Ligand Directed Functional Selectivity of G-Protein Coupled Receptor Signalling

描述（由申请人提供）：所有组织中的细胞对许多外部刺激（例如光、气味、神经递质、激素）的转导是通过激活称为 G 蛋白偶联受体 (GPCR) 的大型基因超家族的成员来进行的，该基因超家族通过大量且不断扩展的细胞内和膜结合蛋白将受体激活与复杂的信号事件级联耦合。 GPCR 转导过程的一个共同特征是异源三聚体 G 蛋白的中介。激动剂与 GPCR 结合后，同源 G1 和 G23 亚基的解离或重排被认为会触发各种酶、离子通道和其他效应分子的刺激或抑制。经典受体理论表明，特定 GPCR 的任何激动剂都会诱导相同的构象变化，并且激动剂之间功效和效力的差异仅反映偶联效率（信号强度）和相对结合亲和力。最近关于许多 GPCR 的证据表明存在配体特异性构象，这些构象优先运输受体激活以选择信号通路的子集，从而通过相同的 GPCR 同种型导致不同激动剂的不同信号级联。就人类 D2 多巴胺受体而言，我们发现了 K 通道和 Ca 通道、腺苷酸环化酶和 MAP 激酶在暴露于激动剂喹吡罗、DHX 和 NPA 后的选择性激活模式（效力和内在活性）。激动剂作用的这种“功能选择性”的潜在分子机制尚不清楚。在拟议的研究中，我们将通过四个具体目标来研究神经内分泌 AtT20 细胞系和原代大鼠神经元中功能性受体信号复合物的潜在机制：（1）定义与五个效应器偶联的 D2 和 D3 多巴胺受体的配体依赖性选择性；腺苷酸环化酶 (AC)、Kir3.0 通道、CaV2.0 通道、p42/44 MAP 激酶 (Erk1/2) 和 2-arrestin 易位。 (2) 评估大鼠中脑神经元天然 D2 受体信号传导的功能选择性参数。 (3) 测试跨膜结构域（TM2、TM5、TM7）和细胞内环中的保守残基（先前显示为其他 GPCR 中的信号开关）对通过 D2 受体的激动剂信号传导的功能选择性的贡献。 (4) 检验特定受体/G1 蛋白复合物赋予激动剂依赖性功能选择性的假设。这些实验将增进对分子间相互作用在确定多巴胺能配体的效力和功效方面的作用的理解，进而对针对几种神经精神和内分泌疾病的药物的开发产生影响。 公共健康相关性：这项研究探索了一种称为“功能选择性”的新型信号传导现象的分子基础，通过这种现象，不同的药物和神经递质可以通过相同的神经递质受体亚型引导非常不同的信号传导结果。我们将重点关注与药物滥用行为以及精神分裂症等几种精神疾病广泛相关的一类重要的多巴胺受体。由于受体是药物治疗的重要靶点，我们的研究结果将指导开发针对这些疾病的更精细、更特异性的药物，且副作用更少。

DESCRIPTION (provided by applicant): Transduction of many external stimuli (e.g. light, odorants, neurotransmitters, hormones) by cells in all tissues occurs through activation of members of a large gene superfamily called G-protein coupled receptors (GPCRs) that couple receptor activation to complex cascades of signaling events through a vast and expanding array of intracellular and membrane bound proteins. A common feature of the transduction process for GPCRs is the intermediation of heterotrimeric G-proteins. Upon agonist binding to GPCRs, dissociation or rearrangement of the cognate G1 and G23 subunits is thought to trigger stimulation or inhibition of various enzymes, ion channels, and other effector molecules. Classical receptor theory suggests that any agonist of a specific GPCR will induce the same conformational changes, and that differences in efficacy and potency among agonists reflect only the coupling efficiency (strength of signal) and relative binding affinity. Recent evidence for a number of GPCRs has alternatively suggested the existence of ligand-specific conformations that preferentially traffic receptor activation to select subsets of signaling pathways resulting in distinct signal cascades for different agonists through the same GPCR isoform. In the case of the human D2 dopamine receptor, we have discovered selective activation patterns (potency and intrinsic activity) for K and Ca channels, adenylyl cyclase, and MAP kinase upon exposure to the agonists quinpirole, DHX, and NPA. The underlying molecular mechanisms for this "functional selectivity" of agonist action are unknown. In the proposed research we will examine mechanisms underlying functional receptor-signal complexes in the neuroendocrine AtT20 cell line and primary rat neurons through four specific aims: (1) To define ligand- dependent selectivity of D2 and D3 dopamine receptors coupled to five effectors; adenylyl cyclase (AC), Kir3.0 channels, CaV2.0 channels, p42/44 MAP kinase (Erk1/2), and 2-arrestin translocation. (2) To assess the parameters of functional selectivity for native D2 receptor signaling in rat midbrain neurons. (3) To test the contribution of conserved residues in transmembrane domains (TM2, TM5, TM7) and intracellular loops previously shown to be signal switches in other GPCRs to functional selectivity of agonist signaling through D2 receptors. (4) To test the hypothesis that specific receptor/G1-protein complexes confer agonist-dependent functional selectivity. These experiments will increase understanding of the role of intermolecular interactions in specifying the potency and efficacy of dopaminergic ligands and, in turn, will have impact on the development of drugs targeted toward several neuropsychiatric and endocrine disorders. PUBLIC HEALTH RELEVANCE: This research explores the molecular basis for a novel signaling phenomenon, termed "functional selectivity", by which different drugs and neurotransmitters can direct very different signaling outcomes through the same isoform of neurotransmitter receptor. We will focus on an important class of dopamine receptors widely implicated in substance abuse behaviors as well as several psychiatric disorders such as schizophrenia. As the receptors are important targets for pharmaco-therapies, the outcome of our research will guide the development of more refined and specific drugs for these disorders with fewer side effects.