Novel mechanisms in the control of cAMP dynamics

GPCR 是最大的哺乳动物膜受体家族，在治疗中具有明确的相关性， 占目前 FDA 批准的药物的三分之一。特别是通过 cAMP 的 GsPCR 涉及许多 生理和病理生理条件。传统上认为仅源自等离子体 膜，这一观点最近受到观察的挑战，观察表明 GsPCR 在内化后可以 维持细胞内区室的 cAMP 信号传导。最重要的是，第二个内吞 cAMP 阶段 与负责产生特定生物的核转录事件严格相关 反应，如慢性炎症、慢性疼痛和偏头痛，这些疾病影响着全世界数百万人。 然而，持续信号传导所涉及的分子机制仍然未知。他们的表征 应加深我们对 GPCR 时空组织和功能的理解，并可能提供 治疗干预的新途径。我们发现了一种新型的不依赖 GαS 的 CAP1-Rap1-AC9 本提案将讨论监管单位及其对持续信号的潜在参与。 该提案的科学前提是基于一种新型的独立于 GαS 的 CAP1-Rap1 的鉴定 复杂的直接调节AC9。利用新型光遗传学执行器和靶向策略，我们将测试 假设在第一个质膜产生的 GαS 依赖性 cAMP 波上，这种新的细胞内 独立于 GαS 的 CAP1-Rap1-AC9 调节单元负责持续的 cAMP 阶段。 提出了四个综合的具体目标来通过实验检验这一假设。在 SA #1 中，我们将描述 AC9 作为 CAP1-Rap1 敏感的 tmAC 同工型，参与细胞中 cAMP 动力学的增强。在南澳#2 我们将识别并表征 AC9 中区分 GαS 和 Rap1 结合的关键残基。在 SA #3 中，我们将 使用 ALFA 标签定位策略将传感器 (AC9-ALFA/Nb-ALFA-H188) 靠近 AC9 以测试 假设 AC9-CAP1-Rap1 三元复合物建立了一个分隔的正反馈回路 负责 GPCR 持续的 cAMP 波。最后，在 SA #4 中，我们将使用 GαS 和 Rap1 为目标 破坏质膜中功能性 GαS 和 Rap1 依赖的环化酶激活事件 和细胞内 AC9 区室。 我们实验室的长期兴趣是了解 cAMP 依赖性的时空调节 信号事件。结合生物化学、光遗传学和纳米抗体靶向的多学科方法 将利用策略来测试 AC9-CAP1-Rap1 在区室化 cAMP 信号传导中的作用。 拟议研究的成功完成将为细胞利用机制提供新的见解 计算（编码/解码）cAMP 信号的中继（即保真度、特异性、效率）并为我们提供 确定新的亚细胞局部靶点以进行药物干预的机会 关键的第二信使途径。

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.