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Versatile and high-fidelity optical biosensor platforms for GPCR signaling

用于 GPCR 信号传导的多功能高保真光学生物传感器平台

基本信息

批准号：
10679863
负责人：
Mikel Garcia-Marcos
金额：
$ 35.48万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10679863
关键词：
Acceleration Achievement Area Asthma Astrocytes Biological Biological Assay Bioluminescence Biosensor Breeding Cardiovascular Diseases Cell Communication Cell Line Cell Surface Receptors Cell model Cells Clinical Cytoplasm Detection Disease Dissociation Drug usage Energy Transfer Event Experimental Models Family Future G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP Binding GTP-Binding Proteins Gene Delivery Goals Guanosine Triphosphate Heterotrimeric G Protein Subunit Heterotrimeric GTP-Binding Proteins Hormones Human Genome Hypersensitivity In Vitro Malignant Neoplasms Marketing Measurement Measures Mediating Membrane Membrane Proteins Methodology Molecular Mus Names Neurons Neurotransmitters Opioid Analgesics Optics Pain management Pathway interactions Pharmaceutical Preparations Pharmacology Physiological Physiological Processes Physiology Primary Cell Cultures Process Protein Family Proteins Reporting Reproducibility Research Research Personnel Resources Signal Transduction System Technology Transfection Transgenic Mice Translations Viral Packaging cell type design dimer drug discovery druggable target expectation extracellular improved in vivo interest neuropsychiatric disorder novel novel therapeutics optical sensor pharmacologic prototype receptor response sensor stoichiometry success technology development tool vector virtual

项目摘要

SIGNIFICANCE: G protein-coupled receptors (GPCRs) are a large family of membrane proteins that initiate cellular responses to a wide range of extracellular signals, like neurotransmitters or hormones, by activating heterotrimeric G proteins inside cells. This makes GPCRs critical for many physiological processes and their dysregulation is frequently associated with disease. GPCRs are not only the family of proteins most widely targeted by clinically approved drugs, with an estimated market value of hundreds of billions of dollars per year, but also the subject of many ongoing campaigns towards novel therapeutics. Moreover, many GPCRs still remain understudied and “undrugged”, which has great untapped potential for future drug discovery. Despite the biomedical importance of these receptors in physiology and pharmacology, the methodologies currently used to measure GPCR signaling activity in cells have limitations that hinder progress. One significant limitation is the use of approaches that compromise the fidelity of the readout because they are too indirect and/or distort the natural stoichiometry of signaling components. Another significant limitation is that many of the approaches are only feasible in cells in which gene delivery is easy, limiting their implementation to a few cell lines instead of more physiologically relevant systems like primary cell cultures. GOAL: Here, we propose to develop technologies that will allow to measure GPCR signaling activity directly and in diverse, physiologically-relevant cell systems without introducing major perturbations on the natural stoichiometry of signaling components. The tools and resources generated in this project will be made available to other investigators without restrictions. SYNOPSIS OF AIMS: We will develop two complementary assay platforms based on optical biosensors that directly detect the activity of heterotrimeric G proteins. In Aim1, we will establish a suite of broadly applicable “compact” vectors compatible with simple transfection or with viral packaging for the expression of G protein activity biosensors. In Aim 2, we will validate mouse transgenic lines for the conditional expression of biosensors of endogenous G protein activity. Collectively, these two platforms will allow the direct assessment of GPCR signaling activity under native expression conditions, in physiologically relevant cellular systems, and in formats that enable increased throughput to permit the parallel interrogation of the druggable GPCR-ome. IMPACT: The achievement of our goal could transform how the large field of research interested in GPCR- mediated cell communication approaches something as fundamental as measuring signaling activity. The technologies to be developed here would advance the field by: (i) enabling the direct characterization of GPCR signaling in systems in which it is currently not possible, (ii) revealing mechanisms of GPCR signaling with unprecedented fidelity in diverse contexts, and/or (iii) facilitating pharmacological or mechanistic interrogation in formats compatible with increased throughput.

意义：G蛋白偶联受体（gpcr）是一大家族的膜蛋白