Transduction Mechanisms of Opioid Agonist Efficacy

描述（由申请人提供）：阿片类药物（如吗啡）激活Gi/o蛋白偶联的μ型阿片受体（mu-OR）。μ-阿片样物质激动剂产生一系列作用，包括镇痛、呼吸抑制、镇静、诱导耐受性和依赖性以及成瘾倾向。药物最大效应的大小取决于其内在功效，或其激活受体的程度。这些研究将通过关注细胞信号转导来研究μ-阿片内在功效的机制。在上一个资助期，研究表明，激动剂激活G蛋白的功效取决于GDP和钠浓度、受体密度、受体-换能器放大、脱敏和脑区等因素。拟议项目的主要目标如下。首先，我们将检验阿片配体的相对疗效取决于特定G-α亚型的假设。将稳定表达mu-OR的CHO-K1细胞（MOR-CHO）与不同Gi/o亚型的百日咳毒素（PTX）不敏感突变体共转染，用PTX处理以消除与内源性Gi蛋白的偶联，并通过[35 S] GTP-γ S结合和腺苷酸环化酶抑制来确定不同完全和部分激动剂的相对效力。 将根据相对于标准完全激动剂的最大刺激对配体进行分级，以区分配体功效的幅度或等级顺序的变化。突变体G-α亚型之间的差异将通过野生型G-α亚基的表达和RNA干扰敲低来确认。这些研究的结果将用于指导使用受体和G蛋白放射自显影方法进行的阿片类药物在大脑中的疗效研究。第二个目的是检验μ阿片样物质配体的相对功效在G-α和G-β γ调节的效应物之间不同的假设。这些研究将比较这些配体在完整MOR-CHO细胞中通过G-α抑制腺苷酸环化酶与通过G-β γ激活促有丝分裂激酶的相对功效。然后将这些配体在24小时内维持促有丝分裂激酶活化的相对效力与它们刺激细胞增殖的相对效力进行比较，并确定它们对程序性细胞死亡和细胞周期进程的影响。由于mu-OR脱敏的差异，将检查急性与持续效应激活的配体功效的差异，并且将使用药理学抑制剂和特异性信号传导蛋白的显性负突变体研究不同配体的有丝分裂与细胞毒性机制的差异。通过阐明阿片配体效应和配体选择性/μ-OR信号转导机制，这些研究可能为阿片药物治疗提供新的方向。

DESCRIPTION (provided by applicant): Opioid drugs such as morphine activate Gi/o-protein-coupled mu-type opioid receptor (mu-OR). mu-opioid agonists produce an array of effects, including analgesia, respiratory depression, sedation, induction of tolerance and dependence, and addiction liability. The magnitude of a drug's maximal effect depends on its intrinsic efficacy, or the degree to which it activates receptors. These studies will investigate the mechanisms of mu-opioid intrinsic efficacy by focusing on cellular signal transduction. In the previous funding period, studies revealed that agonist efficacy to activate G-proteins is dependent on factors including GDP and sodium concentrations, receptor density, receptor-transducer amplification, desensitization and brain region. The major goals of the proposed project are as follows. First, we will test hypothesis that relative efficacy of opioid ligands depends on specific G-alpha subtypes. CHO-K1 cells stably expressing mu-ORs (MOR-CHO) will be co-transfected with pertussis toxin (PTX)-insensitive mutants of different Gi/o subtypes, treated with PTX to eliminate coupling to endogenous Gi-proteins, and the relative efficacies of different full and partial agonists will be determined by [35S]GTP-gammaS binding and adenylyl cyclase inhibition. Ligands will be ranked according to maximal stimulation relative to a standard full agonist to distinguish changes in the magnitude or rank order of ligand efficacy. Differences among mutant G-alpha subtypes will be confirmed by expression of the wild-type G-alpha subunits and by knockdown with RNA interference. Results of these studies will be used to guide studies of opioid efficacy in the brain using receptor and G-protein autoradiographic approaches. The second aim is to test the hypothesis that relative efficacies of mu opioid ligands differ between G-alpha and G-beta gamma regulated effectors. These studies will compare the relative efficacies of these ligands for inhibition of adenylyl cyclase by G-alpha versus activation of mitogenic kinases by G-beta gamma in intact MOR-CHO cells. The relative efficacies of these ligands for sustaining activation of mitogenic kinases over 24 hr will then be compared to their relative efficacies for stimulating cell proliferation, and their effects on programmed cell death and cell cycle progression will be determined. Differences in ligand efficacy for acute versus sustained effector activation due to differences in mu-OR desensitization will be examined, and differences in mechanisms of mitogenesis versus cytotoxicity by different ligands will be investigated with pharmacological inhibitors and dominant negative mutants of specific signaling proteins. By elucidating the mechanisms of opioid ligand efficacy and ligandselective/ mu-OR signaling, these studies may provide new directions in opioid pharmacotherapeutics.