CLONING OF A PUTATIVE NON OPIOID BINDING PROTEIN OF DYNA

目前提出的目的是克隆一种非阿片结合蛋白。内源性阿片肽dynorphinA-(1-17)及其类似物dynorphinA-(2-17)对kappa阿片受体具有高亲和力，但大量证据表明它们也与非阿片蛋白结合。dynorphin的一些特性阻碍了该蛋白的鉴定，包括其与生物组织的高度非特异性结合，对蛋白质水解降解的敏感性以及缺乏对其初级结构的了解。本文提出了两种互补的方法来规避这些问题，这两种方法都被应用于dynorphinA-(2-17)，它不与阿片受体结合，对于假定的非阿片蛋白来说，它将是一个更具选择性的配体。首先，酵母双杂交系统将用于识别任何与DynA-(2-17)相互作用的分子。其次，表达克隆和同胞选择将用于鉴定结合DynA-的克隆（2-17）。DynA-(1-17)及其类似物DynA-(2-17)在脑内无镇痛作用，但能增强吗啡耐受/依赖动物的抗痛觉作用，抑制戒断症状。因此，它在改善长期需要吗啡的患者的疗效以及治疗阿片类药物成瘾方面具有很大的临床前景。鉴定介导这些作用的结合蛋白对这些进展至关重要。

The purpose of the current proposal is to clone a non-opioid binding protein. The endogenous opioid peptide dynorphinA-(1-17) and its analog dynorphinA-(2-17) have high affinity for kappa opioid receptors but considerable evidence indicates that they also bind to a non-opioid protein. Identification of this protein has been hindered by several features of dynorphin, including its high non-specific binding to biological tissue, its sensitivity to proteolytic degradation and a lack of knowledge of its primary structure. Two complementary approaches are proposed here to circumvent these problems, both being applied to dynorphinA-(2-17), which does not bind to opioid receptors and would be a more selective ligand for the putative non-opioid protein. First, the yeast two-hybrid system will be used to identify any molecules interacting with DynA-(2-17). Second, expression cloning and sib selection will be used to identify clones that bind DynA-(2-17). DynA-(1-17) and its analog dynA- (2-17) are not analgesic in the brain but can enhance morphine antinociception and suppress withdrawal symptoms in morphine tolerant/dependent animals. Thus, it has great clinical promise in improving the efficacy of morphine in patients who require the drug chronically, as well as a treatment for opioid addiction. Identification of the binding protein mediating these effects is critical to these advances.