Development and validation of novel optical methods for direct screening of taste receptor activation

直接筛选味觉受体激活的新型光学方法的开发和验证

• 批准号: 10593556
• 负责人: Robert J. Lee
• 金额: $ 24.38万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593556
• 关键词: Address; Adrenergic Receptor; Agonist; Amino Acids; Arrestins; Aspergillus flavus; Aspergillus fumigatus; Bacteria; Binding; Binding Proteins; Biological Assay; Biology; Calcium; Calcium Channel; Calcium Signaling; Cells; Clinical; Complex Mixtures; Couples; Cultured Cells; Cyclic AMP; Data; Development; Epithelial Cells; Epithelium; Exhibits; Family; Fluo 4; Fluorescence; Food; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic Transcription; Glutaminase; Glutamine; Heterotrimeric GTP-Binding Proteins; High Pressure Liquid Chromatography; Immune; Immunologic Receptors; Inhalation; Innate Immune Response; Ligands; Mediating; Methicillin; Methicillin Resistance; Methods; Molecular; Natural Immunity; Nutritional Study; Optical Methods; Optics; Orphan; Pathway interactions; Peptide Hydrolases; Phospholipase C; Phospholipases A; Phosphotransferases; Plant Extracts; Play; Potassium Channel; Protein Isoforms; Pseudomonas aeruginosa; Publications; Publishing; Quinine; Reader; Receptor Activation; Reporting; Research; Role; Sensory; Signal Pathway; Signal Transduction; Signaling Protein; Staphylococcus aureus; Taste Buds; Taste Perception; Testing; Tissues; Tongue; Validation; Vegetables; Visualization; Work; airway epithelium; arm; calcium indicator; clinically relevant; expression vector; fungus; high throughput screening; improved; insight; instrumentation; novel; nutrition; pathogen; pathogenic bacteria; pathogenic fungus; preservation; prevent; rat Gnat3 protein; receptor; respiratory smooth muscle; response; screening; sweet receptor; sweet taste perception; taste transduction; tool; voltage

PROJECT SUMMARY Taste family 1 receptors (T1Rs, forming sweet and umami receptors) and taste family 2 receptors (T2Rs, forming bitter receptors) are important G-protein couple receptors (GPCRs) expressed in taste buds of the tongue. T1Rs and T2Rs also play important chemosensory roles in tissues all over the body, including the airway epithelium and in immune cells. We and other have characterized T1Rs and T2Rs as novel players in innate immunity. They detect bacterial metabolites to stimulate rapid innate immune responses. While current screening methods have revealed a lot about specific agonists that activate specific T1Rs and T2Rs, understanding T1R and T2R activation by complex mixtures (e.g., plant extracts or conditioned media from bacteria) is currently very difficult. Existing screening methods are focused on calcium signaling as a read-out. Many compounds activate calcium signals independently of taste receptors in cultured cells, and the HEK293 cells commonly used express endogenous T2Rs. Calcium-focused screening methods have also likely missed “biased agonists,” that is, agonists which do not activate T2R G protein signaling but activate arrestin signaling. This has been reported for many GPCRs, but no one has yet screened for biased agonists of taste receptors. This is a critical gap in the studies of T1R and T2R biology that will be addressed here. To overcome limitations of calcium-based assays and reveal new insights into T2R-arrestin signaling, we will adapt a fluorescence-based tripartite GFP-based assay (known as the TRIO assay) that directly visualizes heterologously-expressed receptor activation through arrestin binding to the activated receptor. This assay is much less limited by off-target effects of complex mixtures or single compounds compared with current methods. It is also adaptable to high throughput plate-reader instrumentation. This assay is faster (1-2 hrs) than other GPCR assays which rely on transcription as a readout (12-24 hrs). We have utilized this assay to study activation and inhibition of protease-activated and adrenergic receptors in prior studies. Preliminary data suggest this assay works well with T2Rs. We hypothesize that T1R and T2R TRIO assays will reveal novel ligand-T1R/T2R interactions and may be useful to de-orphanize the remaining orphan T2R isoforms. In this proposal, Aim 1 will focus on validation of this optical assay using known bitter and sweet compounds. Aim 2 will use this assay to screen taste receptors against common pathogenic bacteria and fungi to characterize which receptors are activated by which pathogens. New clinically relevant data will be revealed, and the expression constructs validated in this study will become useful tools for taste receptor research. All expression vectors will be made widely available via Addgene after initial publication. This research has important utility for both immune research as well as molecular sensory nutrition and taste research.

项目总结