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Isolation and cloning of VIP receptors.

VIP 受体的分离和克隆。

基本信息

批准号：
03454101
负责人：
ITO Shigeo
金额：
$ 3.84万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454101/
关键词：
vasoactive intestinal peptide canine liver VIP binding experiment PACAP VIP receptors cloning vascactive imtestinal peptide パラサイロイドホルモン受容体 Vasoactive intestinal peptide VIP受溶体イヌ肝臓細胞膜 PCR法

项目摘要

The binding characteristic of vasoactive intestinal peptide (VIP) receptors was examined using radioligand binding assay with iodinated VIP and unlabelled peptides in liver mambranes of the dog. VIP binding to canine liver membranes was rapid, reversible, saturable, specific and dependent on temperature. Guanine nucleotides dose-dependently inhibited VIP binding. Stoichiometric data suggested the presence of two classes of VIP binding sites. The order of potency by competition experiments with unlabelled peptides was: VIP > pituitary adenylate cyclase activating peptide (PACAP)-27 > PACAP-38 >> peptide histidine isoleucine (PHI) = secretin in the dog and PACAP-27 > PACAP-38 > VIP > PHI > secretin in the rat. PHI and secretin were about 5000 times less potent than VIP in the dog but about 100 times less potent in the rat. It was concluded that the VIP receptors in canine liver membranes possesses recognition sites for VIP which were distinct from those in rat liver membranes. To obtain DNA probe for screening. VIP receptor gene of the rat liver was amplified by polymerase chain reaction using synthesized oligonucleotide primers. The products, 540-base-pair fragments, were purified and served as the probe after labelling. A cDNA library (300000 clones) derived from canine liver was screened under low stringency conditions. Six positive colonies were detected. Four out of 6 clones with larger inserts (1-1.5 kbb) were subjected to nucleotide sequence analysis. #35 clone has a nucleic acid sequence identical to parathyroid hormone receptors of the rat and opossum. #102 clone has a nucleic acid sequence similar to rat VIP receptors but not #201 and #513 clones. The analysis of #102 clone is in progress.

用放射性配基结合分析法研究了犬肝膜血管活性肠肽（VIP）受体与碘标记VIP和未标记VIP的结合特性。VIP与犬肝膜的结合是快速的、可逆的、可饱和的、特异性的，并且依赖于温度。鸟嘌呤核苷酸剂量依赖性地抑制VIP结合。化学计量数据表明存在两类VIP结合位点。与未标记肽竞争实验的效力顺序为：VIP>垂体腺苷酸环化酶激活肽（PACAP）-27> PACAP-38>>肽组氨酸异亮氨酸（PHI）=促胰液素（狗）和PACAP-27> PACAP-38> VIP> PHI>促胰液素（大鼠）。PHI和分泌素在狗中的效力比VIP低约5000倍，但在大鼠中的效力低约100倍。结论：犬肝膜VIP受体具有与大鼠肝膜不同的VIP识别位点。获得筛选用DNA探针。用合成的寡核苷酸引物，通过聚合酶链反应扩增了大鼠肝脏VIP受体基因。纯化产物，即540个碱基对的片段，标记后用作探针。在低严格性条件下筛选来自犬肝脏的cDNA文库（300000个克隆）。检测到6个阳性菌落。对6个具有较大插入物（1 - 1.5kbb）的克隆中的4个进行核苷酸序列分析。#35克隆具有与大鼠和负鼠的甲状旁腺激素受体相同的核酸序列。#102克隆具有与大鼠VIP受体相似的核酸序列，但不具有与#201和#513克隆相似的核酸序列。#102克隆的分析正在进行中。