Novel mechanism on subpopulation-dependent biased GPCR signaling in neurons

摘要 肾上腺素能受体(Ars)是一个典型的GPCRs家族，与神经元紊乱、代谢紊乱有关。 综合症和心血管疾病。在中枢神经系统，去甲肾上腺素(NE)调节注意力和警觉性。 β-2AR是谷氨酸突触中普遍存在的突触后去甲肾上腺素效应器，它在那里相互作用 用AMPAR，NMDAR和L型钙通道Cav1.2调节神经元的兴奋性，突触可塑性， 以及记忆和学习。了解与NE相关的精神疾病具有临床意义 抑郁症、注意缺陷多动障碍(ADHD)、焦虑症(如创伤后应激 精神障碍、创伤后应激障碍)和阿尔茨海默病。而β阻滞剂用于治疗各种外周血管疾病 包括心力衰竭、高血压、青光眼、哮喘和慢性阻塞性肺病在内的疾病，其临床用途是 受到包括焦虑和抑郁在内的副作用的阻碍。最近的爆炸性的结晶学研究 配体-gpr相互作用，对特定配体如何导致多效性的理解仍然有限。 GPCR的细胞反应(包括副作用)。在这项研究中，我们假设一个不同的 PKA磷酸化的β2ARs亚群控制着海马神经元LTCC的激活，这可以 被一组有偏向的配体选择性地激活。我们将通过以下具体目标来验证我们的假设： 目的1是检验β2AR可以存在于不同功能亚群的假设 哺乳动物细胞。我们将使用生化分离/分离和超分辨率成像来 研究PKA-pβ2AR和Grk-pβ2AR在不同亚细胞中的分布。目标2是检验这一假设 PKA-pβ2AR通过选择性地调制离子通道活性来传递偏置信号 质膜(PM)。目标3是检验交感神经β阻滞剂作为偏向作用的假设 选择性激活β2AR的PKA磷酸化亚群以激活离子通道的配体 下午。如果成功，这些目标将揭示一个平台，以理解由两个 β2AR的不同亚群，并为设计更有效的β-AR药物提供了新的途径 在临床应用中副作用较少。

Summary Adrenergic receptors (ARs) are a family of prototypical GPCRs linked to neuronal disorders, metabolic syndrome, and cardiovascular diseases. In the CNS, norepinephrine (NE) regulates attention and alertness. The β2AR is emerging as the prevalent postsynaptic NE effector at glutmatergic synapses, where it interacts with AMPAR, NMDAR and L-type Ca2+ channel Cav1.2 to modulate neuronal excitability, synaptic plasticity, and memory and learning. It is clinically relevant to understand NE-linked mental diseases such as depression, attention deficit hyperactivity disorder (ADHD), anxiety disorders (e.g., posttraumatic stress disorder, PTSD) and Alzheimer's disease. While β-blockers are used to treat a variety of peripheral diseases including heart failure, hypertension, glaucoma, asthma, and COPD, their clinical utility is hampered by the side effects including anxiety and depression. Recent explosion of crystallography study of ligand-GPCR interactions, there is still limited understanding on how a specific ligand leads to pleotropic cellular responses (including sides effects) of a GPCR. In this study, we hypothesize that a distinct subpopulation of PKA-phosphorylated β2ARs control LTCC activation in hippocampal neurons, which can be selectively activated by a set of biased ligands. We will test our hypothesis with following specific aims: Aim 1 is to test the hypothesis that β2AR can exist in distinct functional subpopulations in a single mammalian cell. We will use biochemical isolation/fractionation and super-resolution imaging to characterize distinct subcellular distribution of PKA-pβ2AR and GRK-pβ2AR. Aim 2 is to test the hypothesis that PKA-pβ2AR transduce biased signal through selectively modulation of ion channel activity at the plasma membrane (PM). Aim 3 is to test the hypothesis that sympathomimetic β-blockers act as biased ligands that selectively activate PKA-phosphorylated subpopulation of β2AR to activate ion channel at the PM. If successful, these aims will reveal a platform to understand the biased signaling induced by two distinct subpopulations of β2AR, and offering a new avenues for designing more efficacious β-AR drugs with fewer side effects in clinical applications.