健康和生存至关重要的食物线索的检测和区分提供重要的新见解。

DESCRIPTION (provided by applicant): Mammals use several chemosensory systems to detect and encode their chemical environment. How these systems discriminate relevant chemical cues is a major unresolved question. We hypothesize that differences in the stimulus selectivity of different populations of chemosensory cells largely reflects differences in the ligand selectivity and sensitivity of the chemosensory receptors (CRs) expressed therein. Difficulties in obtaining large amounts of receptor protein suitable for biochemical or structural analysis, as well as the small number of CRs for which ligands are known, has hampered efforts to characterize the basis of ligand specificity. One group of CRs, the T1R taste receptors, offers unique advantages that will permit the first systematic analysis of how CR structure/function relationships impact the ability of a chemosensory cell population to detect and discriminate physiologically relevant ligands. We will take advantage of the demonstrated sensitivity of T 1Rs for sweet-tasting ligands, and an extracellular N-terminal ligand-binding domain amenable to biochemical purification and structural characterization, to establish the role of different T1Rs in the detection of sweet tasting stimuli. Aim 1: The structure of the T1R ligand-binding pockets, in the presence and absence of ligands, will be solved by a combination of circular dichroism spectrophotometry and X-ray crystallography of T1R N-terminal domains. Aim 2: To determine the specific contributions of ligand binding to taste function, targeted mutations will be introduced in the ligand-binding pocket of T1R N-terminal domains both in vitro and by gene targeting in mice. Changes in ligand binding kinetics will be measured using isothermal titration calorimetry, while the effects of T1R deletion or mutation on taste function will be assayed by brief-access behavioral tasks where the sensitivity of targeted mice to sweet stimuli will be determined. Together, these studies will provide the first in-depth structural and quantitative analyses of the interactions between chemosensory receptors and their ligands, and will offer important new insights into how individual taste receptors contribute to the detection and discrimination of food cues critical for health and survival.