权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Novel mechanisms in the control of cAMP dynamics

控制 cAMP 动力学的新机制

基本信息

批准号：
10733273
负责人：
DANIEL L ALTSCHULER
金额：
$ 38.96万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
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：
2023-09-01 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10733273
关键词：
Address Adenylate Cyclase Affect Agonist Binding Biochemistry Biological Biological Assay Cell membrane Cells Chemosensitization Chimera organism Chronic Code Complex Cyclic AMP Data Dose Down-Regulation Event FDA approved Family Feedback Fluorescence Resonance Energy Transfer Forskolin G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Generations Genetic Transcription Goals Guanosine Triphosphate Hormones In Vitro Inflammation Intervention Isoproterenol Laboratories Mammalian Cell Mediating Membrane Migraine Modeling Molecular Monitor Mutation Nuclear Pathway interactions Pharmaceutical Preparations Phase Physiological Protein Isoforms Regulation Role Second Messenger Systems Signal Transduction Source Specificity Structure Testing Therapeutic Intervention Transfection cell type chronic pain experimental study insight interdisciplinary approach interest mutant nanobodies new therapeutic target novel optogenetics overexpression pharmacologic receptor response sensor spatiotemporal

项目摘要

GPCRs are the largest family of mammalian membrane receptors with an established relevance in therapy, representing a third of the currently FDA-approved drugs. Particularly GsPCRs via cAMP are involved in many physiological and pathophysiological conditions. Classically considered to originate solely from the plasma membrane, this view was recently challenged by observations showing that GsPCRs, upon internalization, can sustain cAMP signaling from intracellular compartments. Most importantly, this second endocytic cAMP phase was strictly associated with nuclear transcriptional events responsible for the generation of specific biological responses, like chronic inflammation, chronic pain, and migraine, conditions that affect millions worldwide. However, the molecular mechanisms involved in sustained signaling are still unknown. Their characterization should deepen our understanding of the GPCRs’ spatiotemporal organization and function and may provide novel avenues for therapeutic intervention. We have identified a novel GαS-independent CAP1-Rap1-AC9 regulatory unit and its potential involvement in sustained signaling will be addressed in this proposal. The scientific premise for this proposal is based on the identification of a novel GαS-independent CAP1-Rap1 complex directly regulating AC9. Utilizing novel optogenetic actuators and targeting strategies, we will test the hypothesis that upon the first plasma membrane-generated GαS-dependent cAMP wave, this new intracellular GαS-independent CAP1-Rap1-AC9 regulatory unit is responsible for the sustained cAMP phase. Four integrated specific aims are proposed to experimentally test this hypothesis. In SA #1 we will characterize AC9 as the CAP1-Rap1-sensitive tmAC isoform involved in the potentiation of cAMP dynamics in cells. In SA #2 we will identify and characterize key residues in AC9 that discriminate GαS and Rap1 binding. In SA #3, we will use the ALFA-tag targeting strategy to bring sensors (AC9-ALFA/Nb-ALFA-H188) close to AC9 to test the hypothesis that the AC9-CAP1-Rap1 ternary complex establishes a compartmentalized positive feedback loop responsible for the GPCR sustained cAMP wave. Finally, in SA #4, we will use GαS- and Rap1-targeted disruptors to uncouple functional GαS- and Rap1-dependent cyclase activation events in the plasma membrane and intracellular AC9 compartments. Our laboratory’s long-standing interest is to understand the spatiotemporal regulation of cAMP-dependent signaling events. A multidisciplinary approach combining biochemistry, optogenetics, and nanobody-targeted strategies will be exploited to test the role of AC9-CAP1-Rap1 in compartmentalized cAMP signaling. Successful completion of the proposed studies should provide new insights into the mechanism/s cells utilize to compute (code/decode) the relay of the cAMP signal (i.e., fidelity, specificity, efficiency) and provide us opportunities in the identification of new subcellular localized targets for pharmacological intervention of this critical second messenger pathway.

gpcr是哺乳动物膜受体中最大的家族，在治疗中具有明确的相关性，