Isolation and cloning of VIP receptors.

采用碘化肠肽和肝膜未标记肽的放射配体结合试验，研究了血管活性肠肽（VIP）受体的结合特性。VIP与犬肝膜的结合具有快速、可逆、饱和、特异性和温度依赖性。鸟嘌呤核苷酸剂量依赖性抑制VIP结合。化学计量数据表明存在两类VIP结合位点。与未标记肽竞争的效价顺序为：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) =狗分泌素，PACAP-27 > PACAP-38 > VIP > PHI >分泌素。在狗体内，PHI和分泌素的效力大约是VIP的5000倍，而在大鼠体内的效力大约是VIP的100倍。结果表明，犬肝膜VIP受体具有与大鼠肝膜不同的VIP识别位点。获取DNA探针进行筛选。利用合成的寡核苷酸引物，采用聚合酶链反应扩增大鼠肝脏VIP受体基因。产物540碱基对片段被纯化，标记后作为探针。在低严格条件下，从犬肝脏中筛选了一个cDNA文库（30万个克隆）。检测到6个阳性菌落。6个较大插入片段（1-1.5 kbb）的克隆中有4个进行了核苷酸序列分析。35号克隆体的核酸序列与大鼠和负鼠的甲状旁腺激素受体相同。#102克隆具有与大鼠VIP受体相似的核酸序列，但与#201和#513克隆不同。102号克隆的分析正在进行中。

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.