Transduction Mechanisms of Opioid Agonist Efficacy

描述(申请人提供)：阿片类药物如吗啡激活Gi/o蛋白偶联的mu型阿片受体(MU-OR)。MU-阿片激动剂产生一系列的作用，包括镇痛、呼吸抑制、镇静、诱导耐受和依赖以及成瘾倾向。一种药物的最大作用的大小取决于它的内在疗效，或它激活受体的程度。这些研究将从细胞信号转导的角度探讨Mu-阿片类药物的内在疗效机制。在之前的资助期间，研究表明，激动剂激活G蛋白的效果取决于GDP和钠浓度、受体密度、受体转导放大、脱敏和大脑区域等因素。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-ORs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[35S]GTP-Gamma结合和腺苷环化酶抑制来确定不同完全和部分激动剂的相对效果。配基将根据相对于标准全激动剂的最大刺激进行排序，以区分配基效力的大小或等级顺序的变化。突变的G-α亚型之间的差异将通过野生型G-α亚基的表达和RNA干扰敲除来证实。这些研究的结果将被用来指导使用受体和G蛋白放射自显影方法研究阿片类药物在大脑中的疗效。第二个目的是验证假设，即在G-α和G-β伽马调节的效应器之间，MU阿片配体的相对有效性不同。这些研究将比较这些配体在完整的MOR-CHO细胞中由G-α抑制腺酰环化酶与由G-β-伽马激活有丝分裂酶的相对效率。然后将这些配体在维持有丝分裂酶激活超过24小时的相对效率与它们刺激细胞增殖的相对效率进行比较，并确定它们对细胞程序性死亡和细胞周期进程的影响。将研究由于MU-OR脱敏作用的差异而导致的急性和持续效应器激活的配基效力的差异，并将使用药理抑制剂和特定信号蛋白的显性负突变来研究不同配基的有丝分裂和细胞毒性机制的差异。通过阐明阿片配体的作用机制和配体选择/Mu-OR信号转导机制，这些研究可能为阿片类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.