MUTATIONAL LIBRARIES TO STUDY OPIOID RECEPTOR FUNCTION

描述：(申请人摘要) 点突变已被证明是有效的识别 结构型氨基酸参与G蛋白偶联受体功能。 然而，合适的突变体的产生需要先入为主的 关于哪些氨基酸可能相关的假说。对于许多人来说 受体的功能很难假设先验地参与了 特定的氨基酸及其可能的性质 很重要。该方案的目的是产生区域选择性随机 应用聚合酶链式反应策略构建阿片受体突变文库。 最初，这些文库将用于筛选干扰基因的突变 MU-阿片受体隔离。此前我们已经发现内源性的 阿片肽和生物碱的一个子集可以诱导隐匿 MU-阿片受体，而吗啡和许多临床使用的阿片类药物 不要触发受体内化，或仅微弱地触发内化。这 提示具有类似作用的激动剂配体 受体介导的信号转导可能会对 受体交易。这项研究的目的是使用随机突变 了解配体依赖受体分子基础的文库 内部化。在未来，结果应该有助于澄清 内化在阿片类药物诱导发展中的意义 宽容和依赖。

DESCRIPTION: (Applicant's Abstract) Point mutations have proven to be powerful for the identification of structural amino acids involved in Gprotein coupled receptor function. However, the generation of appropriate mutants necessitates a preconceived hypothesis regarding which amino acids may be relevant. For a number of receptor functions it is difficult to assume a priori the involvement of particular amino acids and what properties of the amino acids might be important. This proposal aims to generate region-selective random mutational libraries of the mu-opioid receptor using PCR strategies. Initially these libraries will be used to screen for mutations that perturb mu-opioid receptor sequestration. Previously we have found that endogenous opioid peptides and a subset of alkaloids can induce sequestration of the mu-opioid receptor, whereas morphine and many clinically-used opiate drugs do not trigger receptor internalization, or do so only weakly. This suggests that agonist ligands which have similar effects on receptor-mediated signaling can have dramatically different effects on receptor trafficking. The aim of the study is to use the random mutational libraries to understand the molecular basis for ligand-dependent receptor internalization. In the future, the results should help to clarify the significance of internalization in the development of opiate drug-induced tolerance and dependence.