Novel mechanisms in the control of cAMP dynamics

GPCR是哺乳动物膜受体的最大家族，在治疗中具有确定的相关性， 占目前FDA批准药物的三分之一。特别是通过cAMP的GsPCR参与了许多 生理和病理生理条件。传统上被认为仅来源于血浆 膜，这一观点最近受到挑战的观察表明，GsPCR，在内化， 维持来自细胞内区室的cAMP信号传导。最重要的是，这第二个内吞cAMP阶段 与核转录事件密切相关，核转录事件负责产生特定的生物活性。 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，参与持续信号传导的分子机制仍然是未知的。试者的表征 应该加深我们对GPCR的时空组织和功能的理解，并可能提供 治疗干预的新途径。我们已经鉴定了一个新的Gα S非依赖性CAP 1-Rap 1-AC 9 调节单元及其在持续信号传导中的潜在参与将在本提案中得到解决。 这一建议的科学前提是基于一个新的Gα S独立的CAP 1-Rap 1的鉴定 直接调节AC 9的复合物。利用新型光遗传学致动器和靶向策略，我们将测试 假设在第一个质膜产生的Gα S依赖性cAMP波，这个新的细胞内 Gα S非依赖性CAP 1-Rap 1-AC 9调节单位负责维持cAMP相。 四个集成的具体目标，提出了实验测试这一假设。在SA #1中，我们将描述 AC 9作为CAP 1-Rap 1敏感的tmAC同种型参与细胞中cAMP动力学的增强。在SA #2中 我们将鉴定和表征AC 9中区分GαS和Rap 1结合的关键残基。在SA #3中，我们将 使用ALFA标签靶向策略使传感器（AC 9-ALFA/Nb-ALFA-H188）靠近AC 9，以测试 假设AC 9-CAP 1-Rap 1三元复合物建立了一个区室化的正反馈回路 负责GPCR持续的cAMP波。最后，在SA #4中，我们将使用GαS-和Rap 1-靶向 在质膜中解偶联功能性GαS-和Rap 1-依赖性环化酶激活事件的干扰物 和细胞内AC 9区室。 我们实验室长期以来的兴趣是了解cAMP依赖性的时空调控。 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 将利用这些策略来测试AC 9-CAP 1-Rap 1在区室化cAMP信号传导中的作用。 成功完成拟议的研究应提供新的见解的机制/s细胞利用 为了计算（编码/解码）cAMP信号的中继（即，忠诚、专一、高效），并为我们提供 在确定新的亚细胞定位靶点的药理学干预的机会， 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.