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Direct chemogenetic control of heterotrimeric G protein signaling

异源三聚体 G 蛋白信号传导的直接化学遗传学控制

基本信息

批准号：
10590217
负责人：
Mikel Garcia-Marcos
金额：
$ 45.38万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10590217
关键词：
Adenylate Cyclase Adopted Affinity Agreement Antiviral Agents Binding Biological Assay Biological Process Bioluminescence Biosensor Cell Line Cell Surface Receptors Cell physiology Cells Chemicals Clinical Communication Communities Cyclic AMP Disease Drug Targeting Energy Transfer Engineering Etiology Event FDA approved Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP Binding GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Genetic Goals Guanosine Triphosphate Hepatitis C Heterotrimeric G Protein Subunit Heterotrimeric GTP-Binding Proteins Hormones Ion Channel Left Length Ligands Location Mammalian Cell Measures Mediating Methods Mus Names Neurons Neurosciences Neurotransmitter Receptor Neurotransmitters Optics Outcome Output Pathologic Peptides Performance Pharmaceutical Preparations Pharmacology Phospholipase C Photons Physiological Physiological Processes Proteins Receptor Activation Regulation Research Research Personnel Role Second Messenger Systems Signal Transduction Signaling Protein Specificity Stimulus Technology Therapeutic Transducers base design experimental study human disease improved nervous system disorder neuropsychiatric disorder neuroregulation neurotransmission new technology novel performance tests protein activation receptor receptor-mediated signaling response subcellular targeting tool

项目摘要

ABSTRACT SIGNIFICANCE: G protein-coupled receptors (GPCRs) initiate cellular responses to many different stimuli, like neurotransmitters, hormones or photons. They are critical for many physiological processes and their dysregulation frequently leads to human disease, which is also in agreement with the fact that >30% of FDA- approved drugs target GPCRs. GPCRs are key pharmacological targets in neurological and neuropsychiatric diseases based on their function as metabotropic neurotransmitter receptors with a prominent role in neuromodulation. The main mechanism of action of GPCRs is through activation of heterotrimeric G proteins, which are broadly divided in 4 families (Gs, Gi/o, Gq/11, G12/13). However, the mechanisms and consequences of heterotrimeric G protein signaling have been difficult to elucidate because of the lack of adequate experimental tools to manipulate their activity with high precision and specificity in a cellular context. Our goal is to develop a new class of chemogenetic tool to directly activate heterotrimeric G proteins without perturbing GPCRs or other cellular processes. Chemogenetics, in general, refers to a method by which a protein is engineered to interact with previously unrecognized chemical compounds. The tools to be developed here will allow investigators in this field of research to manipulate and dissect the functional consequences of G protein activation with unprecedented precision, thereby revealing fundamental mechanisms that underlie physiological, pathological, or therapeutic modulation of neurotransmitter responses and other biological processes. BACKGROUND: Upon stimulation, GPCRs promote GTP loading on the Gα-subunit of heterotrimeric G proteins (Gαβγ). In turn, Gα-GTP binds to effector proteins to propagate signaling. In the context of neurotransmission, Gα proteins of the Gs (e.g., Gαs) or the Gq/11 (e.g., Gαq) family are primarily neurostimulatory by virtue of their ability to increase cAMP or intracellular Ca2+, respectively. In contrast, Gα proteins of the Gi/o family (e.g., Gαi) cause neuroinhibition via suppression of cAMP. These effects are mediated through direct binding to and modulation of effector proteins that control second messenger levels— i.e., adenylyl cyclases for Gαs and Gαi, or phospholipases C for Gαq. Gβγ also contributes to neuromodulation through the regulation of ion channels. We have envisioned and partially validated a chemogenetic approach to achieve the direct and specific activation of G proteins without the need of GPCRs. SYNOPSIS OF AIMS: In Aim 1, we will identify the components required to engineer Gαi, Gαs, Gαq, or Gβγ proteins that are activated by chemical compounds that do not have known targets or effects in mammalian cells. These constructs will be evaluated by using optical biosensors that directly detect active G proteins. In Aim 2, we will test the performance of these chemically-activated G proteins by using downstream signaling readouts directly dependent on cognate G protein effectors in cell lines and in neuronal primary cultures. .

摘要