Novel mechanisms in the control of cAMP dynamics

控制 cAMP 动力学的新机制

• 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
• 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

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.

GPCRs是哺乳动物最大的膜受体家族，在治疗中具有既定的相关性， 占目前FDA批准的药物的三分之一。特别是通过cAMP的GsPCR参与了许多 生理和病理生理条件。经典地被认为完全起源于等离子体 膜，这一观点最近受到了挑战的观察表明，GsPCRs，在内化后，可以 维持细胞内隔间的cAMP信号。最重要的是，这第二个内吞阵营阶段 严格地与负责产生特定生物的核转录事件有关 慢性炎症、慢性疼痛和偏头痛等影响全球数百万人的疾病。 然而，参与持续信号传递的分子机制仍不清楚。他们的性格特征 应加深我们对GPCRs的时空组织和职能的理解，并可提供 治疗干预的新途径。我们已经鉴定出一种新的G-αS-非依赖性CAP1-RAP1-AC9 本提案将涉及监管单位及其对持续信令的潜在参与。 这一提议的科学前提是基于鉴定出一种新的GαS独立的CAP1-RAP1 直接调控AC9的复合体。利用新的光生致动器和目标策略，我们将测试 假设在第一个质膜产生的GαS依赖的cAMP波之后，这个新的细胞内 GαS-独立的CAP1-RAP1-AC9调控单位负责持续的cAMP阶段。 提出了四个综合的具体目标来实验验证这一假说。在SA#1中，我们将描述 AC9作为Cap1-Rap1敏感的TMAC异构体，参与细胞内cAMP动力学的增强。在SA#2中 我们将鉴定和鉴定AC9中区分G、α、S和RAP1结合的关键残基。在SA#3中，我们将 使用Alfa-Tag目标策略将传感器(AC9-ALFA/NB-ALFA-H188)带到AC9附近，以测试 假设AC9-Cap1-Rap1三元复合体建立了一个分隔的正反馈环 对gpr持续的cAMP波负责。最后，在SA#4中，我们将使用GαS和RAP1-目标 解偶联质膜GαS和RAP1依赖的环化酶激活事件的干扰物 和细胞内AC9隔室。 我们实验室长期以来的兴趣是了解cAMP依赖的时空调节 信号事件。一种结合生物化学、光遗传学和纳米体靶向的多学科方法 将利用策略来测试AC9-cap1-Rap1在cAMP信号区隔中的作用。 成功完成拟议的研究应该会为细胞利用的机制提供新的见解/S 计算(编码/解码)CAMP信号的中继(即保真度、特异度、效率)并为我们提供 为药物干预寻找新的亚细胞定位靶点的机会 关键的第二信使途径。