来自有尾两栖动物粗皮蝾螈（Taricha granulosa）大脑的δ和μ阿片受体：克隆、异源表达及药理学特性分析

Delta and mu opioid receptors from the brain of a urodele amphibian, the rough-skinned newt Taricha granulosa: cloning, heterologous expression, and pharmacological characterization.

作者信息

Bradford C Samuel, Walthers Eliza A, Stanley David J, Baugh Martha M, Moore Frank L

机构信息

Department of Zoology, Oregon State University, Corvallis, OR 97331, USA.

出版信息

Gen Comp Endocrinol. 2006 May 1;146(3):275-90. doi: 10.1016/j.ygcen.2005.11.002. Epub 2005 Dec 20.

DOI:10.1016/j.ygcen.2005.11.002

PMID:16375901

Abstract

Two full-length cDNAs, encoding delta (delta) and mu (mu) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (kappa) and ORL1 opioid receptors. The newt delta receptor shares 82% amino acid sequence identity with a frog delta receptor and lower (68-70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt mu receptor shares 79% sequence identity with a frog mu receptor, 72% identity with mammalian mu receptors, and 66-69% identity with mu receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt delta receptor possessed a single, high-affinity (Kd = 2.4 nM) binding site for the nonselective opioid antagonist [3H]naloxone. In competition binding assays, the newt delta receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, beta-endorphin, and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) (a delta-selective agonist in mammals), and very low affinity for mu-, kappa-, or ORL1-selective agonists. COS-7 cells expressing the newt mu receptor also possessed a high-affinity (Kd = 0.44 nM) naloxone-binding site that showed highest affinity for beta-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a mu-selective agonist in mammals), and very low affinity for DPDPE and kappa- or ORL1-selective agonists. COS-7 cells expressing either receptor type (delta or mu) showed very high affinity (Kd = 0.1-0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (delta, mu, kappa, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.

摘要

从糙皮蝾螈（Taricha granulosa）的大脑中克隆出了两个全长cDNA，分别编码δ（delta）和μ（mu）阿片受体，这补充了我们实验室之前关于蝾螈大脑κ（kappa）和ORL1阿片受体克隆的工作。蝾螈δ受体与青蛙δ受体的氨基酸序列同一性为82%，与从哺乳动物和斑马鱼克隆的直系同源受体的同一性较低（68 - 70%）。蝾螈μ受体与青蛙μ受体的序列同一性为79%，与哺乳动物μ受体的同一性为72%，与从硬骨鱼克隆的μ受体的同一性为66 - 69%。从瞬时表达蝾螈δ受体的COS - 7细胞中分离出的膜，对非选择性阿片拮抗剂[³H]纳洛酮具有单一的高亲和力（Kd = 2.4 nM）结合位点。在竞争结合试验中，蝾螈δ受体对甲硫氨酸脑啡肽显示出最高亲和力，对亮氨酸脑啡肽、β - 内啡肽和[D - 青霉胺，D - 青霉胺]脑啡肽（DPDPE，一种在哺乳动物中为δ选择性激动剂）的亲和力相对较低，对μ、κ或ORL1选择性激动剂的亲和力非常低。表达蝾螈μ受体的COS - 7细胞也具有一个高亲和力（Kd = 0.44 nM）的纳洛酮结合位点，该位点对β - 内啡肽显示出最高亲和力，对甲硫氨酸脑啡肽、亮氨酸脑啡肽和DAMGO（一种在哺乳动物中为μ选择性激动剂）的亲和力为中度至低度，对DPDPE以及κ或ORL1选择性激动剂的亲和力非常低。表达任何一种受体类型（δ或μ）的COS - 7细胞对非选择性阿片拮抗剂二丙诺啡显示出非常高的亲和力（Kd = 0.1 - 0.3 nM）。糙皮蝾螈表达了在其他脊椎动物类群中发现的相同四种阿片受体亚型（δ、μ、κ和ORL1），但蝾螈中的配体选择性似乎不如在哺乳动物中记录的那样严格。